Patent Application
20230253066
The Anfinsen hypothesis states that a protein's sequence encodes its tertiary structure and underlying function (1). Conversely, a protein's tertiary structure encodes the possible sequences compatible with a particular function. De novo protein design has succeeded in the creation of proteins that fold to various targeted tertiary structures (structure to sequence) (2, 3). Nevertheless, it has been extremely challenging to design proteins that not only fold but also bind to complex small molecules (function/structure to sequence) (2-4). Algorithms optimized for packing apolar protein cores struggle to design polar cavities required for binding hydrophilic molecules (5). Consequently, design of small-molecule-binding proteins has generally required recursive experimental screening and large libraries to engender function, mostly starting with natural proteins rather than de novo structures (
In one aspect, there is provided a system that includes at least one data processor and at least one memory storing instructions. The instructions may cause operations when executed by the at least one data processor. The operations may include: querying a van der Mer (vdM) database to identify a first van der Mer known to interact with a first portion of a first compound, the first van der Mer corresponding to an in silico unit of protein structure that defines, based at least on a statistically preferred orientation of the first portion of the first compound relative to a backbone structure of a protein, a binding site for the first compound relative to the backbone structure of the protein; and generating, based at least on the first van der Mer, a sequence for the protein such that the protein exhibits a binding affinity for the first compound.
In some variations, one or more features disclosed herein including the following features can optionally be included in any feasible combination. The van der Mer database may include a plurality of van der Mers. Each of the plurality of van der Mers may be associated with a portion of a compound and a backbone structure.
In some variations, the plurality of van der Mers may be organized into one or more clusters of van der Mers exhibiting a same or similar interaction with the portion of the compound.
In some variations, the plurality of van der Mers may be clustered based at least on a first set of atomic protein coordinates associated with the portion of the compound and a second set of atomic protein coordinates associated with the backbone structure.
In some variations, the plurality of van der Mers included in the van der Mer database may be identified by searching a database of known protein structures for one or more units of protein structure exhibiting a van der Waals (vdW) contact with the portion of the compound.
In some variations, the one or more units of protein structure exhibiting the van der Waals (vdW) contact with the portion of the compound may be identified as van der Mers based at least on a nature of contact with the portion of the compound.
In some variations, the nature of contact may be one of a hydrogen bond, a close van der Waals contact, and a wide van der Waals contact.
In some variations, the operations may further include: generating a first set of coordinates corresponding to the backbone structure of the protein; generating a second set of coordinates corresponding to the compound or the portion of the compound; and querying, based at least on the first set of coordinates and the second set of coordinates, the van der Mer database.
In some variations, the operations may further include: querying the van der Mer database to identify a second van der Mer known to interact with the first portion of the first compound or a second portion of the first compound; and generating, based at least on the second van der Mer, the sequence for the protein.
In some variations, the backbone structure of the protein may include one of a plurality of backbone structures with a geometry consistent with a known plasticity of a selected protein fold.
In some variations, the sequence of the protein may be further generated by packing additional residues in the binding site.
In some variations, the sequence of the protein may be further generated by packing a core of the protein.
In some variations, the portion of the compound may include a chemical group.
In some variations, the compound may include a ligand.
In some variations, the ligand may include a peptide, a protein, a small molecule, or a small molecule-metal-ion complex.
In some variations, the first van der Mer may be selected instead of a second van der Mer based at least on the first van der Mer being observed in more experimentally determined protein structures than the second van der Mer.
In some variations, the operations may further include: optimizing the sequence of the protein by at least identifying a location of the binding site relative to the backbone structure of the protein associated with a minimum energy function.
In some variations, the optimizing may be performed by applying one or more of an interative algorithm, a heurisitic algorithm, a Monte Carlo sampling algorithm, a dead-end elimination algorithm, a branch and bound algorithm, a pruning algorithm, a simplex algorithm, a memetic algorithm, a differential evolution algorithm, an evolutionary algorithm, a genetic algorithm, a tabu algorithm, a particle swarm algorithm, and a simulated annealing algorithm.
In some variations, the energy function may include a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, and/or protein radius of gyration function.
In some variations, the sequence of the protein is further generated such that the protein exhibits a desired tertiary structure including one or more folds.
In another aspect, there is provided a computer-implemented method that includes: querying a van der Mer (vdM) database to identify a first van der Mer known to interact with a first portion of a first compound, the first van der Mer corresponding to an in silico unit of protein structure that defines, based at least on a statistically preferred orientation of the first portion of the first compound relative to a backbone structure of a protein, a binding site for the first compound relative to the backbone structure of the protein; and generating, based at least on the first van der Mer, a sequence for the protein such that the protein exhibits a binding affinity for the first compound.
In some variations, one or more features disclosed herein including the following features can optionally be included in any feasible combination. The van der Mer database may include a plurality of van der Mers. Each of the plurality of van der Mers may be associated with a portion of a compound and a backbone structure.
In some variations, the plurality of van der Mers may be organized into one or more clusters of van der Mers exhibiting a same or similar interaction with the portion of the compound.
In some variations, the plurality of van der Mers may be clustered based at least on a first set of atomic protein coordinates associated with the portion of the compound and a second set of atomic protein coordinates associated with the backbone structure.
In some variations, the plurality of van der Mers included in the van der Mer database may be identified by searching a database of known protein structures for one or more units of protein structure exhibiting a van der Waals (vdW) contact with the portion of the compound.
In some variations, the one or more units of protein structure exhibiting the van der Waals (vdW) contact with the portion of the compound may be identified as van der Mers based at least on a nature of contact with the portion of the compound.
In some variations, the nature of contact may be one of a hydrogen bond, a close van der Waals contact, and a wide van der Waals contact.
In some variations, the method may further include: generating a first set of coordinates corresponding to the backbone structure of the protein; generating a second set of coordinates corresponding to the compound or the portion of the compound; and querying, based at least on the first set of coordinates and the second set of coordinates, the van der Mer database.
In some variations, the method may further include: querying the van der Mer database to identify a second van der Mer known to interact with the first portion of the first compound or a second portion of the first compound; and generating, based at least on the second van der Mer, the sequence for the protein.
In some variations, the backbone structure of the protein may include one of a plurality of backbone structures with a geometry consistent with a known plasticity of a selected protein fold.
In some variations, the sequence of the protein may be further generated by packing additional residues in the binding site.
In some variations, the sequence of the protein may be further generated by packing a core of the protein.
In some variations, the portion of the compound may include a chemical group.
In some variations, the compound may include a ligand.
In some variations, the ligand may include a peptide, a protein, a small molecule, or a small molecule-metal-ion complex.
In some variations, the first van der Mer may be selected instead of a second van der Mer based at least on the first van der Mer being observed in more experimentally determined protein structures than the second van der Mer.
In some variations, the method may further include: optimizing the sequence of the protein by at least identifying a location of the binding site relative to the backbone structure of the protein associated with a minimum energy function.
In some variations, the optimizing may be performed by applying one or more of an interative algorithm, a heurisitic algorithm, a Monte Carlo sampling algorithm, a dead-end elimination algorithm, a branch and bound algorithm, a pruning algorithm, a simplex algorithm, a memetic algorithm, a differential evolution algorithm, an evolutionary algorithm, a genetic algorithm, a tabu algorithm, a particle swarm algorithm, and a simulated annealing algorithm.
In some variations, the energy function may include a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, and/or protein radius of gyration function.
In some variations, the sequence of the protein is further generated such that the protein exhibits a desired tertiary structure including one or more folds.
In another aspect, there is provided a non-transitory computer readable medium storing instructions that cause operations when executed by at least one data processor. The operations may include: querying a van der Mer (vdM) database to identify a first van der Mer known to interact with a first portion of a first compound, the first van der Mer corresponding to an in silico unit of protein structure that defines, based at least on a statistically preferred orientation of the first portion of the first compound relative to a backbone structure of a protein, a binding site for the first compound relative to the backbone structure of the protein; and generating, based at least on the first van der Mer, a sequence for the protein such that the protein exhibits a binding affinity for the first compound.
In another aspect, there is provided an apparatus that includes: means for querying a van der Mer (vdM) database to identify a first van der Mer known to interact with a first portion of a first compound, the first van der Mer corresponding to an in silico unit of protein structure that defines, based at least on a statistically preferred orientation of the first portion of the first compound relative to a backbone structure of a protein, a binding site for the first compound relative to the backbone structure of the protein; and means for generating, based at least on the first van der Mer, a sequence for the protein such that the protein exhibits a binding affinity for the first compound.
In another aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including identifying van der Mers representing the chemical groups of the compound and amino acid residues of the protein capable of interacting with the chemical groups of the compound in silico, and wherein the protein has secondary and tertiary protein structure when bound to the compound.
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In an aspect is provided a computer-implemented method for identifying a complex of a protein bound to a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
TABLE S1 lists crick parameters for generation of parametric 4-helix bundle ensemble.
TABLE S2 lists PDB accession codes and Cα RMSD values of matches to a 4-helix query (10-residues each helix) of the initial parameterized backbone of ABLE.
TABLE S3 lists data collection and refinement statistics of drug-free- and drug-bound ABLE.
TABLE S4 lists data collection and refinement statistics of H49A mutant of unliganded ABLE.
The terms “a” or “an,” as used in herein means one or more. In addition, the phrase “substituted with a[n]” as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is “substituted with an unsubstituted C1-C20 alkyl, or unsubstituted 2 to 20 membered heteroalkyl,” the group may contain one or more unsubstituted C1-C20 alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.
An amino acid residue in a protein “corresponds” to a given residue when it occupies the same essential structural position within the protein as the given residue.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics, which are not found in nature.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues, wherein the polymer may in embodiments be conjugated to a moiety that does not consist of amino acids. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. A “fusion protein” refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety. In embodiments, the protein includes at least 30 amino acid residues. A protein may be characterized as having a protein backbone. A “protein backbone” is used herein in accordance with its ordinary meaning and refers to the polymer of amino acid residues that create a continuous chain. For example, a protein backbone may refer to the series of amino acid residues covalently linked together, e.g.,
wherein each R independently represents optionally different amino acid side chains. In embodiments, the protein backbone includes core amino acid residues and ligand binding amino acid residues. In embodiments, the protein backbone includes core amino acid residues. In embodiments, the protein backbone includes ligand binding amino acid residues.
The term “amino acid side chain” refers to the functional substituent contained on amino acids. For example, an amino acid side chain may be the side chain of a naturally occurring amino acid. Naturally occurring amino acids are those encoded by the genetic code (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine), as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. In embodiments, the amino acid side chain may be a non-natural amino acid side chain. In embodiments, the amino acid side chain is
wherein the symbol “” corresponds to the attachment of a chemical moiety (e.g., side chain) to the remainder of a molecule or chemical formula (e.g., the amino acid core, or
The term “non-natural amino acid side chain” refers to the functional substituent of compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, allylalanine, 2-aminoisobutryric acid. Non-natural amino acids are non-proteinogenic amino acids that either occur naturally or are chemically synthesized. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples include exo-cis-3-aminobicyclo[2.2.1]hept-5-ene-2-carboxylic acid hydrochloride, cis-2-aminocycloheptanecarboxylic acid hydrochloride,cis-6-amino-3-cyclohexene-1-carboxylic acid hydrochloride, cis-2-amino-2-methylcyclohexanecarboxylic acid hydrochloride, cis-2-amino-2-methylcyclopentanecarboxylic acid hydrochloride, 2-(Boc-aminomethyl)benzoic acid, 2-(Boc-amino)octanedioic acid, Boc-4,5-dehydro-Leu-OH (dicyclohexylammonium), Boc-4-(Fmoc-amino)-L-phenylalanine, Boc-β-Homopyr-OH, Boc-(2-indanyl)-Gly-OH, 4-Boc-3-morpholineacetic acid, 4-Boc-3-morpholineacetic acid, Boc-pentafluoro-D-phenylalanine, Boc-pentafluoro-L-phenylalanine, Boc-Phe(2-Br)—OH, Boc-Phe(4-Br)—OH, Boc-D-Phe(4-Br)—OH, Boc-D-Phe(3-Cl)—OH, Boc-Phe(4-NH2)-OH, Boc-Phe(3-NO2)-OH, Boc-Phe(3,5-F2)-OH, 2-(4-Boc-piperazino)-2-(3,4-dimethoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(2-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(3-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-fluorophenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-(4-methoxyphenyl)acetic acid purum, 2-(4-Boc-piperazino)-2-phenylacetic acid purum, 2-(4-Boc-piperazino)-2-(3-pyridyl)acetic acid purum, 2-(4-Boc-piperazino)-2-[4-(trifluoromethyl)phenyl]acetic acid purum, Boc-β-(2-quinolyl)-Ala-OH, N—Boc-1,2,3,6-tetrahydro-2-pyridinecarboxylic acid, Boc-β-(4-thiazolyl)-Ala-OH, Boc-β-(2-thienyl)-D-Ala-OH, Fmoc-N-(4-Boc-aminobutyl)-Gly-OH, Fmoc-N-(2-Boc-aminoethyl)-Gly-OH, Fmoc-N-(2,4-dimethoxybenzyl)-Gly-OH, Fmoc-(2-indanyl)-Gly-OH, Fmoc-pentafluoro-L-phenylalanine, Fmoc-Pen(Trt)-OH, Fmoc-Phe(2-Br)—OH, Fmoc-Phe(4-Br)—OH, Fmoc-Phe(3,5-F2)-OH, Fmoc-β-(4-thiazolyl)-Ala-OH, Fmoc-β-(2-thienyl)-Ala-OH, 4-(hydroxymethyl)-D-phenylalanine.
The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting protein (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).
As used herein, the term “about” means a range of values including the specified value, which a person of ordinary skill in the art would consider reasonably similar to the specified value. In embodiments, about means within a standard deviation using measurements generally acceptable in the art. In embodiments, about means a range extending to +/−10% of the specified value. In embodiments, about means the specified value.
The terms “bind” and “bound” as used herein is used in accordance with its plain and ordinary meaning and refers to the association between atoms or molecules. The association can be direct or indirect. For example, bound atoms or molecules may be direct, e.g., by covalent bond or linker (e.g. a first linker or second linker), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi or hydrophobic effects), hydrophobic interactions and the like).
The term “compound” refers to a substance formed when two or more chemical elements are chemically bonded (e.g., covalent, ionic, etc.) together (e.g., small molecule, biomolecule, agonist, antagonist, protein). In embodiments, the compound is capable of binding to a protein (e.g., a protein described herein). In embodiments, a compound binds (e.g., covalently or non-covalently) to a protein. Typically, upon binding the compound has an effect on the protein (e.g., structural change of the protein, modulation of signaling pathways). A compound is associated with a set of compound atomic coordinates (e.g., Cartesian coordinates, internal coordinates, polar coordinates, spherical coordinates) which define the compound in space (e.g., Euclidean space). The compound may be endogenous or exogenous. Non-limiting examples of compounds include a catalyst, detectable agent, therapeutic agent, biological agent, cytotoxic agent, diagnostic agent, theranostic (e.g., a combined therapeutic and diagnostic agent), photodynamic therapy (PDT) agent, porphyrin, porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin, chlorophyll, chlorin, phthalocyanine, porphyrazine, corrole, N-confused porphyrin, bacteriochlorophyll, pheophytin, texaphyrin, or related macrocyclic-based component that is capable of binding a metal ion. In embodiments, the compound is a peptide (e.g., 2 to 30 amino acid residues), a protein (e.g., greater than 30 amino acid residues), a small molecule (e.g., a compound with a molecular weight of less than 2000 Daltons), or a small molecule-metal-ion complex (e.g., a metalloporphyrin). In embodiments, the compound is endogenous. In embodiments, the compound is exogenous. In embodiments, the compound is a chemical molecule having a molecular weight of less than 10000 Daltons (e.g., less than 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100).
The term “ligand” refers to an agent (e.g., compound, metal, ion, biomolecule, agonist, antagonist) which is capable of binding to a protein (e.g., a protein described herein). In embodiments, a ligand refers to an agent (e.g., compound, metal, ion, biomolecule) which binds (e.g., covalently or non-covalently) to a protein. Typically, upon binding the ligand has an effect on the protein (e.g., structural change of the protein, modulation of signaling pathways). A ligand is associated with a set of ligand atomic coordinates (e.g., Cartesian coordinates, internal coordinates, polar coordinates, spherical coordinates) which define the ligand in space (e.g., Euclidean space). The ligand may be endogenous or exogenous. Non-limiting examples of ligands include a catalyst, detectable agent, therapeutic agent, biological agent, cytotoxic agent, magnetic resonance imaging (MRI) agent, positron emission tomography (PET) agent, radiological imaging agent, diagnostic agent, theranostic (e.g., a combined therapeutic and diagnostic agent), photodynamic therapy (PDT) agent, porphyrin, porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin, chlorophyll, chlorin, phthalocyanine, porphyrazine, corrole, N-confused porphyrin, bacteriochlorophyll, pheophytin, texaphyrin, or related macrocyclic-based component that is capable of binding a metal ion. In embodiments, the ligand is a peptide (e.g., 2 to 30 amino acid residues), a protein (e.g., greater than 30 amino acid residues), a small molecule (e.g., a compound with a molecular weight of less than 2000 Daltons), or a small molecule-metal-ion complex (e.g., a metalloporphyrin). In embodiments, the ligand is endogenous. In embodiments, the ligand is exogenous. In embodiments, the ligand is a chemical molecule having a molecular weight of less than 10000 Daltons (e.g., less than 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100). In embodiments, the ligand is a compound.
The terms “optimizing” and “optimization” are used in accordance with their ordinary meaning in mathematics and computer science and refers to identifying a favorable outcome subject to certain criteria (e.g., constraints) from a set of available possibilities. Optimizing may employ iterative or heuristic algorithms, such as simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, stimulated annealing algorithm, Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. For example, optimizing typically includes evaluating an energy function (e.g., force field model) and finding the minimum (e.g., global minimum or local minimum). Optimizing may include repeated evaluations of the energy function and may include fixing an atomic coordinate (e.g., fixing an atomic coordinate of at least one ligand binding amino acid residue atomic coordinate), introducing additional amino acid residues into the set of amino acid residues (e.g., the set of ligand binding amino acid residues), restricting the introduction of additional amino acid residues into the set of amino acid residues (e.g., the set of ligand binding amino acid residues), or a geometric transformation (e.g., translation or rotation) of an amino acid residue atomic coordinate (e.g., the atomic coordinate of the ligand binding amino acid residue atomic coordinates). The output of an optimization process may provide a set of compound binding amino acid residues and a corresponding set of compound binding amino acid residue atomic coordinates, and a set of core amino acid residues and a corresponding set of core amino acid residue atomic coordinates, which corresponds to an energetically stabilized protein. In embodiments the outcome of the optimization is the global minimum (e.g., the most energetically stabilized protein). In embodiments the outcome of the optimization is a local minimum (e.g., a minimum energy given the domain). In embodiments the optimization is complete when the derivative of the energy with respect to the position of the atoms, ∂E/∂r, is zero and the Hessian matrix has positive eigenvalues. In embodiments, optimizing includes a plurality of minimization calculations. In embodiments the optimization is a finite number of iterations.
An energy minimization calculation refers to the process of evaluating the energy as a function of the atomic coordinates, V(r). The energy function may include intra- and intermolecular energy terms within the system (e.g., protein) which may be written as Vtotal(r)=Vbonds(r)+Vangles(r) Vdihedral(r) Vimproper(r) Vnonbonding(r) Velectrostatics(r); where Vtotal(r) corresponds to the total energy as a function of the atomic positions; Vbonds(r) corresponds to the energy contribution from bonded atoms, Vangles(r) corresponds to the energy contribution from angles; Vdihedral(r) corresponds to the energy contribution from dihedral torsions; Vimproper(r) corresponds to the energy contribution from out-of-plane torsions; Vnonbonding(r) corresponds to the energy contribution from nonbonding interactions; and Velectrostatics(r) corresponds to the energy contribution from electrostatic interactions. Additional energy function terms may also be included in the total energy function, Vtotal(r), for example additional functions from molecular mechanics, functions from structural bioinformatics (log-odds scores), amino acid sidechain packing functions (e.g., functions and algorithms which vary the identity and rotamer of an amino acid side chain), protein radius of gyration functions, or a penalty function.
The term biomolecule as used herein refers to a molecule present in living organisms (e.g., proteins, carbohydrates, lipids, and nucleic acids, metabolites) and may be endogenous or exogenous in origin.
The term “energetically stabilized protein” is used in accordance with its ordinary meaning in the art, and is understood to refer to a protein which is structurally and thermodynamically stable relative to the protein that has not been energetically stabilized. For example, an energetically stabilized protein is determined to be energetically stabilized by determining the difference in the Gibbs free energy between the folded and unfolded states of the protein, also referred to herein as ΔGfolding. An energetically stabilized protein may be characterized by a well-dispersed NMR spectrum and/or the presence of a significantly folded core. In embodiments, the energetically stabilized protein is an enzyme. In embodiments, the energetically stabilized protein is an apo protein (e.g., a protein that is not bound to a ligand). In embodiments, the energetically stabilized protein is a holo protein (e.g., a protein that is bound to a ligand). In embodiments, the energetically stabilized protein is an apo protein which is capable of becoming a holo protein upon ligand binding. In embodiments, an energetically stabilized protein refers to a protein which is capable of performing a function (e.g., modulating a signal pathway). In embodiments, the energetically stabilized protein resists side-reactions such as aggregation and proteolysis. In embodiments, the energetically stabilized protein has a ΔGfolding of about −5 to about −40 kcal/mol in standard physiological conditions (e.g., temperature range of 20-40 degrees Celsius, atmospheric pressure of 1, pH of 6-8, glucose concentration of 1-20 mM, atmospheric oxygen concentration).
The term “small molecule” or the like as used herein refers, unless indicated otherwise, to a molecule having a molecular weight of less than about 700 Dalton, e.g., less than about 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 100, or 50 Dalton.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like. “Consisting essentially of or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
The term “van der Mer” as used herein refers to an in silico unit of protein structure interacting with a portion of a compound. In embodiments, the van der Mer may be used to map the backbone of an amino acid type to a statistically preferred position when interacting with specific chemical groups. A van der Mer may include a unit of local protein structure that directly links a tertiary structure to key interactions that engender tight and specific binding and defines the placement of key chemical groups in the ligand (compound) relative to the backbone atoms of the contacting amino acid residue (see for example
The term “atomic coordinates” as used herein refers to a set of numbers that define the location of an atom or group of atoms (e.g., covalently bonded atoms in a compound or amino acid backbone, residue, or sidechain) in space (e.g., Euclidean space) in silico. Atomic coordinates may be, for example, Cartesian coordinates, internal coordinates, polar coordinates, spherical coordinates). In embodiments, the atomic coordinates of an atom will be understood to describe the location of all portions of the atom as understood by a person having ordinary skill in the art. For example, the atomic coordinates of an atom may be numbers describing a single point in space, however, it will be understood that the atomic coordinates further include the three dimensional space around the single point that would be occupied by the atom based on the radius of the atom as understood by a person having ordinary skill in the art. For example, when atomic coordinates described covalently attached atoms of a chemical group or amino acid (e.g., portion of backbone or sidechain), it will be understood that the atomic coordinates may explicitly describe single points in space for each atom but the atomic coordinates will be understood to also include the three dimensional space occupied by the atoms and the space occupied by the bonds between the atoms. The term “atomic protein coordinates” refers to atomic coordinates representing the atom(s) of a protein (e.g., backbone atom(s) of a protein, a protein capable of binding a compound). The term “atomic van der Mer coordinates” refers to atomic coordinates representing the atom(s) of a van der Mer, for example the atom(s) of a chemical group of a van der Mer or the atom(s) of an amino acid side chain of a van der Mer or the atom(s) of a portion of a protein backbone of a van der Mer bound to the atom(s) of a side chain of a van der Mer. The term “atomic chemical coordinates” refers to the atomic coordinates representing the atom(s) of a compound (e.g., a compound a protein is capable of binding to) or ligand (e.g., a ligand a protein is capable of binding to). The term “atomic amino acid coordinates” refers to the atomic coordinates representing the atom(s) of an amino acid residue (e.g., portion of the protein backbone and attached sidechain) of a protein in a complex of a protein bound to a compound, wherein the amino acid is not represented (e.g., overlap) by a van der Mer or wherein the amino acid does not interact with a chemical group of the compound bound to the protein.
The term “overlapping” when referring to juxtaposition of atomic coordinates (e.g., atomic van der Mer coordinates and atomic protein coordinates or atomic van der Mer coordinates and atomic chemical coordinates or atomic amino acid coordinates and atomic protein coordinates) refers to the situation wherein atoms (or bonds) represented by atomic coordinates of two different sources (e.g., atomic van der Mer coordinates and atomic protein coordinates or atomic van der Mer coordinates and atomic chemical coordinates or atomic amino acid coordinates and atomic protein coordinates) occupy the same space in three dimensions. It will be understood that the atomic coordinates may provide the location of a single point or multiple single points in space however, overlap will be determined by comparing the locations of the space around such single point(s) that is understood to be occupied by the atom represented by the atomic coordinates or by the atoms and bonds represented by the atomic coordinates of covalently bonded atoms. It will be understood that overlapping may be partial and complete overlap of all portions of an atom or bond are not necessary for overlap to occur.
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including identifying van der Mers representing the chemical groups of the compound and amino acid residues of the protein capable of interacting with the chemical groups of the compound in silico, and wherein the protein has secondary and tertiary protein structure when bound to the compound.
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In embodiments, the method includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the method includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the compound chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the compound identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the compound chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the compound, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In an aspect is provided a computer-implemented method for identifying a complex of a protein bound to a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a compound, including:
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the optimizing includes an energy minimization calculation. In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, identifying atomic van der Mer coordinates of a chemical group of a van der Mer as exposed to bulk solvent is performed using a convex hull algorithm. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the compound includes a charged chemical group at physiological pH. In embodiments, the compound includes a polar chemical group at physiological pH. In embodiments, the method further includes making the protein. In embodiments, the method further includes making the protein using molecular biology techniques. In embodiments, the method further includes making the protein using peptide synthesis. In embodiments, the method further includes making the protein by expressing the protein from an exogenous nucleic acid. In embodiments, the method includes use of a method described in international application no. WO2019/023644. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes an iterative algorithm. In embodiments, the optimizing includes a heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the optimizing includes knobs-into-holes side chain packing. In embodiments, the optimization may begin with an idealized, parameterized backbone. In embodiments, optimization may relax the backbone structure of the protein, for example, by using gradient descent algorithms, while optimizing the protein sequence via rotamer sampling and minimization.
In embodiments, the optimizing includes introducing an additional compound binding amino acid residue into the set of compound binding amino acid residues, deleting a compound binding amino acid residue from the set of compound binding amino acid residues, a geometric transformation of at least one atomic coordinate of the compound binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes introducing an additional compound binding amino acid residue into the set of compound binding amino acid residues (e.g., designating an amino acid residue previously not designated as a compound binding amino acid residue to a compound binding amino acid residue). In embodiments, the optimizing includes replacing a compound binding amino acid residue within the set of compound binding amino acid residues. In embodiments, the optimizing includes deleting a compound binding amino acid residue from the set of compound binding amino acid residues. In embodiments, the optimizing includes a geometric transformation of at least one atomic coordinate of the compound binding amino acid residue atomic coordinates. In embodiments, the optimizing includes a geometric transformation of the atomic coordinates of at least one of the compound binding amino acid residue atomic coordinates. In embodiments, the optimizing includes a geometric transformation of the atomic coordinates of the compound binding amino acid residue atomic coordinates.
In embodiments, the geometric transformation includes a translation (i.e., a geometric transformation that moves a coordinate by the same distance in a given direction) or a rotation of at least one atomic coordinate of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation (e.g., displacing the x coordinate) of at least one atomic coordinate of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least two atomic coordinates of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of all atomic coordinates (e.g., x, y, and z coordinates in Cartesian space) of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least one atomic coordinate of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least two atomic coordinates of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least three atomic coordinates of the compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of all atomic coordinates of the compound binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes a geometric transformation of at least one atomic coordinate of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation or a rotation of at least one atomic coordinate of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least one atomic coordinate of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least two atomic coordinates of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of all atomic coordinates of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least one atomic coordinate of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least two atomic coordinates of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least three atomic coordinates of the non-compound binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of all atomic coordinates of the non-compound binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes 1a) calculating the force on each atom in the protein (e.g., the set of compound binding amino acid residues; the set of non-compound binding amino acid residues; and the compound); 2a) evaluating the calculation to determine if it is the minimum or below an acceptable threshold; 3a) if the force is less than a threshold, the optimization is finished, otherwise perform a geometric transformation (e.g., translation) of at least one atomic coordinate on the atoms in the protein; and 4a) repeat.
In embodiments, the geometric transformation of at least one atomic coordinate includes no greater than a 6 Å displacement of any atomic coordinate. In embodiments, the geometric transformation of at least one atomic coordinate includes no greater than a 3 Å displacement of any atomic coordinate. In embodiments, the displacement is no greater than 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Å displacement of any atomic coordinate. In embodiments, the displacement is no greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 Å displacement of any atomic coordinate.
In embodiments, the set of compound binding amino acids includes at least 50 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 40 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 30 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 20 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 12 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 10 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 8 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 6 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 5 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 4 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 3 amino acid residues. In embodiments, the set of compound binding amino acids includes at least 2 amino acid residues. In embodiments the compound binding amino acids are apolar. In embodiments the compound binding amino acids are hydrophilic.
In embodiments, the set of compound binding amino acids includes 50 amino acid residues. In embodiments, the set of compound binding amino acids includes 40 amino acid residues. In embodiments, the set of compound binding amino acids includes 30 amino acid residues. In embodiments, the set of compound binding amino acids includes 20 amino acid residues. In embodiments, the set of compound binding amino acids includes 12 amino acid residues. In embodiments, the set of compound binding amino acids includes 10 amino acid residues. In embodiments, the set of compound binding amino acids includes 8 amino acid residues. In embodiments, the set of compound binding amino acids includes 6 amino acid residues. In embodiments, the set of compound binding amino acids includes 5 amino acid residues. In embodiments, the set of compound binding amino acids includes 4 amino acid residues. In embodiments, the set of compound binding amino acids includes 3 amino acid residues. In embodiments, the set of compound binding amino acids includes 2 amino acid residues. In embodiments the compound binding amino acids are polar. In embodiments the compound binding amino acids are hydrophilic.
In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, the energy minimization calculation includes a penalty function.
In embodiments, the compound is a porphyrin, porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin, chlorophyll, chlorin, phthalocyanine, porphyrazine, corrole, N-confused porphyrin, bacteriochlorophyll, pheophytin, texaphyrin, or related macrocyclic-based component, that is capable of binding a metal ion. In embodiments, the compound is a detectable agent. In embodiments, the compound is a therapeutic agent, biological agent, cytotoxic agent, magnetic resonance imaging (MRI) agent, positron emission tomography (PET) agent, radiological imaging agent, diagnostic agent, theragnostic, or a photodynamic therapy (PDT) agent. In embodiments, the compound is a therapeutic agent. In embodiments, the compound is a biological agent. In embodiments, the compound is a cytotoxic agent (e.g., an anticancer agent). In embodiments, the compound is a magnetic resonance imaging (MRI) agent. In embodiments, the compound is a positron emission tomography (PET) agent. In embodiments, the compound is a radiological imaging agent. In embodiments, the compound is a diagnostic agent. In embodiments, the compound is a theragnostic agent. In embodiments, the compound is a photodynamic therapy (PDT) agent. In embodiments, the compound is a small molecule.
In embodiments, the compound is a catalyst. In embodiments, the catalyst catalyzes an abiological or bio-orthogonal reaction. In embodiments, the compound is a molecule that exists within a living system (e.g., within an organism or a cell). In embodiments, the compound atomic coordinates are optimized using known methods in the art (e.g., density functional theory using the B3-LYP functional).
In embodiments, the method further includes synthesizing the protein (e.g., utilizing the expression vectors such as the plasmid method described in the Example, such as cloning into the IPTG-inducible pET-11a plasmid). In embodiments, the method further includes expressing the protein.
These compound binding amino acid residues can form the backbone of a protein. Each compound binding amino acid residue within the protein can be associated with a set of compound binding amino acid residue atomic coordinates, which can define the compound binding amino acid residue in space. Furthermore, each atom of the compound can be associated with a set of ligand atomic coordinates, which can define the compound in space. As noted herein, these coordinates can be Cartesian coordinates, internal coordinates, polar coordinates, spherical coordinates, and/or the like.
The set of compound binding amino acid residues, the set of compound binding amino acid residue atomic coordinates, the set of non-compound binding amino acid residues, and the set of non-compound binding amino acid residue atomic coordinates can be optimized. For example, the optimization can be performed using an energy minimization calculation including, for example, a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, and/or the like. Optimizing the set of compound binding amino acid residues, the set of compound binding amino acid residue atomic coordinates, the set of non-compound binding amino acid residues, and the set of non-compound binding amino acid residue atomic coordinates can generate an energetically stabilized protein.
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a ligand (e.g., compound), including identifying van der Mers representing the chemical groups of the ligand (e.g., compound) and amino acid residues of the protein capable of interacting with the chemical groups of the ligand (e.g., compound) in silico, and wherein the protein has secondary and tertiary protein structure when bound to the ligand (e.g., compound).
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a ligand (e.g., compound), including:
In an aspect is provided a computer-implemented method for identifying a complex of a protein bound to a ligand (e.g., compound), including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a ligand (e.g., compound), including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a ligand (e.g., compound), including:
In an aspect is provided a computer-implemented method for identifying a protein capable of binding a ligand (e.g., compound), including:
In embodiments, the ligand is a compound. In embodiments, the compound is a chemical molecule having molecular weight of less than 10000 Daltons (e.g., less than 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100).
In embodiments, the method includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the method includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the ligand chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the ligand identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the ligand chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the ligand, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the ligand chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the ligand chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the ligand chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the ligand includes a charged chemical group at physiological pH. In embodiments, the ligand includes a polar chemical group at physiological pH. In embodiments, the method further includes making the protein. In embodiments, the method further includes making the protein using molecular biology techniques. In embodiments, the method further includes making the protein using peptide synthesis. In embodiments, the method further includes making the protein by expressing the protein from an exogenous nucleic acid. In embodiments, the method includes use of a method described in international application no. WO2019/023644.
In embodiments, the optimizing includes introducing an additional ligand binding amino acid residue into the set of ligand binding amino acid residues, deleting a ligand binding amino acid residue from the set of ligand binding amino acid residues, a geometric transformation of at least one atomic coordinate of the ligand binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes introducing an additional ligand binding amino acid residue into the set of ligand binding amino acid residues (e.g., designating an amino acid residue previously not designated as a ligand binding amino acid residue to a ligand binding amino acid residue). In embodiments, the optimizing includes replacing a ligand binding amino acid residue within the set of ligand binding amino acid residues. In embodiments, the optimizing includes deleting a ligand binding amino acid residue from the set of ligand binding amino acid residues. In embodiments, the optimizing includes a geometric transformation of at least one atomic coordinate of the ligand binding amino acid residue atomic coordinates. In embodiments, the optimizing includes a geometric transformation of the atomic coordinates of at least one of the ligand binding amino acid residue atomic coordinates. In embodiments, the optimizing includes a geometric transformation of the atomic coordinates of the ligand binding amino acid residue atomic coordinates.
In embodiments, the geometric transformation includes a translation (i.e., a geometric transformation that moves a coordinate by the same distance in a given direction) or a rotation of at least one atomic coordinate of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation (e.g., displacing the x coordinate) of at least one atomic coordinate of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least two atomic coordinates of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of all atomic coordinates (e.g., x, y, and z coordinates in Cartesian space) of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least one atomic coordinate of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least two atomic coordinates of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least three atomic coordinates of the ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of all atomic coordinates of the ligand binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes a geometric transformation of at least one atomic coordinate of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation or a rotation of at least one atomic coordinate of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least one atomic coordinate of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of at least two atomic coordinates of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a translation of all atomic coordinates of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least one atomic coordinate of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least two atomic coordinates of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of at least three atomic coordinates of the non-ligand binding amino acid residue atomic coordinates. In embodiments, the geometric transformation includes a rotation of all atomic coordinates of the non-ligand binding amino acid residue atomic coordinates.
In embodiments, the optimizing includes 1a) calculating the force on each atom in the protein (e.g., the set of ligand binding amino acid residues; the set of non-ligand binding amino acid residues; and the ligand); 2a) evaluating the calculation to determine if it is the minimum or below an acceptable threshold; 3a) if the force is less than a threshold, the optimization is finished, otherwise perform a geometric transformation (e.g., translation) of at least one atomic coordinate on the atoms in the protein; and 4a) repeat.
In embodiments, the geometric transformation of at least one atomic coordinate includes no greater than a 6 Å displacement of any atomic coordinate. In embodiments, the geometric transformation of at least one atomic coordinate includes no greater than a 3 Å displacement of any atomic coordinate. In embodiments, the displacement is no greater than 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Å displacement of any atomic coordinate. In embodiments, the displacement is no greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 Å displacement of any atomic coordinate.
In embodiments, the set of ligand binding amino acids includes at least 50 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 40 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 30 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 20 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 12 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 10 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 8 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 6 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 5 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 4 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 3 amino acid residues. In embodiments, the set of ligand binding amino acids includes at least 2 amino acid residues. In embodiments the ligand binding amino acids are apolar. In embodiments the ligand binding amino acids are hydrophilic.
In embodiments, the set of ligand binding amino acids includes 50 amino acid residues. In embodiments, the set of ligand binding amino acids includes 40 amino acid residues. In embodiments, the set of ligand binding amino acids includes 30 amino acid residues. In embodiments, the set of ligand binding amino acids includes 20 amino acid residues. In embodiments, the set of ligand binding amino acids includes 12 amino acid residues. In embodiments, the set of ligand binding amino acids includes 10 amino acid residues. In embodiments, the set of ligand binding amino acids includes 8 amino acid residues. In embodiments, the set of ligand binding amino acids includes 6 amino acid residues. In embodiments, the set of ligand binding amino acids includes 5 amino acid residues. In embodiments, the set of ligand binding amino acids includes 4 amino acid residues. In embodiments, the set of ligand binding amino acids includes 3 amino acid residues. In embodiments, the set of ligand binding amino acids includes 2 amino acid residues. In embodiments the ligand binding amino acids are polar. In embodiments the ligand binding amino acids are hydrophilic.
In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, the energy minimization calculation includes a penalty function.
In embodiments, the ligand is a porphyrin, porphycene, rubyrin, rosarin, hexaphyrin, sapphyrin, chlorophyll, chlorin, phthalocyanine, porphyrazine, corrole, N-confused porphyrin, bacteriochlorophyll, pheophytin, texaphyrin, or related macrocyclic-based component, that is capable of binding a metal ion. In embodiments, the ligand is a detectable agent. In embodiments, the ligand is a therapeutic agent, biological agent, cytotoxic agent, magnetic resonance imaging (MRI) agent, positron emission tomography (PET) agent, radiological imaging agent, diagnostic agent, theragnostic, or a photodynamic therapy (PDT) agent. In embodiments, the ligand is a therapeutic agent. In embodiments, the ligand is a biological agent. In embodiments, the ligand is a cytotoxic agent (e.g., an anticancer agent). In embodiments, the ligand is a magnetic resonance imaging (MRI) agent. In embodiments, the ligand is a positron emission tomography (PET) agent. In embodiments, the ligand is a radiological imaging agent.
In embodiments, the ligand is a diagnostic agent. In embodiments, the ligand is a theragnostic agent. In embodiments, the ligand is a photodynamic therapy (PDT) agent. In embodiments, the ligand is a small molecule.
In embodiments, the ligand is a catalyst. In embodiments, the catalyst catalyzes an abiological or bio-orthogonal reaction. In embodiments, the ligand is a molecule that exists within a living system (e.g., within an organism or a cell). In embodiments, the ligand atomic coordinates are optimized using known methods in the art (e.g., density functional theory using the B3-LYP functional). In embodiments, the ligand is a small molecule. In embodiments, the ligand is a metal cofactor. In embodiments, the ligand is a metal ion. In embodiments, the ligand is a protein. In embodiments, the ligand is a compound.
In embodiments, the method further includes synthesizing the protein (e.g., utilizing the expression vectors such as the plasmid method described in the Example, such as cloning into the IPTG-inducible pET-11a plasmid). In embodiments, the method further includes expressing the protein.
These ligand binding amino acid residues can form the backbone of a protein. Each ligand binding amino acid residue within the protein can be associated with a set of ligand binding amino acid residue atomic coordinates, which can define the ligand binding amino acid residue in space. Furthermore, each atom of the ligand can be associated with a set of ligand atomic coordinates, which can define the ligand in space. As noted herein, these coordinates can be Cartesian coordinates, internal coordinates, polar coordinates, spherical coordinates, and/or the like.
The set of ligand binding amino acid residues, the set of ligand binding amino acid residue atomic coordinates, the set of non-ligand binding amino acid residues, and the set of non-ligand binding amino acid residue atomic coordinates can be optimized. For example, the optimization can be performed using an energy minimization calculation including, for example, a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, and/or the like. Optimizing the set of ligand binding amino acid residues, the set of ligand binding amino acid residue atomic coordinates, the set of non-ligand binding amino acid residues, and the set of non-ligand binding amino acid residue atomic coordinates can generate an energetically stabilized protein.
In some example embodiments, challenges associated with designing de novo a protein capable of binding to a small molecule (or another ligand) are addressed by mapping the tertiary structure of a protein directly to the sequences of amino acids that encode the folding and the binding exhibited by the protein. For example, a protein may be designed de novo by creating an ensemble of backbones with geometries consistent with the known plasticity of a selected protein fold. For each backbone, one or more van der Mers (vdMs) that interact with a portion of the ligand, such as one or more targeted chemical groups within the small molecule, may be identified. As each van der Mer is a structural unit occupying a specific residue position on the backbone of a protein, the identification of van der Mers may also determine the binding sites of the small molecule (or other ligand). Thus, the backbone geometry of the protein may be dictated by a maximum binding affinity to the desired small molecule (or other ligand). Once the binding sites are identified, additional residues within the binding sites and the protein core may be packed. The resulting protein may therefore exhibit a tertiary structure and sequence that support the desired function of binding to the small molecule (or other ligand).
In some example embodiments, the design engine 110 may be configured to support the de novo design of a protein exhibiting a binding affinity for a ligand including, for example, a peptide (e.g., 2 to 30 amino acid residues), a protein (e.g., greater than 30 amino acid residues), a small molecule (e.g., a compound with a molecular weight of less than 2000 Daltons), a small molecule-metal-ion complex (e.g., a metalloporphyrin), and/or the like. The design engine 110 may design the protein by creating an ensemble of backbones with geometries consistent with the known plasticity of a selected protein fold. In the example of the protein design system 100 shown in
For each backbone, the design engine 110 may identify one or more van der Mers (vdMs) that interact with a portion of the ligand, such as one or more targeted chemical groups within the small molecule. Each van der Mer may be an in silico unit of local protein structure known to interact with a portion of the ligand. Moreover, each van der Mer may occupy a specific residue position (e.g., a statistically preferred position) on the backbone structure of the protein when the van der Mer is interacting with the portion of the ligand (e.g., the targeted chemical groups of the small molecule). Thus, the van der Mers identified by the design engine 110 may provide a direct link between the tertiary structures of the protein to a desired function, such as a binding affinity for the ligand. For example, the van der Mers that are identified by the design engine 110 may define the binding sites of the ligand with additional residues within the binding sites and the protein core being packed accordingly.
In some example embodiments, the one or more van der Mers known to interact with the portion of the ligand, such as the targeted chemical groups of a small molecule, may be identified by the design engine 110 querying the van der Mer database 120. The van der Mer database 120 may include a selection of van der Mers, each of which known to exhibit an interaction with a portion of a ligand (e.g., a targeted chemical group such as aspartic acid (Asp), carboxamide (CONH2), and/or the like). The selection of van der Mers may be curated by searching a database of known protein structures (e.g., from the Protein Data Bank (PDB) and/or the like). For example, a unit of a protein structure may be identified as a van der Mer of a chemical group if the amino-acid residues contained therein are in van der Waals (vdW) contact with the given chemical group. Furthermore, a unit of a protein structure may be identified as the van der Mer of a chemical group based on the nature of the contact (e.g., H-bond, close vdW contact, wide vdW contact). For instance, van der Mers of the chemical group carboxamide may be identified by iterating through Asn and Gln residues in each unique known protein chain (e.g., in the Portein Data Bank (PDB) and/or the like). Van der Mers of the chemical group carboxamide may be those residues that are within van der Waals contact with the sidechain's carboxamide (e.g., CB, CG, OD1, ND2, HD21, HD22 atoms of Asn) by forming H-bonded interactions.
In some example embodiments, the van der Mers included in the van der Mer database 120 may be organized into clusters of related van der Mers that exhibit similar observed interactions with a chemical group. For example, the van der Mers of a chemical group may be clustered based on the coordinates of the protein backbone and the coordinates of the chemical group bound to the protein. This clustering may facilitate subsequent searches through the van der Mer database 120. For instance, only 31 clusters of Asp/carboxamide vdMs are needed to capture half of the observed interactions. Each van der Mer cluster may be associated with a cluster score (C), which provides a quantitative measure for how representative that cluster's interaction geometry is for that residue type across the known protein structures (e.g, in the Protein Data Bank (PDB) and/or the like). The score may be determined based on the placement of a chemical group relative to a protein backbone, since the coordinates of the backbone and chemical group are the only coordinates involved in the clustering. A positive cluster score may indicate that the location of the chemical group relative to the backbone, represented by the cluster, is enriched relative to other locations of the cluster group. Thus, in some example embodiments, the design engine 110 may select van der Mers having a positive cluster score when identifying van der Mers for the de novo design of a protein.
As noted, the identification of one or more van der Mers exhibiting an interaction with a portion of a ligand (e.g., a targeted chemical group of a small molecule) may determine the binding sites for the ligand on the backbone of a protein being designed to exhibit a binding affinity for the ligand. This is because each van der Mer may occupy a specific residue position on the backbone structure of the protein when the van der Mer is interacting with the portion of the ligand (e.g., the targeted chemical groups of the small molecule). The position of each van der Mer may correspond to the statistically preferred orientation of the portion of the ligand relative to the backbone structure of the protein when the van der Mer is interacting with the portion of the ligand. The remainder of the protein may be designed by the design engine 110 packing additional residues within the binding sites and packing the protein core. The resulting protein may exhibit a tertiary structure and amino acid sequence that supports the desired binding affinity to a particular ligand.
The design engine 110 may creating an ensemble of protein backbones with geometries consistent with the known plasticity of a selected protein fold (802). For example, the design engine 110 may receive, from the client device 130, one or more user inputs identifying a designable protein fold. The design engine 110 may create an ensemble of protein backbones with geometries that are consistent with the known plasticity of the designable protein fold.
The design engine 110 may determine the binding sites for a ligand by identifying, for each protein backbone from the ensemble of protein backbones, one or more van der Mers that interact with a portion of the ligand (804). In some example embodiments, the design engine 110 may identify one or more van der Mers known to interact with the portion of the ligand, such as the targeted chemical groups of a small molecule, by querying the van der Mer database 120.
The van der Mer database 120 may include a selection of van der Mers, each of which known to exhibit an interaction with a portion of a ligand (e.g., a targeted chemical group such as aspartic acid (Asp), carboxamide (CONH2), and/or the like). The van der Mers included in the van der Mer database 120 may be organized into clusters of related van der Mers that exhibit similar observed interactions with a chemical group. For example, the van der Mers of a chemical group may be clustered based on the coordinates of the protein backbone and the coordinates of the chemical group bound to the protein. By organizing van der Mers into clusters, queries to the van der Mer database 120 may be executed faster and with less computational resources.
The design engine 110 may complete the design for each protein by packing additional residues within the binding site and/or the protein core (806). In some example embodiments, upon identifying the binding sites for the ligand, the remainder of the protein structure may be designed by the design engine 110 packing additional residues within the binding sites and packing the protein core. The design engine 110 may apply a variety of algorithms to pack each protein structure. For example, the protein structure may be packed with hydrophobic amino acid residues and/or hydrophilic amino acid residues.
As shown in
The memory 920 is a computer readable medium such as volatile or non-volatile that stores information within the computing system 900. The memory 920 can store data structures representing configuration object databases, for example. The storage device 930 is capable of providing persistent storage for the computing system 900. The storage device 930 can be a floppy disk device, a hard disk device, an optical disk device, or a tape device, or other suitable persistent storage means. The input/output device 940 provides input/output operations for the computing system 900. In some example embodiments, the input/output device 940 includes a keyboard and/or pointing device. In various implementations, the input/output device 940 includes a display unit for displaying graphical user interfaces.
The memory 920 is a computer readable medium such as volatile or non-volatile that stores information within the computing system 900. The memory 920 can store data structures representing configuration object databases, for example. The storage device 930 is capable of providing persistent storage for the computing system 900. The storage device 930 can be a floppy disk device, a hard disk device, an optical disk device, or a tape device, or other suitable persistent storage means. The input/output device 940 provides input/output operations for the computing system 900. In some example embodiments, the input/output device 940 includes a keyboard and/or pointing device. In various implementations, the input/output device 940 includes a display unit for displaying graphical user interfaces.
In some example embodiments, the computing system 900 can be used to execute various interactive computer software applications that can be used for organization, analysis and/or storage of data in various formats. Alternatively, the computing system 900 can be used to execute any type of software applications. These applications can be used to perform various functionalities, e.g., planning functionalities (e.g., generating, managing, editing of spreadsheet documents, word processing documents, and/or any other objects, etc.), computing functionalities, communications functionalities, etc. The applications can include various add-in functionalities or can be standalone computing products and/or functionalities. Upon activation within the applications, the functionalities can be used to generate the user interface provided via the input/output device 940. The user interface can be generated and presented to a user by the computing system 900 (e.g., on a computer screen monitor, etc.).
One or more aspects or features of the subject matter described herein can be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs, field programmable gate arrays (FPGAs) computer hardware, firmware, software, and/or combinations thereof. These various aspects or features can include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which can be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.
These computer programs, which can also be referred to as programs, software, software applications, applications, components, or code, include machine instructions for a programmable processor, and can be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus and/or device, such as for example magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and/or data to a programmable processor. The machine-readable medium can store such machine instructions non-transitorily, such as for example as would a non-transient solid-state memory or a magnetic hard drive or any equivalent storage medium. The machine-readable medium can alternatively or additionally store such machine instructions in a transient manner, such as for example, as would a processor cache or other random access memory associated with one or more physical processor cores.
To provide for interaction with a user, one or more aspects or features of the subject matter described herein can be implemented on a computer having a display device, such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) or a light emitting diode (LED) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer. Other kinds of devices can be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, including acoustic, speech, or tactile input. Other possible input devices include touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive track pads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.
In an aspect is provided a system for identifying a protein capable of binding a compound including at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including identifying van der Mers representing the chemical groups of the compound and amino acid residues of the protein capable of interacting with the chemical groups of the compound in silico, and wherein the protein has secondary and tertiary protein structure when bound to the compound.
In an aspect is provided a system for identifying a protein capable of binding a compound, including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In embodiments, the system includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the system includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the compound chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the compound identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the compound chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the compound, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In an aspect is provided a system for identifying a complex of a protein bound to a compound, including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a compound, including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a compound, including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a compound, including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, identifying atomic van der Mer coordinates of a chemical group of a van der Mer as exposed to bulk solvent is performed using a convex hull algorithm. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the compound includes a charged chemical group at physiological pH. In embodiments, the compound includes a polar chemical group at physiological pH. In embodiments, the system includes use of a system described in international application no. WO2019/023644. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a compound including when executed by at least one data processor, causes operations including identifying van der Mers representing the chemical groups of the compound and amino acid residues of the protein capable of interacting with the chemical groups of the compound in silico, and wherein the protein has secondary and tertiary protein structure when bound to the compound.
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a compound, which when executed by at least one data processor, causes operations including:
In embodiments, the non-transitory computer-readable storage medium including program code includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the non-transitory computer-readable storage medium including program code includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the compound chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the compound identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the compound bound to the compound. In embodiments, the cluster score is the natural logarithm of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the compound chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the compound, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a complex of a protein bound to a compound, which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a compound, which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a compound, which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a compound, which, when executed by at least one data processor, causes operations including:
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, identifying atomic van der Mer coordinates of a chemical group of a van der Mer as exposed to bulk solvent is performed using a convex hull algorithm. In embodiments, the cluster score is the natural logarithm of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping compound van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the compound includes a charged chemical group at physiological pH. In embodiments, the compound includes a polar chemical group at physiological pH. In embodiments, the non-transitory computer-readable storage medium including program code includes use of a method described in international application no. WO2019/023644. In embodiments, the overlap of the van der Mers and the compound chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the compound chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the compound chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the compound chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In an aspect is provided a system for identifying a protein capable of binding a ligand (e.g., compound) including at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including identifying van der Mers representing the chemical groups of the ligand (e.g., compound) and amino acid residues of the protein capable of interacting with the chemical groups of the ligand (e.g., compound) in silico, and wherein the protein has secondary and tertiary protein structure when bound to the ligand (e.g., compound).
In an aspect is provided a system for identifying a protein capable of binding a ligand (e.g., compound), including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a complex of a protein bound to a ligand (e.g., compound), including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a ligand (e.g., compound), including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a ligand (e.g., compound), including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a system for identifying a protein capable of binding a ligand (e.g., compound), including: at least one data processor; and at least one memory storing instructions which, when executed by the at least one data processor, result in operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a ligand (e.g., compound) including when executed by at least one data processor, causes operations including identifying van der Mers representing the chemical groups of the ligand (e.g., compound) and amino acid residues of the protein capable of interacting with the chemical groups of the ligand (e.g., compound) in silico, and wherein the protein has secondary and tertiary protein structure when bound to the ligand (e.g., compound).
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a ligand (e.g., compound), which when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a complex of a protein bound to a ligand (e.g., compound), which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a ligand (e.g., compound), which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a ligand (e.g., compound), which, when executed by at least one data processor, causes operations including:
In an aspect is provided a non-transitory computer-readable storage medium including program code for identifying a protein capable of binding a ligand (e.g., compound), which, when executed by at least one data processor, causes operations including:
In embodiments, the ligand is a compound. In embodiments, the compound is a chemical molecule having molecular weight of less than 10000 Daltons (e.g., less than 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100). In embodiments, the ligand is a small molecule. In embodiments, the ligand is a metal cofactor. In embodiments, the ligand is a metal ion. In embodiments, the ligand is a protein. In embodiments, the ligand is a compound.
In embodiments, the system includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the system includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the ligand chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the ligand identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the ligand chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the ligand, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the ligand chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the ligand chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the ligand chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, identifying atomic van der Mer coordinates of a chemical group of a van der Mer as exposed to bulk solvent is performed using a convex hull algorithm. In embodiments, the cluster score is a function (e.g., including but not limited to a natural log or logistical function) of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the ligand includes a charged chemical group at physiological pH. In embodiments, the ligand includes a polar chemical group at physiological pH. In embodiments, the system includes use of a system described in international application no. WO2019/023644. In embodiments, the overlap of the van der Mers and the ligand chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the ligand chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the ligand chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In embodiments, the non-transitory computer-readable storage medium including program code includes generating a plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the non-transitory computer-readable storage medium including program code includes generating all possible sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand in step (f). In embodiments, the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and optimization of the overlap between the atomic chemical coordinates of the ligand chemical groups and the atomic van der Mer coordinates representing the independent chemical groups of the ligand identified in steps (b) to (d) is performed without duplication of the sets. In embodiments, the plurality of independent sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand are independently different from each other and are scored. In embodiments, the scoring includes calculating a cluster score for each of the plurality of sets of atomic coordinates of an in silico complex of the protein capable of binding the ligand bound to the ligand. In embodiments, the cluster score is the natural logarithm of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, step (a) includes generating a plurality of independent sets of atomic protein coordinates representing independent backbone structures of the protein. In embodiments, the plurality of independent backbone structures of the protein have a similar overall three dimensional fold. In embodiments, the plurality of independent backbone structures of the protein have an RMSD of less than 3 angstrom. In embodiments, the ligand chemical groups and van der Mer chemical groups are polar groups. In embodiments, steps (g) and (h) include use of a method described in international application no. WO2019/023644. In embodiments, step (c) includes identifying all portions of the backbone structure of the protein having atomic protein coordinates that are capable of overlapping with the atomic van der Mer coordinates of the first portion of a protein backbone of the first van der Mer. In embodiments, step (d) includes repeating steps (b) and (c) for all van der Mer in the van der Mer database independently representing all chemical groups of the ligand, an additional independent amino acid side chain and an additional independent portion of a protein backbone bound to said independent additional amino acid side chain. In embodiments, the overlap of the van der Mers and the ligand chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the ligand chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the ligand chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
In embodiments, the optimizing includes an iterative or heuristic algorithm. In embodiments, the optimizing includes a simplex algorithm, memetic algorithm, differential evolution algorithm, evolutionary algorithm, genetic algorithm, tabu algorithm, particle swarm algorithm, or stimulated annealing algorithm. In embodiments, the optimizing includes a Monte Carlo sampling algorithm, dead-end elimination algorithm, branch and bound algorithm, or a pruning algorithm. In embodiments, the energy minimization calculation includes a molecular mechanics function, a structural bioinformatics function, an amino acid sidechain packing function, a protein radius of gyration function, or a combination thereof. In embodiments, identifying atomic van der Mer coordinates of a chemical group of a van der Mer as exposed to bulk solvent is performed using a convex hull algorithm. In embodiments, the cluster score is the natural logarithm of the ratio of 1) the number of members in an independent set of geometrically overlapping van der Mer of one chemical group and one amino acid to 2) the average number of members in all independent sets van der Mer of the chemical group and the amino acid. In embodiments, the members in an independent set of geometrically overlapping ligand van der Mer of one chemical group are identified by having an RMSD below a threshold, wherein the RMSD is calculated using the atomic coordinates of the chemical group and the backbone atoms of the amino acid residue of a van der Mer. In embodiments, the RMSD threshold is 0.5 angstrom. In embodiments, the preferred amino acid of step (g) is an amino acid in a van der Mer having a cluster score greater than 2. In embodiments, identifying an amino acid residue for each protein backbone residue having atomic amino acid coordinates that are not overlapping with the atomic van der Mer coordinates of a van der Mer is performed using The Rosetta Software. In embodiments, the van der Mer database is a collection of independent van der Mer each including a unique set of atomic van der Mer coordinates describing the three dimensional positions of a chemical group interacting in silico with an amino acid residue, further wherein the interacting was identified in an empirically determined protein and chemical group complex. In embodiments, the protein is a 4-helix bundle protein. In embodiments, the ligand includes a charged chemical group at physiological pH. In embodiments, the ligand includes a polar chemical group at physiological pH. In embodiments, the non-transitory computer-readable storage medium including program code includes use of a method described in international application no. WO2019/023644. In embodiments, the overlap of the van der Mers and the ligand chemical groups are not selected one at a time, but rather in pairs, triplets or higher order combinations. In embodiments, van der Mers at multiple sites are selected and the RMSD between these van der Mer chemical groups and the ligand chemical groups is computed. In embodiments, the RMSD between van der Mer chemical groups and the ligand chemical groups is precomputed and saved in lookup tables. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in pairs. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in triplets. In embodiments, the overlap of the van der Mers and the ligand chemical groups are selected in combinations greater than three. In embodiments an identified van der Mer is a cluster having a 0.5 angstrom RMSD between members. In embodiments an identified van der Mer is a sub-cluster of a van der Mer. In embodiments an identified van der Mer is a van der Mer cluster member. In embodiments an identified van der Mer is a van der Mer representative. In embodiments an identified van der Mer is a cluster having a 0.1 angstrom RMSD between members.
A defined structural unit enables de novo design of small-molecule-binding proteins. A new representation of protein—chemical-group interactions enables design of proteins that bind the drug, apixaban. Computational code and design scripts are available in the supplement and github. Coordinates and data files of ABLE structures have been deposited to the PDB with accession codes: 6W6X (drug-free ABLE), 6W70 (apixaban-bound ABLE), 6X8N (H49A ABLE mutant).
The de novo design of proteins that bind highly functionalized small molecules represents a great challenge. To enable computational design of binders, we developed a unit of protein structure—a van der Mer (vdM)— that maps the backbone of each amino acid to statistically preferred positions of interacting chemical groups. Using vdMs, we designed six de novo proteins to bind the drug apixaban; two bound with low and sub-micromolar affinity. X-ray crystallography and mutagenesis confirmed a structure with a precisely designed cavity that forms favorable interactions in the drug-protein complex. vdMs may enable design of functional proteins for applications in sensing, medicine, and catalysis.
Here, we accomplish the reverse of the Anfinsen hypothesis by simultaneously designing structure and binding function from scratch, targeting a small-molecule drug with significant polarity and structural complexity. To do this, we developed a unit of local protein structure that directly links a tertiary structure to key interactions that engender tight and specific binding. These findings illuminate the principles underlying the emergence and evolution of complex function in proteins, and provide a methodology for designing useful proteins.
We targeted the factor Xa inhibitor, apixaban, an organic compound with five rotatable bonds and eight heteroatoms. Our first objective was to compute a tertiary structure capable of cooperatively binding the polar groups of apixaban. Instead of repurposing natural binding proteins or folds that have been shown to bind a similar ligand, in this work we use de novo 4-helix bundles because they are mathematically parameterized (10, 11), designable (12), and share no similarity to the fold of factor Xa. 4-helix bundles generally do not bind small molecules and instead bind metal ions or metalloporphyrins by strong coordinate bonds (10, 13-16). However, 4-helix bundles are tubular and can be designed to have high thermodynamic stability (11, 13) to compensate for the energetically demanding process of building binding cavities replete with buried polar functionality (17). Thus, the design of a de novo helical bundle that binds the drug apixaban critically tests the design method.
The design of proteins relies on optimal packing of interior sidechains in discrete conformations called rotamers (2, 3, 18-22). However, the design of ligand-binding proteins additionally requires sidechains that interact favorably with the target small molecule. Previous design strategies have approached this problem by computationally appending the target ligand to rotamers with idealized interaction geometries that—although comprised of billions of conformations—sample only a small fraction of the possible conformational space (6, 8, 23). These strategies rarely deliver sub-millimolar binders from the initial computational design so subsequent steps rely on experimental random mutagenesis and screening of libraries.
We wondered how much of the vast, possible conformational space of protein-chemical-group interactions is actually sampled in observed protein structures, and if sampling interactions directly from this distribution might aid the design of high-affinity binders. While previous analyses have focused on local sidechain contacts with chemical groups (24), we sought a structural unit that directly maps backbone coordinates to chemical-group locations, the link between the protein fold and binding function. We developed a unit of protein structure analogous to rotamers—van der Mers—that defines the placement of key chemical groups in the ligand relative to the backbone atoms of the contacting residue (
The use of vdMs contrasts with procedures that place ligands at idealized locations relative to the terminal atoms of a sidechain (6, 8, 23, 25), which results in vast numbers of ligand—rotamer combinations that might never occur in proteins. Instead, vdMs sample locations of chemical groups relative to the backbone that have been experimentally vetted to achieve binding, regardless of ideality of the interaction. They also implicitly consider interactions with ordered or bulk water, which might influence their interaction geometries. Moreover, unlike ligand-appended and inverse rotamers used in earlier approaches (6, 8, 23, 25), vdMs may derive from contacts with either mainchain, sidechain or both in a multivalent interaction. Finally, the prevalence of a given vdM in the Protein Data Bank (PDB) can be used in scoring functions, similarly to scoring rotamers, which may assist automated selection of binding-site residues for design.
To maximize the number of observed protein—chemical-group contacts, we created vdMs using the chemical groups of amino acids that comprise the protein (e.g., CONH2 of Gln and Asn, N—H and C═O of backbone amide). To avoid bias from local structure, we counted only the interactions that were distant in the linear polypeptide chain, as described in the supplement. The set of chemical groups can also be expanded to include those from small-molecule drugs, metal ions, and cofactors, although these are not as pervasive in crystal structures.
We rank vdMs by their prevalence in the PDB using a log-odds score, C (
Proteins use the same set of 20 amino acids to fold as well as to recognize a vast array of highly functionalized ligands. We therefore hypothesized that the interaction modes used by amino acids to stabilize their tertiary structures would also be used to achieve tight binding of ligands, even those containing structurally distinct heterocyclic chemical groups. To test this hypothesis, we examined the streptavidin—biotin complex (
Our analysis of the streptavidin—biotin complex suggests that binding sites can be designed by considering folds that position vdMs to collectively bind the distinct chemical groups found in a target small-molecule ligand. Moreover, the vdMs of the binding site should be maximally prevalent in the PDB. We developed a search algorithm, called Convergent Motifs for Binding Sites (COMBS), to discover favorable poses of a ligand that satisfy these criteria.
Our design strategy consists of several hierarchic steps, which prioritize the most essential and difficult features to avoid sampling regions in sequence/structure space with little chance of success (
We focused on apixaban's carboxamide (both the C═O and —NH2), as well as two additional carbonyls (
Sidechains from vdMs in six selected binding poses were fixed, and their H-bonding interactions with apixaban were constrained in all subsequent steps of sequence design using Rosetta. After insertion of interhelical loops, we used a flexible-backbone design protocol (13) (
We designed six proteins of varying length, topology, ligand position, ligand burial, and keystone interactions (
Binding of apixaban to ABLE restricts the drug's conformation, resulting in a redshift of its electronic absorbance spectrum (
ABLE readily crystalized with apixaban and diffracted to 1.3 Å resolution. Two very closely related monomers are observed in the asymmetric unit (
To assess the extent of preorganization of the protein, we also solved the drug-free structure to 1.3 Å resolution (
Insights from the Structure and Function of ABLE
Two of the three keystone interactions identified by COMBS contribute significantly to binding affinity. Substitution of His49 or Gln14 to alanine individually decreases affinity by approximately 1 kcal/mol (˜3-fold,
We also examined the structural consequences of substituting His49 to Ala by solving the crystal structure of the unliganded H49A mutant protein (
Substitution of the third keystone residue, Thr112, to Ala resulted in little change in affinity (
Flexible-backbone sequence design of ABLE recruited two Tyr residues that interact with apixaban (
Finally, we wondered if ab initio folding predictions (26) might distinguish between successful versus unsuccessful designs. Of the six designs, only two—ABLE and LABLE—were predicted by folding simulations to maintain uncollapsed binding sites (
Previously, the design of de novo proteins that bind in a shape-selective manner to rigid, flat, hydrophobic dyes or lipidic metabolites has been possible, but binding flexible molecules replete with polar atoms has been more challenging (4, 8, 31-33). Natural proteins bind highly functionalized ligands by first accruing the ability to weakly bind fragments within the context of a particular fold (34-36). To mimic this process, we developed the vdM structural unit to directly link the protein fold to statistically preferred binding modes of chemical groups. We sampled vdMs on the backbone of a designable 4-helix bundle to create constellations of chemical groups that, when matched with the shape of apixaban, defined the binding site. This contrasts with previous approaches that search for positional matching of whole ligands, sampled using idealized interaction geometries. Such approaches are highly sensitive to small changes in the interaction geometries, thus requiring an enormous amount of sampling to discover possible binding solutions, many of which may contain interactions not observed in the PDB.
vdMs sample from the experimentally-vetted distribution of observed protein structures. vdMs are surprisingly sparse and discrete (
COMBS and vdMs can now be used for a variety of protein engineering applications, and in full partnership with experimental optimization strategies for exploring sequence space. We anticipate that vdMs can also be used to predict chemical-group hotspots of proteins with fixed sequence. vdMs may also enable design of protein—protein interfaces in a self-consistent manner. Finally, because vdMs sample from the distribution of evolved interaction geometries observed in protein structures, it is tempting to view the chemical-group constellations constructed by vdMs as a structural hypothesis of the evolutionary path to acquire binding within the context of a given fold.
We downloaded protein structures from the RCSB with 30% sequence homology, X-ray diffraction resolution ≤2.0 Å, and Robs≤0.3. We used the program Reduce (37) to add hydrogens to the structures and to perform any necessary rotamer-flips of Asn, Gln, and His residues. We then used the program Molprobity (38) to obtain the Molprobity score for each structure. We subsequently constructed biological assemblies of the PDBs with Molprobity score ≤2, using the program Prody (39). The final list of accession codes/chain IDs for van der Mer (vdM) searching can be found in the supporting file, Data S1. The non-redundant structural database contains a total of 8743 PDBs with 9189 unique chains. Note that while we used biological assemblies to search for vdMs, we only searched through the non-redundant chains in the structure, such that contacts could be found across subunits of the assembly, without artificial duplication of vdMs.
We approximated chemical groups (CGs) as fragments of amino-acid sidechain or mainchain, in order to increase sampling statistics. For example, our database contains 348,067 residues contacting a carboxamide derived from Asn or Gln sidechains. Of these, 189,849 residues have interactions with carboxamide that are distant in sequence (>7 amino acids away in the linear polypeptide chain), which avoids bias from nonspecific proximity effects. In this work, we further winnowed the number of interacting residues by considering only H-bonded interactions (85,750 residues). To define a vdM, we next categorize the interactions by residue type (e.g., 5,785 Tyr residues H-bond with a carboxamide).
We used the program Probe (40) to determine which amino-acid residues are in van der Waals (vdW) contact with a given chemical group (CG), as well as the nature of the contact (H-bond, close vdW contact, wide vdW contact). For example, to search for vdMs of carboxamide, we iterated through every Asn and Gln residue in each unique protein chain in the database. For each Asn and Gln residue in the chain, we used Probe to detect other residues in the biological assembly that are within vdW contact of the sidechain's carboxamide (e.g., CB, CG, OD1, ND2, HD21, HD22 atoms of Asn). To find vdMs of carbonyl (C═O), we used the backbone carbonyl of Gly and Ala residues. We then used only the subset of vdMs that formed H-bonded interactions. These vdMs were grouped in two ways: by superposition on mainchain for sampling, and by superposition on mainchain and chemical group coordinates for scoring (see text below and
We scored vdMs based on their prevalence in the non-redundant protein structural database. Instead of aligning vdMs exactly by amino-acid backbone atoms, we performed a pair-wise all-against-all superposition of backbone (N, Cα, C) and CG atoms for every vdM of a particular amino-acid type. Using both backbone and CG in the superposition helps to alleviate the lever-arm effect, where small changes in backbone coordinates lead to large changes in the location of a CG. The all-against-all pairwise RMSD matrix was used to cluster vdMs by RMSD <0.5 Å, using a greedy clustering algorithm. Much of the interaction space sampled by proteins in our database is captured in a small number of these clusters. For example, only 31 clusters of Asp/carboxamide vdMs are needed to capture half of the observed interactions (
A single cluster may use a variety of sidechain rotamers to position the chemical group in the same location relative to the residue's backbone atoms, and the sidechain dihedral angles of vdMs appear to follow the same distribution as canonical rotamers (
We defined a cluster score (C) of a vdM as a quantitative measure for how representative that cluster's interaction geometry is for that residue type in the PDB. The score is based on placement of a CG relative to the protein backbone, since backbone and CG coordinates are the only coordinates involved in clustering. Sidechain conformation (rotamer) is not explicitly considered in the clustering and therefore not in C. We compare the size of the cluster k to the average cluster size of that vdM type by C(k)=In N(k)/N
where N(k) is the number of members in cluster k and
N
|
is the average cluster size (
For sampling we used more fine-grained clusters, which would allow sampling over finer elements of conformational space (
We aimed to create a highly stable protein that not only folds to the desired structure but also binds a ligand, which further restrains the sequence space in addition to the requirements for folding. We therefore sought to use a highly designable scaffold that can accommodate many sequences but is still tractable to computationally design from scratch. Consequently, we parametrically generated a small set [32] of antiparallel 4-helix bundles using Crick parameters that are similar to those describing natural heme-binding proteins, such as helical bundles in cytochrome BC1, and to those describing non-natural porphyrin-binding proteins, such as the de novo bundle PS1 (13). Using the CCCP server (10), we sampled parameters on a grid that varied the bundle radius from 7.9 Å to 8.2 Å, and covaried the superhelical phases of two helices by 14°, resulting in bundles that had wide interfaces that varied between 108 and 120° (interhelical Cα distances of ˜8.2-9.8 Å). These parameters were chosen because they result in highly designable backbones that can accommodate a variety of sequences (see structural bioinformatics below), as well as provide a variable-sized binding cavity for the ligand. Bundle parameters can be found in Table S1.
We used the program Master (41) to query a structural database of approximately 20,000 protein crystal structures filtered at 50% sequence homology and with resolution <2.5 Å (Robs<0.3). A four-helix query of the database (10 residues each helix) returned 319 unique proteins with structural matches with Cα RMSD <2 Å (Table S2). A query of the tightly interfaced helix-helix pair (10 residues each helix) of the parametric backbone returned 1466 unique proteins with structural matches with Cα RMSD <0.7 Å.
The backbone of ABLE was defined by parametric design (28, 42), using a simple algebraic expression with a handful of adjustable parameters to define a highly symmetrical backbone with reasonable bond lengths and angles. The resulting backbone nevertheless served as a scaffold for design of proteins that bind a highly complex and asymmetric ligand. Curious about other proteins that might use this scaffold functionally, we probed the structural similarity of this backbone to natural four-helix bundle proteins in the PDB. We found hundreds of structural matches to a wide variety of proteins both natural and designed, with natural proteins ranging from the meiotic synaptonemal protein complex (43) to a superoxide oxidase (44); and with de novo proteins designed to form internal hydrogen bonds (45) or to bind porphyrins (13) (Table S2). One very recent structure (pdb 5xub) of a domain from a chemotaxis protein (46), deposited subsequently to the design of ABLE, binds citrate in approximately the same location of a four-helix bundle as the location of apixaban in ABLE. This collection of bundles illustrates the emergence of diverse complex functions from relatively minor (<2 Å Cα RMSD) tweaks to an otherwise fully symmetrical scaffold.
We used the conformation of apixaban from the co-crystal structure with factor Xa (pdb 2p16,
The collective process of generating vdMs, loading vdMs on a backbone, sampling ligand poses, and selecting protein—ligand interactions is called COMBS (convergent motifs for binding sites). Below, we describe the process by which COMBS finds binding sites that achieve H-bonded interactions with the ligand apixaban (
The design process starts with the coordinates of a poly-glycine backbone only. We used a restricted set of residues (H, S, T, Y, W) for sampling buried vdMs of carboxamide and carbonyl in the interior of the protein bundle and used a more expanded set for intermediate and exterior positions (H, S, T, Y, W, Q, N, D, E, R, K). We defined interior, intermediate, and exterior positions with a convex hull algorithm (48). We first make an all-Ala version of the protein, which defines the positions of Cβ atoms. The convex hull algorithm uses Cα and Cβ coordinates of the protein to define two surfaces. If the Cβ atom lies on the surface of the Cβ hull, that residue is exposed. If a Cβ atom lies in the interior of the Cα surface, then that residue is either buried or intermediate. Intermediate residues are those that are also part of the Cβ hull. The algorithm can limit the size of the radius of the sphere (alpha sphere) that is used to define the exterior surface, which limits the surface coarseness. We used an alpha-sphere size of 9 Å.
Sampling of vdMs on a Backbone
We sample vdMs by aligning a set of vdM representatives (see above) to a backbone position. This has the effect of placing a chemical group (CG) in space relative to the backbone (sidechain is also placed). Similar to the program Probe, we use van der Waals radii of the atoms to define clashes of vdM sidechain and CG with the surrounding mainchain atoms, taking into account close approaches due to H-bonding. We do not sample vdMs one at a time in a conventional rotamer-sampling algorithm, but instead load them simultaneously onto a backbone scaffold to concurrently enumerate all possible CG locations (see Nearest neighbors graph of CGs). Multiple vdMs can occupy the same residue position on the backbone.
For sampling, we divided vdMs into 4 interaction types: 1) those making only backbone Cα and/or N—H contacts with the CG (called bbNH vdMs); 2) those making only backbone C═O contacts with the CG (called bbCO vdMs), 3) those making only sidechain contacts with the CG (called SC vdMs); and 4) those making both mainchain and sidechain contacts with the CG (called φψ vdMs). For each parametrically generated helical bundle, we aligned vdMs of each category to the backbone by superposing, respectively, by 1) Cα, N, H atoms, 2) Cα, C, O atoms, 3) N, Cα, C atoms, and 4) N, Cα, C atoms. This allows for a finer sampling of vdMs that have interactions that are dependent on only φ, only ψ, or both φ and ψ. bbNH vdMs are φ-dependent, and bbCO vdMs are ψ-dependent. For sampling, we treated SC vdMs as φ/ψ independent, although φ/ψ dependence of the rotamer is implicitly considered when we remove any vdMs that clash with the mainchain. Because φψ vdMs are inherently φ/ψ dependent, we only sampled them from vdMs with φ/ψ in a bin of ±30° of φ/ψ of the scaffold residue onto which they were aligned.
We sampled vdMs over a 14-residue span of each ˜40 residue helix. We loaded vdMs onto 14×4 residue positions and created an array of CG coordinates for construction of a nearest neighbors graph, which we used to discover vdMs that are consistent with the position of a ligand.
We construct a nearest-neighbors graph from the CG coordinates of the vdMs once they have been superimposed onto the backbone scaffold. For carboxamide, we used an RMSD of 1.0 Å for the CG (Cb, Cg, Od1, Nd2 atoms of Asn, and Cg, Cd, Oe1, Ne2 atoms of Gln). For carbonyl (backbone C and O atoms of Gly and Ala), we used an RMSD of 0.7 Å. We used the nearest-neighbors implementation in the Python package sci-kit learn. This allows for very fast lookups of neighbors given query coordinates, which we take from placed ligands (see below). The neighbors tell us precisely which vdMs place a chemical group within the RMSD threshold of the query coordinates, as well as the RMSD distance of each from the query. The next step in the design process is to determine which of these neighboring vdMs possess sidechains that do not clash with the placed ligand, and then to score the clash-free remainder by C (see above).
Previous computational approaches to sample ligand positions have focused on either geometric overlap of entire ligands (6, 49, 50) or on ligand placement with one user-defined contact (23). For example, after sampling ligand-appended rotamers on protein backbones, candidate binding sites were defined as those that placed the full ligand in the same region of space (6). These approaches suffer from the lever-arm effect, where small deviations in protein— ligand contact geometry amplify to large changes of the ligand position remote from the contacting region. Massive amounts of sampling are required to overcome the lever-arm effect (4, 6, 8, 23), yet only a fraction of the total possible conformational space is available for sampling on a reasonable timeframe, even on large computing clusters. COMBS instead uses a set of ligand-superimposed vdMs to initially place a ligand in the binding site (see below) but then looks for nearest-neighbors vdMs of the ligand's chemical groups, instead of matches to full ligand locations. COMBS currently searches through static conformers only, such that searching through multiple conformers of a ligand requires the generation of a different set of ligand-superimposed vdMs for each conformer. Searches through multiple conformers can then be run in parallel.
To generate ligand placements relative to the protein backbone, we first curate a set of vdMs with the ligand superimposed by the CG. We remove all vdM/ligand combinations that are clashing after superposition. We then load this set of ligand-superimposed vdMs onto the backbone scaffold in the same way we load vdMs. This has the advantage of placing the ligand with a least one vdM-derived CG contact, that of its superimposed vdM. We remove any ligand-superimposed vdMs with ligand or sidechain that is clashing with the backbone. We further remove any ligand-superimposed vdMs based on ligand burial. For design of ABLE and LABLE, we required at least 60 percent of apixaban's apolar heavy atoms to be buried in the interior of the protein, as defined by the convex hull (see above).
With the coordinates of the other CGs within the ligand now defined relative to the backbone, we use these coordinates as queries to the nearest-neighbors graph of carboxamide and carbonyl. We look for overlap of the ligand's CGs in their respective nearest-neighbors graphs instead of overlap of an entire ligand in order to reduce the lever arm effect, which amplifies small deviations in local geometry to affect large swings in distant parts of a ligand. The use of CG graphs allows us to find binding interactions for a particular ligand location consistent with small local deviations in the interactions that would otherwise be missed by a search for full ligand overlap. By sampling the ligand position with superposed ligands onto vdMs, we experience the lever arm effect only once (during the superposition), instead of multiple times (one time per CG) in the ligand.
We selected poses of apixaban based on ligand burial and satisfaction of H-bonding constraints to its buried CGs. We required that the two carbonyls and the carboxamide of apixaban be engaged in a vdM-derived H-bond if buried in the interior of the protein. We selected individual vdMs (among all the nearest neighbors) for a ligand pose based on maximizing C while avoiding vdW clashes between vdM sidechains. We chose 6 poses based on apixaban burial and ΣC that explored three distinct placements of apixaban (
After vdM-derived ligand placement and H-bonded interactions were found to apixaban, we performed a custom protocol for flexible-backbone sequence design in the program Rosetta (26) (linux version 2018.33.60351). We froze the identities and rotamers of the H-bonded residues, and constrained the H-bond distances using a harmonic potential. We generated a parameter file for apixaban for use in Rosetta, which defines its partial charges (see supplemental text). We did not allow the ligand conformer to be flexible during design.
We automatically generated Rosetta residue files based on burial and secondary structure of each position in the backbone. To do so, we applied the convex hull algorithm described above, as well as the secondary structure assignment program DSSP, to the entire PDB dataset (9,000 proteins) to create burial and secondary structure propensities for each residue type, based on backbone coordinates only. The propensity is defined as p=faa(burial, ss)/faa where faa(burial, ss) is the frequency that amino acid aa occurs in that burial assignment (exposed, intermediate, or buried) with secondary structure ss, and faa is the frequency of the amino acid aa in the database. We used residues at each position that had a burial and secondary structure propensity p≥0.9. For 3 of the 6 designs, including that of ABLE, we allowed Ala, Ser, Thr, and Val residues at solvent exposed positions during design to lower the surface polarity in order to promote crystallization. Scripts for flexible-backbone sequence design can be found in supplementary text below. The outputted backbones (500 total) varied on average from their starting structure by ˜1 Å Cα RMSD. We selected designs for advancement to the next stage of computation by considering the packing of the core residues (pstat score in Rosetta) and the overall energy (ref2015 weights).
Loops connecting helices are selected from a database of natural α-helical protein structures and spliced onto the backbone to minimize Cα distance with the helices (51). The loop sequences were allowed to vary in the flexible backbone design process, with the set of possible residues selected in the automated fashion describe above.
We used a simple Monte Carlo protocol to bias the desired folded topology, by searching for charged surface residues that stabilize the desired topology and destabilize the reverse topology (52). The protocol results in a surface pattern of negatively and positively charged residues. We modified the Rosetta residue file to account for this surface patterning by disallowing the opposite charge at positions specified by the surface pattern (The residues were still allowed to be neutral and polar). We find that this protocol results in bundles that exhibit well-defined ab initio folding funnels with single minima (e.g.
We selected final, single-chain designs (among 500 total outputted models for each of the 6 designs) by considering the packing of the core residues (pstat score in Rosetta) and the overall energy (ref2015 weights). We used the convex hull algorithm mentioned above and a custom python script based on the program Probe to detect any buried residues with polar atoms not engaged in an H-bond, such as Tyr or Trp residues. We selected designs that did not feature any “unsatisfied” H-bonding residues. Computational models of the designs are freely available at the online repository zenodo (https:/doi.org/10.5281/zenodo.3718920).
Rosetta ab initio folding (53) was performed on the final designed sequences. The command line input for folding simulations can be found in the supplementary text. RMSD was calculated to all Cα atoms of the input model. Of the 6 designed sequences, only 2 were predicted to fold to a structure that maintained an open, solvent-accessible binding site, ABLE and LABLE (
The code for COMBS is available at github (https://github.com/npolizzi/combs_pub). The scripts for flexible-backbone sequence design in Rosetta can be found in supplementary text.
The genes coding for the 6 protein sequences were ordered from GenScript, and were cloned into the IPTG-inducible pet-11a plasmid (cloning site NdeI-BamHI). The sequence of each design also coded for an N-terminal 6×His-tag followed by a TEV protease cleavage sequence.
where the “/” defines the cleavage site of TEV protease. TEV-cleaved ABLE is 126 residues. The plasmids were transfected into E. coli BL21(DE3) cells (Invitrogen), which were grown in LB/ampicillin media until OD @ 600 nm=0.6. The cells were then induced with IPTG and allowed to grow for 4 more hours. Cells were then centrifuged and frozen. The frozen cell pellets were thawed and lysed by sonication, purified by Ni NTA affinity column (Invitrogen), and purified protein was confirmed by gel electrophoresis. The buffer was exchanged to a TEV protease buffer (5 mM DTT, 50 mM Tris, 0.5 mM EDTA, pH 8.0), and proteins were incubated with His-tagged TEV protease for 1 day at room temperature. The cleaved protein was collected from the flow-through of a Ni NTA column and concentrated in a stock of 50 mM NaPi, 100 mM NaCl, pH 7.4 buffer. Both TEV-cleaved and His-tagged proteins were used in experiments, as they showed no significant differences in binding. ABLE had an approximate yield of 200 mg/L.
Apixaban-peg-FITC was synthesized from apixaban acid (ApxCOO−) by coupling with Boc-(PEG)2-amine followed by deprotection and reaction with FITC (Scheme S1). To a solution of apixaban acid (200 mg, 0.43 mmol) in DMF (2 mL) were added Boc-(PEG)2-amine (108 mg, 0.43 mmol), DIPEA (174 uL, 1 mmol) and HCTU (169 mg, 041 mmol). The mixture was stirred at room temperature for 3 h and diluted with ethyl acetate. The organic layer was successively washed with 1 M HCl, sat. NaHCO3, and brine. After drying over Na2SO4, the mixture was concentrated under reduced pressure and a solution of TFA in DCM (50%, 5 mL) was added. The mixture was stirred for 1 h and the volatiles were removed under reduced pressure. To the solution of crude amine in DMF (4 mL) were added DIPEA (134 uL) and FITC (136 mg). After stirring 2 h, the mixture was diluted with ethyl acetate and washed with sat. NaHCO3. The organic layer was concentrated and purified by RP-HPLC. 1H NMR (DMSO-d6, 300 MHz) 8.30 (1H, br s), 7.73 (1H, d, J=8.0 Hz), 7.49 (2H, d, J=8.9 Hz, 1H), 7.33 (2H, d, J=8.8 Hz), 7.26 (2H, d, J=8.8 Hz), 7.16 (1H, d, J=8.3 Hz), 6.98 (2H, d, J=9 Hz), 6.67 (2H, s), 6.56 (m, 4H), 4.04 (m, 2H), 3.79 (s, 3H), 3.5-3.53 (m, 12H), 3.42-3.35 (m, 2H), 3.25-3.15 (m, 2H), 2.38 (2H, br s), 1.85-1.83 (4H, m) ESI-MS (MR+) 980.5
We used spectral titration and fluorescence polarization experiments to determine the binding dissociation constants for ABLE. ABLE was purified via HPLC (C4 reverse phase column), lyophilized, and reconstituted in buffer (50 mM NaPi, 100 mM NaCl, pH 7.4). Aliquots of apixaban from 2 mM, 1 mM, or 0.5 mM stocks in DMSO were serially added to 2 mL solutions of ABLE at 20 μM, 10 μM, and 5 μM concentration, respectively. (Final DMSO concentration was kept below 2%.) Absorbance changes at 305 nm, due to the restricted torsional conformation of apixaban in the bound state, were fit to Equation 1 using a single-site, protein-ligand binding model for the [Apx·ABLE] complex (Equation 2) (KD, and N, where Δε305
s the change in extinction coefficient at 305 nm of the bound complex relative to free apixaban and protein. We used ε305
the extinction coefficient of apixaban at 305 nm, as a local fitting parameter for each concentration, and the results of these locally fit extinction coefficients were within experimental error of each other (also agreeing with that measured for apixaban alone). Results of the fit are listed in
We performed fluorescence anisotropy experiments (54, 55) of ABLE and LABLE using a FITC fluorophore conjugated to apixaban as the fluorescent probe (
Electronic absorption spectra were collected using a HP 8453 spectrophotometer in 1 cm quartz optical cells. The noise level of the instrument was maintained at 0.1 mOD.
CD spectra were collected on a Jasco J-810 CD spectrometer in a 0.1 cm path length quartz cuvette (
We determined oligomerization state by size exclusion chromatography on an Akta Pure FPLC using a Superdex 75 5/150 analytical column. Both drug-free- and drug-bound ABLE eluted at elution volumes equivalent to its molecular weight (
We screened crystallization conditions for unliganded- and liganded ABLE in 96-well hanging drop trays from Hampton Research. His-tag-cleaved ABLE was concentrated in water at 30 mg/mL. For preparation of drug-bound ABLE, we added 1.1 equivalents of apixaban from a concentrated DMSO stock, resulting in a DMSO concentration of 12%. Both drug-bound- and drug-free ABLE readily crystallized in multiple conditions from Hampton Peg Ion 2 screen and the ammonium sulfate (AmSO4) screen. We looped the crystals and submerged them in paratone cryoprotectant before freezing them in liquid nitrogen. Diffraction data was collected remotely using an Eiger 16M detector at the 24-IDE (NE-CAT) beamline of the Advanced Photon Source at Fermi Lab. Multiple conditions gave high-quality diffraction with resolution below 2 Å. The well condition that gave the best diffraction for both drug-bound and drug-free ABLE was 2.6 M AmSO4, 0.1 M Na acetate. Crystals of both proteins diffracted to 1.3 Å resolution in this condition. Reflections were processed and merged using RAPD (https://rapd.nec.aps.anl.gov/). The structures were solved by molecular replacement with Phaser in Phenix, using the design model with apixaban removed. The structures were iteratively refined in Phenix and Coot. Diffraction data and refinement statistics of apixaban-bound- and drug-free ABLE are shown in Table S3. Crystals of the H49A mutant of ABLE were grown in a 24-well hanging drop plate with well solution 0.03 M Citric acid, 0.07 M BIS-TRIS propane/pH 7.6 with 20% w/v Polyethylene glycol 3,350 (Hampton PEG/Ion 2 screen condition 40). Crystals were looped in paratone and frozen in liquid nitrogen, and diffraction data to 1.6 Å resolution was collected on a PILATUS3 6M detector at the 8.3.1 beamline at the Advanced Light Source and Lawrence Berkeley National Labs. Reflections were processed and merged via XDS program and the structure was solved by molecular replacement with Phaser in Phenix, using the drug-free ABLE protein structure as the search model. The structure was iteratively refined in Phenix and Coot. Diffraction data and refinement statistics of unliganded H49A ABLE are shown in Table S4.
Contents of Constraint File (vdM_Hbonds.cst) for ABLE
Contents of Apixaban parameters file (APX.params) for use in Rosetta
PDB accession codes and chain IDs of the proteins used to compile vdM databases. List of 4-character PDB accession codes followed by a one-letter chain ID. Protein chains searched for curation of van der Mers. For each PDB, the biological assembly was constructed and protein contacts with the labeled chain were assessed.
1a1xA; 1a2 pA; 1a92A; 1abaA; 1ae9A; 1at0A; 1atzA; 1b0bA; 1b5eA; 1b6aA; 1b93A; 1bgfA; 1bm8A; 1bquA; 1bu8A; 1bx7A; 1byiA; 1bz4A; 1c1dA; 1c4oA; 1c4qA; 1c5eA; 1c75A; 1c7cA; 1c7kA; 1cc8A; 1ceoA; 1cfbA; 1chdA; 1cmcA; 1cqmA; 1cruA; 1cs6A; 1cv8A; 1cxqA; 1czaN; 1d0dA; 1d2 nA; 1d2sA; 1d2tA; 1d2zA; 1d2zB; 1d4oA; 1d5tA; 1dcsA; 1dj0A; 1dk8A; 1dkiA; 1d15A; 1d1yA; 1dmgA; 1doiA; 1dowA; 1dowB; 1dqgA; 1dusA; 1dwkA; 1dypA; 1dzfA; 1e29A; 1e2wA; 1e58A; 1e71A; 1eajA; 1eaqA; 1earA; 1egpA; 1egpB; 1e16A; 1e1kA; 1e1uA; 1e1wA; 1es5A; 1euvA; 1euwA; 1ev1A; 1ez3A; 1ezgA; fNiA; 1fDIA; 1f1eA; 1f1 mA; 1f1uA; 1f2tA; 1f2tB; 1f39A; 1f46A; 1f5 nA; 1f7A; 1f9zA; 1fhgA; 1fm0E; 1fm0D; 1fo8A; 1fobA; 1fp2A; 1fpoA; 1fxdA; 1fxoA; 1fyeA; 1g1jA; 1g1tA; 1g2bA; 1g3kA; 1g3 pA; 1g5aA; 1g60A; 1g61A; 1g66A; 1g6gA; 1g6uA; 1g8aA; 1ga6A; 1ga8A; 1gciA; 1gcqC; 1g12A; 1g12C; 1g14A; 1g14B; 1gn1A; 1gnyA; 1go3E; 1go3F; 1goiA; 1gp0A; 1gp6A; 1gppA; 1gpqA; 1gr3A; 1gsaA; 1gttA; 1guiA; 1gutA; 1gv2A; 1gv3A; 1gv9A; 1gvdA; 1gvfA; 1gvjA; 1gvnB; 1gvnA; 1gweA; 1gxmA; 1gxrA; 1gxyA; 1h0bA; 1h0hB; 1h12A; 1h16A; 1h2eA; 1h2sB; 1h2vZ; 1h4lA; 1h4aX; 1h6hA; 1h6kA; 1h6kX; 1h72C; 1h7eA; 1h80A; 1h8 pA; 1h97A; 1h98A; 1h99A; 1hdhA; 1hdoA; 1hh8A; 1hm1A; 1hq0A; 1hx6A; 1hxiA; 1hyoA; 1hztA; 1i0rA; 1i12A; 1i24A; 1i27A; 1i2hA; 1i2tA; 1i4uA; 1i71A; 1i7wB; 1i8aA; 1i9sA; 1iapA; 1igqA; 1ihgA; 1ihjA; 1iibA; 1ijbA; 1ikoP; 1ikpA; 1in4A; 1in1A; 1iomA; 1iq6A; 1iqzA; 1isuA; 1ituA; 1itxA; 1iu1A; 1iuqA; 1ix9A; 1ixhA; 1izcA; 1j0pA; 1j2jB; 1j2rA; 1j30A; 1j34C; 1j3aA; 1j3wA; 1j5uA; 1j5wA; 1j77A; 1j8uA; 1j97A; 1j98A; 1j9bA; 1jayA; 1jc1A; 1jd5A; 1jg1A; 1jhgA; 1jiwI; 1jjfA; 1jkeA; 1j10A; 1jm1A; 1jniA; 1jnrA; 1jnrB; 1jp4A; 1jpeA; 1jqeA; 1juhA; 1juvA; 1jvwA; 1jw9B; 1jx6A; 1jyhA; 1jztA; 1k2xB; 1k2xA; 1k3iA; 1k4 nA; 1k5cA; 1k5nB; 1k5 nA; 1k6dA; 1k77A; 1k7cA; 1ka1A; 1kb0A; 1kcqA; 1kdgA; 1keaA; 1kgdA; 1khiA; 1k19A; 1ki1A; 1k1xA; 1kmtA; 1knmA; 1koeA; 1kp6A; 1kq3A; 1kqfA; 1kqfB; 1kqfC; 1kqpA; 1kr4A; 1kwfA; 1kwgA; 1kwmA; 1kzfA; 1kzqA; 1l2 pA; 1l3kA; 1l3 pA; 1l6rA; 1l91A; 1l9xA; 1lamA; 1lf7A; 1lj8A; 1lj9A; 1ljoA; 1l1fA; 1lm1A; 1lniA; 1lo7A; 1lqtA; 1lqvA; 1lqvC; 1lr0A; 1lr5A; 1lr7A; 1ls1A; 1luaA; 1lwbA; 1m0dA; 1m0uA; 1m0wA; 1m15A; 1mifA; 1m1qA; 1m22A; 1m2dA; 1m3uA; 1m65A; 1m6sA; 1m70A; 1m9zA; 1maiA; 1mbmA; 1mc2A; 1mgqA; 1mgtA; 1mhnA; 1mixA; 1mj4A; 1mj5A; 1mkkA; 1mkzA; 1mo9A; 1mofA; 1mpgA; 1mpxA; 1mrzA; 1mugA; 1munA; 1muwA; 1mv1A; 1mw9X; 1mwqA; 1mxrA; 1n08A; 1n0qA; 1n12A; 1n13B; 1n13A; 1n2 mA; 1n62A; 1n62B; 1n62C; 1n71A; 1n7hA; 1n7sD; 1n7sB; 1n7sC; 1n8vA; 1n93X; 1n9 pA; 1na3A; 1nb9A; 1nbaA; 1nc7A; 1ne2A; 1ne7A; 1nepA; 1nfpA; 1ng2A; 1ng6A; 1njhA; 1nkiA; 1nifA; 1nnfA; 1nnhA; 1nniA; 1nnwA; 1noxA; 1nr0A; 1nrjB; 1nrjA; 1ns5A; 1ntvA; 1ntyA; 1nwaA; 1nwwA; 1nwzA; 1nxcA; 1nxmA; 1nycA; 1nykA; 1nz0A; 1nziA; 1nzjA; 1o13A; 1o1yA; 1o22A; 1o2dA; 1o3uA; 1o4wA; 1o4yA; 1o50A; 1o54A; 1o5uA; 1o6aA; 1o75A; 1o7iA; 1o8xA; 1o91A; 1o94A; 1o9gA; 1o9iA; 1oaaA; 1obbA; 1oc7A; 1odmA; 1odvA; 1of8A; 1ofcX; 1of1A; 1ofwA; 1oh0A; 1oh4A; 1oi0A; 1oi2A; 1oi6A; 1oi7A; 1ojhA; 1ok0A; 1okiA; 1oksA; 1onwA; 1ooeA; 1opkA; 1oq1A; 1oqjA; 1oqvA; 1oqwA; 1or0B; 1or0A; 1oruA; 1otkA; 1ou8A; 1ov3A; 1ow1A; 1ow3A; 1ox3A; 1oygA; 1oywA; 1oz2A; 1oz9A; 1oznA; 1p0hA; 1p0zA; 1p1jA; 1p1mA; 1p3cA; 1p3dA; 1p57A; 1p5vB; 1p5zB; 1p6oA; 1p99A; 1p9aG; 1p9gA; 1pb7A; 1pbjA; 1pdoA; 1pe9A; 1pg6A; 1pgxA; 1pj5A; 1pmhX; 1pn0A; 1pn2A; 1pprM; 1ppyA; 1pqhA; 1pswA; 1ptqA; 1pu6A; 1pucA; 1puoA; 1pvgA; 1pwaA; 1pwbA; 1pz4A; 1pzsA; 1q08A; 1q0pA; 1q0rA; 1q35A; 1q40B; 1q42A; 1q4uA; 1q5yA; 1q5zA; 1q6hA; 1q6oA; 1q74A; 1q71A; 1q71B; 1q8bA; 1q8dA; 1q9uA; 1qb0A; 1qbaA; 1qbzA; 1qcsA; 1qcxA; 1qgeD; 1qgeE; 1qkrA; 1qksA; 1qnxA; 1qq5A; 1qreA; 1qs1A; 1qsaA; 1qupA; 1qusA; 1qwdA; 1qwgA; 1qwoA; 1qwrA; 1qwyA; 1qz9A; 1r0dA; 1r0r1; 1r0uA; 1r1tA; 1r29A; 1r45A; 1r4vA; 1r4xA; 1r5 mA; 1r62A; 1r6wA; 1r6xA; 1r77A; 1r7aA; 1r7jA; 1r89A; 1r9hA; 1ra0A; 1rc9A; 1rfeA; 1rfsA; 1rfyA; 1rg8A; 1rhfA; 1ri6A; 1rjdA; 1rk8A; 1rk8C; 1rkiA; 1rkuA; 1r1hA; 1r1iA; 1rm6B; 1rmgA; 1rp0A; 1rqbA; 1rtqA; 1rttA; 1ru4A; 1rutX; 1rw1A; 1rwhA; 1rwjA; 1rwrA; 1rxqA; 1ry9A; 1ryiA; 1ryqA; 1rz3A; 1sidA; 1s29A; 1s4kA; 1s5aA; 1s5dA; 1s7qA; 1s8 nA; 1s99A; 1s9rA; 1s9uA; 1sauA; 1sbxA; 1sdiA; 1sfxA; 1sg4A; 1sgvA; 1sgwA; 1sh8A; 1shuX; 1sjyA; 1skzA; 1smxA; 1sngA; 1so7A; 1sr4A; 1sr4C; 1sraA; 1ss4A; 1stmA; 1sumB; 1suuA; 1syyA; 1szhA; 1sznA; 1szwA; 1t7A; 1t0bA; 1t0fA; 1t0fC; 1t0hB; 1t0hA; 1t0tV; 1tijA; 1tivA; 1t3yA; 1t44G; 1t4aA; 1t61A; 1t6cA; 1t6oA; 1t6oB; 1t6t1; 1t8kA; 1t92A; 1tafB; 1tafA; 1tazA; 1tbfA; 1tc5A; 1te2A; 1tfeA; 1tgrA; 1th7A; 1tigA; 1tjvA; 1tkeA; 1tovA; 1tp6A; 1tr0A; 1ts9A; 1tt8A; 1tu1A; 1tu9A; 1tuaA; 1tuhA; 1tvfA; 1twdA; 1txgA; 1tzpA; 1u07A; 1u0kA; 1u53A; 1u5dA; 1u5fA; 1u5uA; 1u60A; 1u69A; 1u6zA; 1u7iA; 1u7kA; 1u71A; 1u7 pA; 1u84A; 1u8vA; 1u9cA; 1ua4A; 1uaiA; 1ucrA; 1ucsA; 1uebA; 1uekA; 1ufyA; 1ui0A; 1uiiA; 1uixA; 1uj2A; 1ujcA; 1ukfA; 1ukuA; 1u1kA; 1uirA; 1uoyA; 1upsA; 1urqA; 1us0A; 1us3A; 1us5A; 1uscA; 1useA; 1ut7A; 1uujA; 1uuqA; 1uuyA; 1uwkA; 1ux6A; 1uxyA; 1v05A; 1v0aA; 1v2zA; 1v30A; 1v37A; 1v4 pA; 1v5iB; 1v5vA; 1v6tA; 1v70A; 1v76A; 1v7wA; 1v7zA; 1v8cA; 1v8hA; 1v9fA; 1v9yA; 1vbkA; 1vbwA; 1vcdA; 1vc1A; 1vctA; 1vd6A; 1ve4A; 1vj0A; 1vj1A; 1vjnA; 1vjvA; 1vk1A; 1vk4A; 1vkcA; 1vkeA; 1vkfA; 1vkhA; 1vkiA; 1vkkA; 1vkuA; 1vkwA; 1vkyA; 1v11A; 1v14A; 1v15A; 1v17A; 1v1aA; 1v1rA; 1v1yA; 1vmbA; 1vmgA; 1vmhA; 1vp8A; 1vpbA; 1vpkA; 1vpmA; 1vprA; 1vq3A; 1vqsA; 1vr6A; 1vr8A; 1vr9A; 1vraA; 1vyiA; 1vykA; 1vyrA; 1w07A; 1w0hA; 1w0pA; 1w1hA; 1w23A; 1w2wA; 1w2wB; 1w2yA; 1w4sA; 1w53A; 1w5fA; 1w5rA; 1w66A; 1w6sA; 1w6sB; 1w7cA; 1w851; 1w8oA; 1w8sA; 1w99A; 1w9aA; 1w9eA; 1wb0A; 1wb4A; 1wckA; 1wd3A; 1wd5A; 1wddA; 1wddS; 1wdjA; 1wehA; 1whiA; 1whzA; 1wiwA; 1wjxA; 1wkaA; 1wkcA; 1wkoA; 1wkyA; 1w18A; 1w1uA; 1w1zA; 1wmdA; 1wmhA; 1wmhB; 1wmwA; 1wn2A; 1wnaA; 1woqA; 1wouA; 1wovA; 1wpaA; 1wpbA; 1wpnA; 1wqjI; 1wqjB; 1wr8A; 1wrdA; 1wtjA; 1wu9A; 1wvqA; 1wvvA; 1wwiA; 1wwzA; 1wyxA; 1wz3A; 1wzdA; 1wmA; 1wzoA; 1x2iA; 1x54A; 1x6oA; 1x6vA; 1x7dA; 1x7vA; 1x8qA; 1x91A; 1x9dA; 1x9iA; 1x9uA; 1xawA; 1xd3A; 1xdnA; 1xdzA; 1xe1A; 1xe7A; 1xerA; 1xffA; 1xfkA; 1xfsA; 1xg0C; 1xg0B; 1xg0A; 1xg7A; 1xgkA; 1xgsA; 1xiwA; 1xiwB; 1xjuA; 1xkiA; 1xkpA; 1xkpC; 1xkpB; 1xmkA; 1xmtA; 1xnfA; 1xo5A; 1xocA; 1xovA; 1xqaA; 1xqoA; 1xrkA; 1xruA; 1xsvA; 1xt5A; 1xt8A; 1xtpA; 1xu1A; 1xu1R; 1xubA; 1xv2A; 1xw3A; 1y02A; 1y07A; 1y0bA; 1y0hA; 1y0kA; 1y0pA; 1y0uA; 1y12A; 1y2 mA; 1y43B; 1y43A; 1y63A; 1y71A; 1y7wA; 1y81A; 1y88A; 1y89A; 1y8aA; 1y91A; 1y9qA; 1y9zA; 1yacA; 1yar0; 1yb0A; 1yb3A; 1ybkA; 1ybxA; 1ybzA; 1yc9A; 1ycdA; 1yd0A; 1ydyA; 1ye8A; 1yf9A; 1yfqA; 1ygtA; 1yhfA; 1yhtA; 1yj7A; 1ykdA; 1ykiA; 1y1eA; 1y1mA; 1y1xA; 1ym3A; 1ymtA; 1yn3A; 1ynbA; 1yoaA; 1yocA; 1yphE; 1yphC; 1ypyA; 1yq5A; 1yqhA; 1yqsA; 1yrkA; 1ysiX; 1ysrA; 1yt3A; 1yt8A; 1yu0A; 1yu5X; 1yu6C; 1yuzA; 1ywfA; 1ywmA; 1yxiA; 1yyhA; 1yzfA; 1yzmA; 1z05A; 1z0jB; 1z0kB; 1z0nA; 1z0pA; 1z0sA; 1z21A; 1z2nX; 1z3eA; 1z3eB; 1z4eA; 1z4rA; 1z67A; 1z6 mA; 1z6 nA; 1z6oA; 1z6oM; 1z70X; 1z72A; 1z7aA; 1z96A; 1z91A; 1z9tA; 1za0A; 1za4A; 1zarA; 1zceA; 1zczA; 1ze1A; 1zgkA; 1zgxA; 1zgxB; 1zhvA; 1zhxA; 1zi8A; 1zjcA; 1zk5A; 1zkeA; 1zkiA; 1zkpA; 1zl0A; 1zidA; 1z1hB; 1zm8A; 1zmaA; 1zn6A; 1zpsA; 1zpvA; 1zq9A; 1zr6A; 1zruA; 1zs9A; 1zswA; 1zsyA; 1zt3A; 1ztdA; 1zuoA; 1zv1A; 1zvaA; 1zvtA; 1zxxA; 1zy7A; 1zzgA; 2a14A; 2a15A; 2a26A; 2a35A; 2a3 mA; 2a3 nA; 2a40B; 2a40C; 2a4aA; 2a50B; 2a50A; 2a5 dB; 2a61A; 2a6sA; 2a6yA; 2a6zA; 2a71A; 2a9dA; 2a9iA; 2a9sA; 2aanA; 2abkA; 2absA; 2ae0X; 2aeeA; 2aexA; 2ah2A; 2ah5A; 2ahfA; 2ahuA; 2aibA; 2akzA; 2amhA; 2an1A; 2anxA; 2ao9A; 2ap3A; 2apjA; 2apoB; 2apoA; 2ar1A; 2asfA; 2askA; 2au3A; 2axcA; 2axqA; 2axwA; 2aydA; 2ayhA; 2azwA; 2b06A; 2b0aA; 2b0cA; 2b0tA; 2b0vA; 2b18A; 2b1eA; 2b1kA; 2b1yA; 2b3fA; 2b4hA; 2b4vA; 2b5aA; 2b69A; 2b7kA; 2b82A; 2b8iA; 2b8 mA; 2b9eA; 2b9wA; 2bayA; 2bbaA; 2bbeA; 2bbrA; 2bcmA; 2bdrA; 2bezC; 2bezF; 2bf6A; 2bfdB; 2bfdA; 2bfwA; 2bh8A; 2bhuA; 2bi0A; 2biiA; 2bjdA; 2bjiA; 2bjqA; 2bk9A; 2bkaA; 2bkfA; 2bkrA; 2bkxA; 2b10A; 2b18A; 2b19A; 2b1fB; 2b1nA; 2bm5A; 2bmoA; 2bmoB; 2bn1A; 2bnmA; 2bo4A; 2bo9B; 2bogX; 2brfA; 2bryA; 2bsjA; 2bt6A; 2btiA; 2bu3A; 2bueA; 2bv2A; 2bvfA; 2bwrA; 2bz1A; 2bzvA; 2c0aA; 2c0gA; 2c0nA; 2c0zA; 2c1iA; 2c21A; 2c2iA; 2c2 pA; 2c2uA; 2c3fA; 2c3 nA; 2c42A; 2c43A; 2c46A; 2c4bA; 2c4eA; 2c4xA; 2c5aA; 2c5qA; 2c60A; 2c78A; 2c81A; 2c92A; 2c9wA; 2ca1A; 2cayA; 2cb8A; 2cb9A; 2cc0A; 2cc6A; 2cchB; 2cdcA; 2cf7A; 2cgqA; 2ch5A; 2chcA; 2cisA; 2ciuA; 2ciwA; 2cj4A; 2cjjA; 2cjtA; 2ckkA; 2c13A; 2cm5A; 2co3A; 2covD; 2cs7A; 2cu1A; 2cvbA; 2cveA; 2cw9A; 2cwyA; 2cwzA; 2cx1A; 2cx7A; 2cxhA; 2cxiA; 2cxkA; 2cxxA; 2cxyA; 2cy5A; 2czqA; 2d0A; 2d0B; 2d1cA; 2d1sA; 2d1zA; 2d2eA; 2d3dA; 2d4 pA; 2d58A; 2d59A; 2d5 mA; 2d68A; 2d7cC; 2d81A; 2d8dA; 2db7A; 2dbnA; 2dbyA; 2dc3A; 2dc4A; 2ddfA; 2ddrA; 2ddxA; 2de3A; 2de6A; 2dejA; 2dg1A; 2dgaA; 2djfB; 2djfA; 2dkaA; 2dkhA; 2dkjA; 2dkoB; 2dkoA; 2dkvA; 2dm9A; 2dokA; 2dp9A; 2dpfA; 2dp1A; 2dq1A; 2ds5A; 2dskA; 2dsyA; 2dt4A; 2dtcA; 2dtjA; 2du1A; 2duyA; 2dvkA; 2dvmA; 2dwkA; 2dxaA; 2dxqA; 2dxuA; 2dy0A; 2dy1A; 2dyiA; 2dyjA; 2e11A; 2e1nA; 2e1vA; 2e26A; 2e2oA; 2e3hA; 2e3 nA; 2e4tA; 2e5fA; 2e6fA; 2e7zA; 2e8eA; 2eabA; 2eb1A; 2ebbA; 2efvA; 2eg4A; 2egdA; 2egjA; 2eh3A; 2ehpA; 2ehzA; 2ei5A; 2eiyA; 2ej8A; 2ejnA; 2e1cA; 2endA; 2eo4A; 2epiA; 2ep1X; 2eq7C; 2eq8C; 2erfA; 2er1A; 2ervA; 2erwA; 2essA; 2et1A; 2etbA; 2etvA; 2ev1A; 2ew0A; 2ewhA; 2ewtA; 2ez1A; 2fD1A; 2fDcA; 2f1kA; 2f2bA; 2f46A; 2f4 pA; 2f60K; 2f62A; 2f68X; 2f6eA; 2f6rA; 2f7bA; 2f7vA; 2f9iA; 2f9iB; 2faoA; 2fb5A; 2fb6A; 2fbaA; 2fbnA; 2fboJ; 2fbqA; 2fcjA; 2fc1A; 2fcoA; 2fctA; 2fcwB; 2fcwA; 2fd5A; 2fdrA; 2fdvA; 2feaA; 2fefA; 2fexA; 2ff3A; 2ff4A; 2ffuA; 2ffyA; 2fh1A; 2fh7A; 2fhfA; 2fhpA; 2fhzB; 2fhzA; 2fi1A; 2fipA; 2fiuA; 2fj8A; 2fk5A; 2fk8A; 2fk9A; 2fkkA; 2f14A; 2fm9A; 2fmaA; 2fmmE; 2fnaA; 2fnoA; 2fnuA; 2fo3A; 2fomB; 2fomA; 2fozA; 2fp1A; 2fp7A; 2fp7B; 2fprA; 2fq3A; 2fq4A; 2fqpA; 2fqxA; 2fr2A; 2fr5A; 2freA; 2frgP; 2fsqA; 2fsrA; 2fsxA; 2fM0A; 2ftrA; 2fu4A; 2fueA; 2fukA; 2fupA; 2furA; 2fvhA; 2fvvA; 2fvyA; 2fwvA; 2fy6A; 2fy7A; 2fyfA; 2fygA; 2fzpA; 2fzvA; 2g0cA; 2g0iA; 2g0wA; 2g1uA; 2g30A; 2g3aA; 2g3bA; 2g40A; 2g45A; 2g5gX; 2g5xA; 2g62A; 2g7cA; 2g7oA; 2g81I; 2g84A; 2g8sA; 2g9wA; 2ga1A; 2gaiA; 2gakA; 2gauA; 2gaxA; 2gb4A; 2ge7A; 2gecA; 2genA; 2geyA; 2gf6A; 2gffA; 2gfhA; 2gfnA; 2gfqA; 2ggcA; 2ghsA; 2gibA; 2gj4A; 2gkeA; 2gkgA; 2gkpA; 2g19A; 2g1zA; 2gmwA; 2gmyA; 2gnoA; 2gomA; 2gpiA; 2gq0A; 2gqtA; 2gqwA; 2gr8A; 2gs5A; 2gsoA; 2gu3A; 2gu9A; 2guiA; 2gviA; 2gwgA; 2gwmA; 2gwnA; 2gxgA; 2gyqA; 2gz4A; 2gz6A; 2h0uA; 2h1cA; 2h1tA; 2h1vA; 2h2tB; 2h7oA; 2h88A; 2h88B; 2h88C; 2h88D; 2h8eA; 2h8gA; 2h8oA; 2h98A; 2ha8A; 2hbaA; 2hboA; 2hbwA; 2hc8A; 2hc9A; 2hcfA; 2hcmA; 2hd9A; 2hdoA; 2hdwA; 2hdzA; 2hekA; 2heuA; 2hewF; 2heyR; 2hf2A; 2hfsA; 2hhcA; 2hhgA; 2hhzA; 2hi0A; 2hinA; 2hiqA; 2hiyA; 2hjeA; 2hjnA; 2hkjA; 2hkvA; 2h17A; 2h1jA; 2h1rA; 2h1yA; 2hngA; 2hoxA; 2hp0A; 2hpjA; 2hpsA; 2hq7A; 2hq9A; 2hqqA; 2hqsA; 2hqsC; 2hqxA; 2hqyA; 2hrzA; 2hs1A; 2hsbA; 2hsiA; 2hsjA; 2ht9A; 2htaA; 2htdA; 2hu9A; 2huhA; 2hujA; 2hv8D; 2hvwA; 2hw4A; 2hx0A; 2hxiA; 2hxvA; 2hy1A; 2hy5A; 2hy5C; 2hy5B; 2hy7A; 2hytA; 2hzqA; 2i0kA; 2i0zA; 2i2oA; 2i3dA; 2i49A; 2i4lA; 2i51A; 2i5hA; 2i5iA; 2i5uA; 2i5v0; 2i6hA; 2i71A; 2i7aA; 2i7fA; 2i8bA; 2i8dA; 2i8tA; 2i9aA; 2i9cA; 2i9iA; 2i9wA; 2i9xA; 2ia1A; 2ia7A; 2iabA; 2iaiA; 2iayA; 2ib0A; 2ibdA; 2ib1A; 2ibnA; 2ic6A; 2icgA; 2ichA; 2icuA; 2id3A; 2id6A; 2id1A; 2ie1A; 2ieqA; 2iewA; 2if6A; 2ifxA; 2ig8A; 2igiA; 2igpA; 2ih3C; 2ihtA; 2ii1A; 2ii2A; 2iidA; 2ij2A; 2ijqA; 2ikbA; 2iksA; 2i1rA; 2im9A; 2imfA; 2imhA; 2imjA; 2imqX; 2imrA; 2imzA; 2in3A; 2inwA; 2ionA; 2ip1A; 2ip6A; 2iqjA; 2iruA; 2is9A; 2isbA; 2it2A; 2it9A; 2iu1A; 2iu5A; 2iumA; 2iuwA; 2ivfA; 2ivfB; 2ivfC; 2ivyA; 2iw0A; 2iw1A; 2iwaA; 2ixdA; 2ixsA; 2iy2A; 2iy9A; 2iyjA; 2iyvA; 2izxA; 2j0aA; 2j1aA; 2j1vA; 2j2jA; 2j5gA; 2j5yA; 2j6aA; 2j6bA; 2j6yA; 2j73A; 2j7qA; 2j89A; 2j8bA; 2j8hA; 2j8kA; 2j91A; 2j97A; 2j9oA; 2j9wA; 2jaeA; 2jayA; 2jc9A; 2jdaA; 2jdcA; 2jdjA; 2je6B; 2je6A; 2je6I; 2je8A; 2jekA; 2jenA; 2jfrA; 2jg0A; 2jgsA; 2jh1A; 2jhnA; 2jjqA; 2jjsC; 2jkbA; 2jkuA; 2j1iA; 2j1jA; 2mcmA; 2mhrA; 2msbA; 2n1vA; 2nm1A; 2nnuA; 2nnuB; 2no4A; 2np5A; 2nptA; 2nptB; 2nq3A; 2ngtA; 2nqwA; 2nr5A; 2nr7A; 2nrkA; 2nrrA; 2nrtA; 2nszA; 2nt0A; 2ntpA; 2nujA; 2nvaA; 2nw8A; 2nwfA; 2nx2A; 2nx4A; 2nxcA; 2nxfA; 2nxvA; 2nxwA; 2nyiA; 2nz7A; 2nzcA; 2nz1A; 2o0 mA; 2o0yA; 2o16A; 2o1aA; 2o1kA; 2o1qA; 2o28A; 2o2gA; 2o2kA; 2o2vB; 2o2xA; 2o30A; 2o3fA; 2o4dA; 2o4jA; 2o4tA; 2o4xB; 2o57A; 2o5hA; 2o5uA; 2o5vA; 2o62A; 2o66A; 2o6fA; 2o61A; 2o6 pA; 2o6xA; 2o70A; 2o7rA; 2o8 nA; 2o8 pA; 2o8qA; 2o90A; 2o9sA; 2oa2A; 2oafA; 2ob0A; 2ob3A; 2ob5A; 2obpA; 2oczA; 2od0A; 2od4A; 2od5A; 2od6A; 2odfA; 2odkA; 2od1A; 2oebA; 2ofkA; 2ofzA; 2og2A; 2og4A; 2ogfA; 2oh1A; 2oh3A; 2ohwA; 2oikA; 2oiwA; 2oixA; 2oizD; 2oizA; 2oj6A; 2ojhA; 2okfA; 2okgA; 2okqA; 2oktA; 2okuA; 2o1nA; 2o1rA; 2o1tA; 2o1wA; 2omdA; 2omkA; 2om1A; 2onfA; 2ooaA; 2oocA; 2oojA; 2ookA; 2opjA; 2op1A; 2opoA; 2opwA; 2oqbA; 2oqkA; 2oqmA; 2oqzA; 2orwA; 2os0A; 2osoA; 2osxA; 2otmA; 2ou3A; 2ou5A; 2ou6A; 2ouwA; 2ov0A; 2ov9A; 2ovgA; 2ovjA; 2owaA; 2ownA; 2owpA; 2ox6A; 2oxgB; 2oxgA; 2ox1A; 2oy7A; 2oy9A; 2oyaA; 2oyoA; 2oyzA; 2ozgA; 2ozhA; 2oznA; 2oznB; 2oztA; 2ozvA; 2p09A; 2p0aA; 2p0kA; 2p0nA; 2p0sA; 2p0wA; 2p14A; 2p17A; 2p18A; 2p1mA; 2p1mB; 2p25A; 2p2sA; 2p2vA; 2p35A; 2p3hA; 2p3 pA; 2p3wA; 2p3yA; 2p4eA; 2p4oA; 2p4 pA; 2p51A; 2p57A; 2p58A; 2p58C; 2p58B; 2p5kA; 2p5 mA; 2p65A; 2p6vA; 2p6wA; 2p7iA; 2p8gA; 2p8iA; 2p8jA; 2p8tA; 2p97A; 2p9wA; 2pagA; 2pb7A; 2pbcA; 2pbdV; 2pbiA; 2pb1A; 2pc1A; 2pcnA; 2pd1A; 2pdrA; 2pebA; 2petA; 2pfiA; 2pfzA; 2pgeA; 2pgfA; 2pgoA; 2ph0A; 2phnA; 2pieA; 2pjsA; 2pjzA; 2pk8A; 2pkeA; 2pkfA; 2pkhA; 2p1iA; 2pn0A; 2pn1A; 2pn2A; 2pn6A; 2pndA; 2pneA; 2pnwA; 2pokA; 2posA; 2ppqA; 2ppvA; 2ppxA; 2pq7A; 2pqvA; 2pr5A; 2pr7A; 2prvA; 2pstX 2pt6A; 2pttB; 2pttA; 2pu3A; 2puzA; 2pv4A; 2pv7A; 2pwwA; 2pyqA; 2pytA; 2pywA; 2pyxA; 2pzeA; 2q03A; 2q0sA; 2q0tA; 2q0yA; 2q12A; 2q1sA; 2q24A; 2q2fA; 2q2hA; 2q30A; 2q35A; 2q3xA; 2q5cA; 2q5wD; 2q6kA; 2q73A; 2q79A; 2q7bA; 2q7sA; 2q86A; 2q88A; 2q8kA; 2q8 pA; 2q8rE; 2q9kA; 2q9rA; 2q9vA; 2qb7A; 2qbwA; 2qckA; 2qe6A; 2qe8A; 2qe9A; 2qebA; 2qecA; 2qedA; 2qeeA; 2qeuA; 2qf4A; 2qf7A; 2qfaA; 2qfaB; 2qfaC; 2qfeA; 2qg3A; 2qg8A; 2qgmA; 2qguA; 2qgyA; 2qhfA; 2qhkA; 2qhoB; 2qhpA; 2qhqA; 2qhsA; 2qhtA; 2qibA; 2qihA; 2qikA; 2qipA; 2qiwA; 2qiyA; 2qiyC; 2qj1A; 2qjvA; 2qjwA; 2qjzA; 2qkdA; 2qkhB; 2qkhA; 2qkpA; 2q18A; 2q1tA; 2q1wA; 2q1xA; 2qm8A; 2qm1A; 2qmqA; 2qngA; 2qniA; 2qnkA; 2qn1A; 2qntA; 2qo1A; 2qp2A; 2qpwA; 2qpxA; 2qq5A; 2qqiA; 2qqmA; 2qqyA; 2qr6A; 2qruA; 2qs9A; 2qsbA; 2qsiA; 2qskA; 2qsxA; 2qt1A; 2qtdA; 2qtqA; 2qtwB; 2qtzA; 2qudA; 2qupA; 2qv5A; 2qvgA; 2qvkA; 2qw5A; 2qwoB; 2qwuA; 2qy6A; 2qycA; 2qywA; 2qzcA; 2qzqA; 2qztA; 2r01A; 2r09A; 2r0hA; 2r0xA; 2r0yA; 2r16A; 2r1iA; 2r2cA; 2r2oA; 2r2zA; 2r37A; 2r3aA; 2r3bA; 2r44A; 2r4fA; 2r4gA; 2r4iA; 2r4qA; 2r58A; 2r5oA; 2r5uA; 2r6qA; 2r6vA; 2r6zA; 2r78A; 2r7hA; 2r85A; 2r8eA; 2r8uA; 2r9fA; 2ra9A; 2rafA; 2rasA; 2rauA; 2rb7A; 2rbbA; 2rbcA; 2rbdA; 2rbgA; 2rbkA; 2rc3A; 2rccA; 2rciA; 2rczA; 2rdcA; 2rdgA; 2rdqA; 2re2A; 2reeA; 2rfaA; 2rffA; 2rfmA; 2rfqA; 2rfrA; 2rg8A; 2rgqA; 2rh0A; 2rh2A; 2rh3A; 2rhkA; 2rhkC; 2rhmA; 2rhwA; 2rijA; 2ri1A; 2riqA; 2rivB; 2rj2A; 2rjiA; 2rkhA; 2rk1A; 2rkqA; 2rkvA; 2r1dA; 2sakA; 2tpsA; 2uu8A; 2uurA; 2uuyB; 2uv4A; 2uvkA; 2uvoA; 2uw1A; 2uxwA; 2uyoA; 2uytA; 2uz1A; 2v03A; 2v05A; 2v0cA; 2v1mA; 2v1oA; 2v27A; 2v2fF; 2v2fA; 2v2kA; 2v33A; 2v3gA; 2v3iA; 2v4xA; 2v57A; 2v6vA; 2v6xB; 2v75A; 2v76A; 2v7fA; 2v7kA; 2v7sA; 2v89A; 2v8fA; 2v8fC; 2v8iA; 2v8tA; 2v94A; 2v9vA; 2vacA; 2vb1A; 2vbkA; 2vchA; 2vc1A; 2vcnA; 2vdjA; 2ve8A; 2vfoA; 2vfrA; 2vfxA; 2vgxA; 2vh3A; 2vhjA; 2vhkA; 2vifA; 2vjwA; 2vk8A; 2vkjA; 2vk1A; 2v1gA; 2v1iA; 2v1qA; 2vm5A; 2vn4A; 2vngA; 2vn1A; 2vo8A; 2vo9A; 2vovA; 2vpaA; 2vpbB; 2vpbA; 2vpnA; 2vptA; 2vq2A; 2vqgA; 2vqpA; 2vqxA; 2vrsA; 2vrwB; 2vsmA; 2vt1A; 2vt1B; 2vtwA; 2vunA; 2vveA; 2vvmA; 2vvwA; 2vw8A; 2vwsA; 2vxgA; 2vxnA; 2vxtI; 2vxzA; 2vy8A; 2vyoA; 2vzcA; 2vzpA; 2vzsA; 2vzyA; 2w0bA; 2w0iA; 2w15A; 2w1rA; 2w1sA; 2w1vA; 2w2aA; 2w2kA; 2w2rA; 2w31A; 2w39A; 2w3gA; 2w3 pA; 2w3qA; 2w3yA; 2w3zA; 2w40A; 2w4eA; 2w50A; 2w56A; 2w5eA; 2w5fA; 2w5qA; 2w61A; 2w6aA; 2w7aA; 2w87A; 2w8tA; 2w8xA; 2w91A; 2w9yA; 2waaA; 2wanA; 2waoA; 2wawA; 2wb3A; 2wb6A; 2wbfX; 2wbmA; 2wbnA; 2wbqA; 2wbxA; 2wciA; 2wcrA; 2wdcA; 2wdsA; 2we5A; 2wf7A; 2wfiA; 2wfoA; 2wfpA; 2wg7A; 2wgpA; 2wh7A; 2wi8A; 2wiyA; 2wj5A; 2wj9A; 2wjeA; 2wjnM; 2wjnL; 2wjnH; 2wjnC; 2wjrA; 2wk1A; 2wkkA; 2wkqA; 2w1cA; 2w1rA; 2w1uA; 2wm3A; 2wm8A; 2wn9A; 2wnfA; 2wnpF; 2wnsA; 2wnwA; 2wnxA; 2wnyA; 2wo1A; 2woyA; 2wp7A; 2wpvA; 2wpvB; 2wq4A; 2wqfA; 2wqkA; 2wsdA; 2wshA; 2wt0A; 2wtaA; 2wteA; 2wtgA; 2wtmA; 2wuqA; 2wv3A; 2wvfA; 2wvxA; 2wweA; 2wwxB; 2wxuA; 2wy8Q; 2wyhA; 2wz8A; 2wz9A; 2wzbA; 2wzoA; 2wzvA; 2x0kA; 2x0qA; 2x1dA; 2x26A; 2x2sA; 2x2uA; 2x32A; 2x3cA; 2x3gA; 2x3jA; 2x31A; 2x3 mA; 2x3 nA; 2x46A; 2x49A; 2x4dA; 2x4jA; 2x4kA; 2x4lA; 2x55A; 2x5cA; 2x5fA; 2x5hA; 2x5 nA; 2x5oA; 2x5 pA; 2x5rA; 2x5xA; 2x5yA; 2x61A; 2x7 mA; 2x7qA; 2x8hA; 2x8sA; 2x98A; 2x9oA; 2x9xA; 2x9zA; 2xbgA; 2xc1A; 2xcjA; 2xdgA; 2xdjA; 2xdpA; 2xdwA; 2xe4A; 2xedA; 2xepA; 2xetA; 2xf7A; 2xfdA; 2xfgB; 2xfnA; 2xfrA; 2xfvA; 2xg5B; 2xgrA; 2xhfA; 2xhgA; 2xhnA; 2xi8A; 2xi9A; 2xigA; 2xijA; 2xioA; 2xkiA; 2x1gA; 2xm5A; 2xmxA; 2xmzA; 2xn6A; 2xn6B; 2xnqA; 2xocA; 2xodA; 2xo1A; 2xovA; 2xppA; 2xppB; 2xpwA; 2xqoA; 2xrhA; 2xryA; 2xsaA; 2xseA; 2xsgA; 2xskA; 2xsqA; 2xswA; 2xt2A; 2xtpA; 2xtyA; 2xu3A; 2xu8A; 2xuvA; 2xvsA; 2xvyA; 2xw6A; 2xwsA; 2xwtC; 2xwvA; 2xx1A; 2xxmA; 2xxpA; 2xxzA; 2xy2A; 2xyiA; 2xyiB; 2xyqA; 2xz2A; 2xz8A; 2xz9A; 2xzeA; 2xzeQ; 2xziA; 2y0oA; 2y27A; 2y2 mA; 2y2zA; 2y32A; 2y3cA; 2y43A; 2y44A; 2y53A; 2y5fL; 2y6uA; 2y6xA; 2y71A; 2y78A; 2y7bA; 2y7 pA; 2y8gA; 2y8kA; 2y8 nA; 2y8nB; 2y8uA; 2y9uA; 2ya0A; 2yanA; 2yavA; 2yb1A; 2ybqA; 2ybyA; 2yc3A; 2ycdA; 2yd6A; 2yeqA; 2yfaA; 2yfoA; 2yfrA; 2yfuA; 2yg2A; 2yg9A; 2ygnA; 2ygoA; 2yh9A; 2yhaA; 2yhgA; 2yhsA; 2yhwA; 2yi1A; 2yimA; 2yjgA; 2yk4A; 2y1eA; 2y1eB; 2y1nA; 2ymmA; 2ymuA; 2ymvA; 2ymyA; 2yn0A; 2yn5A; 2ynaA; 2ynyA; 2ynzA; 2yp6A; 2ypoA; 2yqyA; 2yqzA; 2yskA; 2yv4A; 2yv9A; 2yviA; 2yvtA; 2yw3A; 2ywiA; 2yw1A; 2yxnA; 2yxoA; 2yxzA; 2yyhA; 2yyyA; 2yzjA; 2yzkA; 2yzqA; 2yzsA; 2yztA; 2yzyA; 2z0bA; 2z0dA; 2z0jA; 2z0qA; 2z0tA; 2z14A; 2zlcA; 2z26A; 2z30B; 2z3hA; 2z3zA; 2z5eA; 2z62A; 2z6dA; 2z6iA; 2z6oA; 2z6rA; 2z72A; 2z7fI; 2z84A; 2z8fA; 2z8xA; 2z98A; 2z9wA; 2za4B; 2zb1A; 2zcaA; 2zcwA; 2zdpA; 2zexA; 2zf9A; 2zfdA; 2zfdB; 2zfuA; 2zfyA; 2zfzA; 2zhjA; 2zkmX; 2znrA; 2zonG; 2zouA; 2zp1A; 2zpmA; 2zptX; 2zq5A; 2zqeA; 2zqmA; 2zqoA; 2zs0A; 2zsjA; 2zuvA; 2zuxA; 2zvcA; 2zw2A; 2zwaA; 2zx2A; 2zxqA; 2zxyA; 2zycA; 2zzdB; 2zzdA; 2zzjA; 3a02A; 3a07A; 3a0sA; 3a0yA; 3a1fA; 3a1sA; 3a21A; 3a2qA; 3a2vA; 3a3dA; 3a4uB; 3a54A; 3a57A; 3a5fA; 3a5 pA; 3a6rA; 3a72A; 3a8gA; 3a8gB; 3a9fA; 3a9iA; 3a91A; 3a9sA; 3aa0B; 3aa0A; 3aa0C; 3ab8A; 3abdA; 3abdX; 3achA; 3acxA; 3adgA; 3adoA; 3aehA; 3afoA; 3ag7A; 3agnA; 3agyA; 3ahcA; 3ahnA; 3ai4A; 3ai5A; 3aiaA; 3aiiA; 3aj3A; 3aj4A; 3aj7A; 3ajdA; 3ajfA; 3ak8A; 3akbA; 3akeA; 3akhA; 3a12A; 3amnA; 3anpA; 3aofA; 3apqA; 3aq2A; 3aqiA; 3aqjA; 3arxA; 3as5A; 3as1A; 3atsA; 3au8A; 3awuA; 3awuB; 3ax2A; 3axbA; 3axgA; 3ay2A; 3ayjA; 3ayvA; 3azoA; 3b02A; 3b08B; 3b0fA; 3b0gA; 3b0pA; 3b0xA; 3b1bA; 3b2yA; 3b40A; 3b42A; 3b4 nA; 3b4qA; 3b4uA; 3b5eA; 3b5 mA; 3b5oA; 3b64A; 3b6eA; 3b6hA; 3b79A; 3b7cA; 3b7eA; 3b8bA; 3b8fA; 3b81A; 3b9tA; 3b9wA; 3ba3A; 3bb0A; 3bb7A; 3bb9A; 3bc1B; 3bc8A; 3bc9A; 3bcwA; 3bcyA; 3bd1A; 3bdeA; 3bdiA; 3bduA; 3bdvA; 3be6A; 3bemA; 3bexA; 3bf5A; 3bf7A; 3bfmA; 3bfoA; 3bg2A; 3bgeA; 3bguA; 3bgyA; 3bh4A; 3bh7B; 3bhdA; 3bhgA; 3bhnA; 3bhqA; 3biqA; 3biyA; 3bj4A; 3bjdA; 3bjeA; 3bjkA; 3bjnA; 3bk5A; 3bkbA; 3bkpA; 3bkrA; 3bkwA; 3bkxA; 3b19A; 3b1nA; 3b1zA; 3bm7A; 3bmvA; 3bmxA; 3bmzA; 3bn0A; 3bn7A; 3bnjA; 3bo6A; 3bodA; 3boeA; 3bofA; 3bonA; 3bosA; 3bp3A; 3bpjA; 3bpkA; 3bptA; 3bpvA; 3bpzA; 3bq3A; 3bqkA; 3bqxA; 3brcA; 3bs4A; 3bs5A; 3bs5B; 3bs6A; 3bt5A; 3butA; 3buuA; 3bv6A; 3bv8A; 3bvuA; 3bwhA; 3bw1A; 3bwsA; 3bwvA; 3bwxA; 3bwzA; 3bx4B; 3by4A; 3byqA; 3bzhA; 3bztA; 3bzwA; 3c05A; 3c0cA; 3c0fB; 3c18A; 3c1aA; 3c1dA; 3c1qA; 3c1vA; 3c24A; 3c26A; 3c2qA; 3c2uA; 3c37A; 3c3 pA; 3c4bA; 3c4 mA; 3c4mC; 3c4sA; 3c57A; 3c5eA; 3c5hA; 3c5 nA; 3c5rA; 3c5vA; 3c6aA; 3c6kA; 3c6vA; 3c6wA; 3c7fA; 3c7xA; 3c85A; 3c8cA; 3c8eA; 3c8iA; 3c81A; 3c8uA; 3c8wA; 3c8zA; 3c9aA; 3c9fA; 3c9hA; 3c9 pA; 3c9uA; 3c9zA; 3ca7A; 3caiA; 3canA; 3cawA; 3cbnA; 3cbwA; 3cc1A; 3cc8A; 3ccdA; 3ccfA; 3ccgA; 3ce7A; 3cecA; 3cetA; 3cexA; 3cg6A; 3cggA; 3cgxA; 3ch0A; 3chjA; 3chmA; 3ci3A; 3ci6A; 3cimA; 3cinA; 3citA; 3cj1A; 3cjdA; 3cjeA; 3cjmA; 3cjnA; 3cjsB; 3cjsA; 3cjyA; 3ck1A; 3ck6A; 3ckcA; 3ckjA; 3ckkA; 3ckmA; 3c15A; 3c1mA; 3cmbA; 3cmgA; 3cnhA; 3cnqP; 3cnuA; 3cnvA; 3cnyA; 3covA; 3cp0A; 3cp5A; 3cp7A; 3cq1A; 3cqvA; 3cryA; 3cs1A; 3csgA; 3ct5A; 3ct6A; 3ctpA; 3ctzA; 3cu2A; 3cu3A; 3cu9A; 3cuzA; 3cv0A; 3cveA; 3cvjA; 3cvoA; 3cwiA; 3cwnA; 3cwrA; 3cwvA; 3cwwA; 3cx2A; 3cxkA; 3cxnA; 3cypB; 3cz1A; 3cz7A; 3czpA; 3czxA; 3czzA; 3d00A; 3d0A; 3d02A; 3d06A; 3d0fA; 3d0jA; 3d0wA; 3d1pA; 3d1rA; 3d22A; 3d21A; 3d2qA; 3d30A; 3d32A; 3d33A; 3d3bJ; 3d3bA; 3d3yA; 3d40A; 3d4eA; 3d59A; 3d5 pA; 3d6jA; 3d6 mA; 3d6mB; 3d79A; 3d7aA; 3d7iA; 3d7jA; 3d8tA; 3d9 nA; 3d9xA; 3da0A; 3da8A; 3dacA; 3dacB; 3da1A; 3daoA; 3db0A; 3db7A; 3dcdA; 3dcxA; 3dcyA; 3dczA; 3dd7A; 3dd7B; 3ddcB; 3ddhA; 3ddjA; 3ddtA; 3defA; 3de1B; 3deoA; 3dewA; 3df7A; 3df8A; 3dffA; 3dgpB; 3dgpA; 3dhaA; 3dhoA; 3dhuA; 3di4A; 3dj1A; 3dk9A; 3dkmA; 3d1cA; 3d1qI; 3d1qR; 3d1uA; 3dm8A; 3dmcA; 3dmeA; 3dmgA; 3dn7A; 3dnjA; 3dnpA; 3dnuA; 3dnxA; 3do6A; 3do8A; 3douA; 3dqgA; 3dqpA; 3dqyA; 3draB; 3drfA; 3drzA; 3ds4A; 3ds8A; 3dsbA; 3dskA; 3dsmA; 3dsoA; 3dssB; 3dssA; 3dttA; 3dtzA; 3dupA; 3dv9A; 3dwgC; 3dxeA; 3dxeB; 3dx1A; 3dxyA; 3dyjA; 3dz1A; 3dzaA; 3e03A; 3e05A; 3e0iA; 3e0xA; 3e0zA; 3e10A; 3e11A; 3e15A; 3e18A; 3e19A; 3e23A; 3e2dA; 3e2oA; 3e2qA; 3e2vA; 3e3uA; 3e46A; 3e48A; 3e4gA; 3e4vA; 3e57A; 3e58A; 3e5uA; 3e6qA; 3e7hA; 3e7rL; 3e8 mA; 3e8oA; 3e8tA; 3e96A; 3e99A; 3e9fA; 3e9kA; 3e9vA; 3ea6A; 3eafA; 3ebbA; 3ebtA; 3ebyA; 3ec4A; 3ec6A; 3ecfA; 3echA; 3echC; 3ed4A; 3edfA; 3edhA; 3edoA; 3edvA; 3ee4A; 3eeaA; 3eehA; 3eerA; 3eesA; 3eetA; 3ef8A; 3efgA; 3efyA; 3eg4A; 3egaA; 3eggC; 3egoA; 3egvB; 3ehgA; 3ehrA; 3eifA; 3einA; 3ej9A; 3ej9B; 3ejgA; 3ejkA; 3ejvA; 3ek3A; 3ekgA; 3e1bA; 3e1fA; 3e1kA; 3en0A; 3en8A; 3eo6A; 3eo7A; 3eofA; 3eoiA; 3eojA; 3ep6A; 3ep6B; 3eqxA; 3er6A; 3er7A; 3erbA; 3es1A; 3es4A; 3es1A; 3esmA; 3essA; 3etnA; 3etoA; 3etvA; 3etzA; 3euaA; 3euoA; 3eurA; 3evpA; 3evyA; 3ewnA; 3ewyA; 3exeA; 3exmA; 3exnA; 3exqA; 3ey8A; 3eyeA; 3eytA; 3ezuA; 3fDdA; 3f0hA; 3f0iA; 3f14A; 3f2eA; 3f2zA; 3f3kA; 3f40A; 3f42A; 3f43A; 3f44A; 3f47A; 3f4aA; 3f4 mA; 3f4sA; 3f52A; 3f59A; 3f5bA; 3f5hA; 3f5oA; 3f5rA; 3f62A; 3f6cA; 3f6oA; 3f6vA; 3f6wA; 3f6yA; 3f75P; 3f7cA; 3f7eA; 3f71A; 3f7qA; 3f7wA; 3f7xA; 3f81A; 3f8bA; 3f8kA; 3f8 mA; 3f8tA; 3f8xA; 3f95A; 3f9sA; 3f9xA; 3fajA; 3fanA; 3fb9A; 3fb1A; 3fbuA; 3fcnA; 3fd3A; 3fdbA; 3fdhA; 3fdjA; 3fdrA; 3fedA; 3fegA; 3fDA; 3ff1A; 3f2A; 3ffrA; 3fg1A; 3fg8A; 3fg9A; 3fgeA; 3fgrB; 3fgrA; 3fgvA; 3fgyA; 3fh1A; 3fhdA; 3fh1A; 3fiaA; 3fidA; 3fiA; 3fj1A; 3fj2A; 3fjsA; 3fjuB; 3fjvA; 3fkaA; 3f12A; 3f1aA; 3f1jA; 3fn0A; 3fm2A; 3fmcA; 3fmyA; 3fn2A; 3fn5A; 3fncA; 3fndA; 3fo3A; 3fo5A; 3fo8D; 3fojA; 3fotA; 3fp3A; 3fp5A; 3fp7J; 3fpcA; 3fpfA; 3fprA; 3fpwA; 3fpzA; 3fqgA; 3fqmA; 3fr7A; 3frhA; 3frqA; 3fryA; 3fsaA; 3fsdA; 3fseA; 3fsgA; 3fsoA; 3fssA; 3fstA; 3ft1A; 3ftdA; 3fuyA; 3fvbA; 3fwkA; 3fwuA; 3fwyA; 3fwzA; 3fx7A; 3fxaA; 3fxgA; 3fxhA; 3fxqA; 3fybA; 3fymA; 3fynA; 3fyqA; 3fzeA; 3fzgA; 3fzyA; 3g0kA; 3g0mA; 3g0tA; 3g14A; 3g16A; 3g1jA; 3g1pA; 3g21A; 3g23A; 3g2eA; 3g2 mA; 3g36A; 3g3sA; 3g3tA; 3g40A; 3g46A; 3g48A; 3g5bA; 3g5jA; 3g5oA; 3g5oB; 3g5sA; 3g5tA; 3g68A; 3g7 nA; 3g7 pA; 3g7qA; 3g7rA; 3g7uA; 3g85A; 3g89A; 3g8yA; 3g8zA; 3g98A; 3ga3A; 3ga4A; 3ga8A; 3gaeA; 3gagA; 3gb5A; 3gbwA; 3gd0A; 3gd6A; 3gdbA; 3gdcA; 3gdhA; 3gdwA; 3ge3A; 3ge3B; 3ge3C; 3ge3E; 3gewB; 3gf3A; 3gf6A; 3gfaA; 3gg7A; 3gg9A; 3ghdA; 3ghjA; 3gi7A; 3giuA; 3giwA; 3gj8B; 3gjyA; 3gk6A; 3gkeA; 3gkjA; 3gkrA; 3gmfA; 3gmgA; 3gmiA; 3gmsA; 3gmxA; 3gn6A; 3gneA; 3gn1A; 3gnzP; 3go2A; 3go5A; 3go9A; 3gocA; 3gohA; 3gonA; 3gp4A; 3gpiA; 3gpkA; 3gqhA; 3gqqA; 3gqvA; 3gr3A; 3grdA; 3grhA; 3gmA; 3gruA; 3grzA; 3gs9A; 3guzA; 3gv1A; 3gveA; 3gwbA; 3gwcA; 3gwiA; 3gwkC; 3gwqA; 3gwyA; 3gx8A; 3gxhA; 3gy9A; 3gybA; 3gycA; 3gydA; 3gykA; 3gzaA; 3gzbA; 3gzkA; 3gzrA; 3h01A; 3h05A; 3h09A; 3h0hA; 3h0nA; 3h0oA; 3h0uA; 3h1dA; 3h1nA; 3h20A; 3h2bA; 3h2gA; 3h2sA; 3h2yA; 3h31A; 3h36A; 3h3hA; 3h31A; 3h4lA; 3h4oA; 3h4tA; 3h4xA; 3h51A; 3h51A; 3h5 nA; 3h6jA; 3h6 nA; 3h6pC; 3h6 pA; 3h74A; 3h7aA; 3h7cX; 3h7iA; 3h87A; 3h87C; 3h8gA; 3h8tA; 3h8uA; 3h8zA; 3h95A; 3h9cA; 3h9 mA; 3ha2A; 3ha9A; 3hbnA; 3hc1A; 3hcgA; 3hcjA; 3hdjA; 3hdxA; 3he5B; 3he5A; 3hfoA; 3hftA; 3hfwA; 3hh1A; 3hhiA; 3hhyA; 3hi2B; 3hi7A; 3hj4A; 3hj9A; 3hjeA; 3hkwA; 3h11A; 3h18A; 3h1xA; 3h1zA; 3hm4A; 3hmzA; 3hn0A; 3hn3A; 3hn5A; 3hn7A; 3ho6A; 3hoiA; 3ho1A; 3hp7A; 3hpcX; 3hq1A; 3hqxA; 3hr0A; 3hr6A; 3hrgA; 3hr1A; 3hroA; 3hrpA; 3hs3A; 3hssA; 3hsyA; 3ht1A; 3htnA; 3htvA; 3htyA; 3hu5A; 3huhA; 3huuA; 3hvwA; 3hvyA; 3hwpA; 3hwuA; 3hx3A; 3hx8A; 3hx9A; 3hx1A; 3hxsA; 3hynA; 3hz6A; 3hzpA; 3i09A; 3i0yA; 3i0zA; 3i10A; 3i18A; 3i1aA; 3i2kA; 3i2 nA; 3i2vA; 3i36A; 3i3fA; 3i3gA; 3i45A; 3i4gA; 3i4oA; 3i4zA; 3i57A; 3i5cA; 3i7 mA; 3i83A; 3i84A; 3i8bA; 3i94A; 3iarA; 3ib5A; 3ib7A; 3ibmA; 3ibzA; 3ic3A; 3ic4A; 3icvA; 3idfA; 3iduA; 3idwA; 3ie4A; 3ieeA; 3ieiA; 3iezA; 3ifnP; 3ig9A; 3ighX; 3igrA; 3ihsA; 3ihtA; 3ihuA; 3ihvA; 3ii2A; 3ii7A; 3iibA; 3iiiA; 3iijA; 3iisM; 3ij3A; 3ij6A; 3ijdA; 3ijmA; 3ijwA; 3ikwA; 3i1wA; 3i1xA; 3im1A; 3im3A; 3im6A; 3imhA; 3imkA; 3imoA; 3iosA; 3ioxA; 3ip0A; 3ipcA; 3iq2A; 3iqtA; 3iquA; 3ir4A; 3irbA; 3irpX; 3irsA; 3is6A; 3isaA; 3isqA; 3isrA; 3isxA; 3it3A; 3it4B; 3it4A; 3iteA; 3itfA; 3itqA; 3iu0A; 3iu5A; 3iu6A; 3iufA; 3iugA; 3iukA; 3iuoA; 3iupA; 3iusA; 3iuwA; 3iuzA; 3iv0A; 3iveA; 3ivfA; 3ivvA; 3iwfA; 3iwtA; 3ix3A; 3ixcA; 3ix1A; 3ixsA; 3ixsB; 3jq0A; 3jq1A; 3jqwA; 3jqyA; 3jr7A; 3jrvA; 3jrvC; 3jsyA; 3jszA; 3jtmA; 3jtwA; 3jtxA; 3jtzA; 3ju3A; 3ju4A; 3judA; 3juiA; 3jumA; 3juuA; 3jx9A; 3jxoA; 3jygA; 3jyoA; 3jysA; 3jyzA; 3jz0A; 3jz9A; 3jzyA; 3k01A; 3k05A; 3k0bA; 3k0zA; 3k1 1A; 3k12A; 3k13A; 3k1hA; 3k1tA; 3k1zA; 3k29A; 3k2oA; 3k2vA; 3k2zA; 3k3cA; 3k4iA; 3k50A; 3k5jA; 3k67A; 3k69A; 3k6 mA; 3k6oA; 3k6qA; 3k6yA; 3k7cA; 3k7iB; 3k7 pA; 3k7xA; 3k8uA; 3k8wA; 3k9oA; 3ka5A; 3ka7A; 3kb9A; 3kbgA; 3kbqA; 3kbyA; 3kc2A; 3kciA; 3kcpA; 3kcwA; 3kd3A; 3kd4A; 3kdwA; 3ke7A; 3kebA; 3keoA; 3kepA; 3kevA; 3kewA; 3kffA; 3kfoA; 3kg0A; 3kg4A; 3kgkA; 3kgrA; 3kgwA; 3kgyA; 3kgzA; 3kh1A; 3kh7A; 3kh8A; 3khiA; 3kizA; 3kk4A; 3kkfA; 3kkgA; 3kkiA; 3kkwA; 3kkzA; 3k1kA; 3k1qA; 3km5A; 3kmaA; 3kmhA; 3kmuA; 3kmvA; 3knvA; 3kogA; 3ko1A; 3kopA; 3korA; 3kosA; 3kp8A; 3kpbA; 3kpeA; 3kpeB; 3kq0A; 3kq5A; 3kqiA; 3ks6A; 3ksmA; 3ksxA; 3kt7A; 3kt9A; 3ktaA; 3ktaB; 3ktcA; 3ktoA; 3ku3A; 3ku3B; 3kvhA; 3kvtA; 3kweA; 3kw1A; 3kwrA; 3kwsA; 3kwuA; 3kxwA; 3kyjA; 3kyzA; 3kzdA; 3kzpA; 3kzxA; 3l00A; 3l0qA; 3l15A; 3l1eA; 3l1 nA; 3l1wA; 3l2hA; 3l34A; 3l39A; 3l3fX; 3l3uA; 3l4lA; 3l46A; 3l4aA; 3l4eA; 3l4hA; 3l4 nA; 3l4rA; 3l51B; 3l51A; 3l5iA; 3l60A; 3l6aA; 3l6bA; 3l6gA; 3l7xA; 3l81A; 3l8dA; 3l8eA; 3l8hA; 3l8qA; 3l8uA; 3l9aX; 3l9fA; 3l9uA; 3l9wA; 3laaA; 3lagA; 3lasA; 3lbeA; 3lccA; 3lcrA; 3ld7A; 3ldcA; 3ldvA; 3le4A; 3ledA; 3leqA; 3letA; 3lewA; 3lf9A; 3lfjA; 3lfrA; 3ftA; 3lg3A; 3lgbA; 3lgdA; 3lgiA; 3lheA; 3lhiA; 3lhnA; 3lhoA; 3lhrA; 3lhxA; 3li9A; 3lidA; 3ljkA; 3ljuX; 3lk5A; 3lk7A; 3lkeA; 3lkkA; 3lkmA; 3l17A; 3l1bA; 3l1cA; 3l1oA; 3l1 pA; 3l1uA; 3l1vA; 3lm2A; 3lm3A; 3lm4A; 3lmaA; 3lmoA; 3lmzA; 3ln1A; 3lo8A; 3logA; 3lopA; 3louA; 3lpeB; 3lpeA; 3lpwA; 3lpzA; 3lq0A; 3lqbA; 3lqhA; 3lqyA; 3lr0A; 3lrkA; 3lrtA; 3ls9A; 3lsnA; 3lssA; 3ltiA; 3lufA; 3lu1A; 3lumA; 3lurA; 3luuA; 3lvuA; 3lwaA; 3lwxA; 3lx3A; 3lx4A; 3lxqA; 3lxrF; 3ly0A; 3ly1A; 3ly7A; 3lydA; 3lygA; 3lyhA; 3lyyA; 3lzaA; 3lz1A; 3m07A; 3m0mA; 3m0zA; 3m1dA; 3m1eA; 3m1tA; 3m1uA; 3m1xA; 3m3hA; 3m3 pA; 3m4iA; 3m4rA; 3m5rA; 3m66A; 3m6jA; 3m6wA; 3m6zA; 3m70A; 3m73A; 3m7aA; 3m7fB; 3m7oA; 3m86A; 3m8eA; 3m8jA; 3m97X; 3m91A; 3m9qA; 3mabA; 3naoA; 3mazA; 3mbkA; 3mc3A; 3mcfA; 3mcwA; 3mcxA; 3mczA; 3md7A; 3md9A; 3mdmA; 3mdpA; 3mdqA; 3mduA; 3me5A; 3me7A; 3meaA; 3mewA; 3mf7A; 3mgdA; 3mggA; 3mi1A; 3mjfA; 3mjoA; 3mk1A; 3m11A; 3m11B; 3mm5A; 3mm5B; 3mmgA; 3mmhA; 3mmyA; 3mmyB; 3mn7S; 3mozA; 3mp6A; 3mprA; 3mq0A; 3mq2A; 3mqdA; 3mqhA; 3mqqA; 3mqzA; 3mstA; 3mswA; 3msxB; 3mt0A; 3mtqA; 3mtrA; 3mtwA; 3mu7A; 3mtujA; 3mvcA; 3mvnA; 3mvpA; 3mvsA; 3mvuA; 3mw8A; 3mwbA; 3mwpA; 3mwqA; 3mwxA; 3mwzA; 3mx7A; 3mxnA; 3mxnB; 3mxoA; 3mxzA; 3myfA; 3myuA; 3myxA; 3mz2A; 3mzfA; 3mzoA; 3n01A; 3n08A; 3n0rA; 3n0uA; 3n10A; 3n1eA; 3n27A; 3n29A; 3n2wA; 3n3 mA; 3n4jA; 3n6tA; 3n6yA; 3n6zA; 3n72A; 3n75A; 3n77A; 3n79A; 3n8bA; 3n90A; 3n9bA; 3n9uC; 3na6A; 3nbcA; 3nbmA; 3nd1A; 3ndcA; 3ndqA; 3ne0A; 3ne8A; 3nehA; 3neuA; 3nf5A; 3nfiA; 3nfkA; 3nfqA; 3nftA; 3nfwA; 3ng2A; 3ng7X; 3nggA; 3nheA; 3ni0A; 3nirA; 3nj2A; 3njdA; 3njeA; 3njnA; 3nk4A; 3nk4C; 3nkdA; 3nkeA; 3nkgA; 3nk1A; 3nksA; 3n19A; 3nnbA; 3no0A; 3no2A; 3no3A; 3no4A; 3no6A; 3no7A; 3nohA; 3nojA; 3nokA; 3noqA; 3npdA; 3npfA; 3nqiA; 3ngnA; 3nr5A; 3nreA; 3nrfA; 3nrhA; 3nrrA; 3nrvA; 3nrwA; 3nrxA; 3ns6A; 3nswA; 3nsxA; 3nt1A; 3ntuA; 3ntvA; 3nuaA; 3nufA; 3nuqA; 3nvsA; 3nvtA; 3nvwC; 3nvwB; 3nvxA; 3nwcA; 3nwoA; 3ny7A; 3nycA; 3nymA; 3nytA; 3nyvA; 3nyyA; 3nzeA; 3nz1A; 3nzmA; 3nmA; 3nztA; 3nzzA; 3o0fA; 3o0qA; 3o0yA; 3o10A; 3o12A; 3o14A; 3o1cA; 3o22A; 3o2eA; 3o2hA; 3o2rA; 3o2tA; 3o2uA; 3o3 mA; 3o3mB; 3o3uN; 3o3xA; 3o48A; 3o53A; 3o5qA; 3o6cA; 3o6 nA; 3o6 pA; 3o70A; 3o7bA; 3o7iA; 3o8bA; 3o8 mA; 3o94A; 3oa5A; 3oa8A; 3oa8B; 3oajA; 3obeA; 3obqA; 3oc9A; 3ocjA; 3ocmA; 3ocrA; 3ocuA; 3od1A; 3odtA; 3oe3A; 3oepA; 3of4A; 3of5A; 3of7A; 3ofgA; 3og2A; 3og9A; 3ogaA; 3oghA; 3ognA; 3oheA; 3ohgA; 3ohrA; 3oi8A; 3oioA; 3oisA; 3oizA; 3oj0A; 3oj7A; 3okpA; 3okxA; 3o10A; 3o13A; 3om0A; 3omdA; 3omtA; 3omyA; 3on2A; 3on4A; 3on9A; 3ondA; 3onjA; 3oo8A; 3oopA; 3oosA; 3oouA; 3ooxA; 3op6A; 3op7A; 3op8A; 3op9A; 3oqiA; 3oqpA; 3oqvA; 3oruA; 3oseA; 3osqA; 3osrA; 3ostA; 3ot1A; 3ot2A; 3otiA; 3otmA; 3otnA; 3ou2A; 3ougA; 3ouiA; 3ouvA; 3ov8A; 3ov9A; 3ovkA; 3owaA; 3owcA; 3owrA; 3owtC; 3owvA; 3oxhA; 3oxpA; 3oyvA; 3oz2A; 3p02A; 3p0bA; 3p0fA; 3p0yA; 3p1vA; 3p1xA; 3p24A; 3p2hA; 3p2tA; 3p3cA; 3p3vA; 3p42A; 3p4gA; 3p4hA; 3p4lA; 3p5 pA; 3p6bA; 3p6iA; 3p61A; 3p8aA; 3p8kA; 3p9aA; 3p9 nA; 3p9vA; 3pa6A; 3pajA; 3pasA; 3pb6X; 3pc3A; 3pc6A; 3pc7A; 3pd7A; 3pddA; 3pdfA; 3pdtA; 3pe6A; 3pe7A; 3pe9A; 3pesA; 3pf2A; 3pf6A; 3pf7A; 3pf9A; 3pfeA; 3pfgA; 3pfoA; 3pfsA; 3pfyA; 3pg0A; 3pg6A; 3pguA; 3ph9A; 3phsA; 3pi7A; 3picA; 3pidA; 3pijA; 3pimA; 3piwA; 3pj0A; 3pj1A; 3pjpA; 3pjvD; 3pjyA; 3pkvA; 3pkzA; 3p10A; 3p18A; 3p1wA; 3pm2A; 3pmcA; 3pmmA; 3pmoA; 3pn3A; 3pnaA; 3pnnA; 3pnxA; 3pnzA; 3po8A; 3pohA; 3pojA; 3powA; 3pp2A; 3pp5A; 3pp9A; 3pp1A; 3ppmA; 3pqcA; 3pr6A; 3pr9A; 3proC; 3ps0A; 3pshA; 3psmA; 3pstA; 3pt1A; 3pt3A; 3pt5A; 3ptyA; 3pu9A; 3puaA; 3pveA; 3pwfA; 3pywA; 3pzfA; 3pzjA; 3q13A; 3q18A; 3q1cA; 3q1nA; 3q1xA; 3q27A; 3q2eA; 3q2uA; 3q3qA; 3q49B; 3q60A; 3q64A; 3q6bA; 3q6sA; 3q7cA; 3q7 mA; 3q7rA; 3q8gA; 3q98A; 3qaoA; 3qayA; 3qb8A; 3qbmA; 3qbtB; 3qbyA; 3qc0A; 3qc5X; 3qc7A; 3qdhA; 3qd1A; 3qe2A; 3qekA; 3qf2A; 3qf7A; 3qf7C; 3qf1A; 3qfmA; 3qftA; 3qguA; 3qh4A; 3qh6A; 3qhbA; 3qhoA; 3qhpA; 3qhqA; 3qi7A; 3qioA; 3qitA; 3qktA; 3q16A; 3q19A; 3q1eA; 3q1jA; 3qnmA; 3qnsA; 3qooA; 3qorA; 3qp3A; 3qp4A; 3qp6A; 3qpaA; 3qraA; 3qr1A; 3qs2A; 3qsgA; 3qsjA; 3qs1A; 3qtaA; 3qu3A; 3qu5A; 3qufA; 3qv1A; 3qvpA; 3qvsA; 3qw3A; 3qw9A; 3qwgA; 3qw1A; 3qxbA; 3qxhA; 3qy1A; 3qy3A; 3qy7A; 3qyfA; 3qzbA; 3r0nA; 3r0vA; 3r1pA; 3r2vA; 3r3qA; 3r3rA; 3r4zA; 3r5dA; 3r5tA; 3r5zA; 3r62A; 3r6aA; 3r6dA; 3r6fA; 3r72A; 3r7aA; 3r87A; 3r89A; 3r8eA; 3r8jA; 3r8yA; 3r90A; 3r9fA; 3r9 mA; 3ragA; 3rayA; 3razA; 3rbsA; 3rc1A; 3rcoA; 3rd5A; 3rd7A; 3rdeA; 3rdoA; 3rdvA; 3rdyA; 3regA; 3renA; 3retA; 3rfeA; 3rfiA; 3rftA; 3rg9A; 3rgaA; 3rh0A; 3rhbA; 3rhgA; 3rhtA; 3rioA; 3rjtA; 3rjuA; 3rjvA; 3rk6A; 3rkgA; 3rk1A; 3r15A; 3r1gA; 3r1kA; 3r1oA; 3r1sA; 3rm3A; 3rmhA; 3rmjA; 3rmqA; 3rmuA; 3m1A; 3mqA; 3mqB; 3mrA; 3robA; 3rofA; 3rotA; 3rp8A; 3rpcA; 3rpdA; 3rpfA; 3rpfC; 3rpjA; 3rppA; 3rpwA; 3rpzA; 3rq4A; 3rq9A; 3rqiA; 3rgtA; 3rqzA; 3rr6A; 3rriA; 3rrxA; 3rtaA; 3rt1A; 3ru6A; 3ruiA; 3rv1A; 3rvcA; 3rwaA; 3rwnA; 3rx9A; 3rxyA; 3ry4A; 3rykA; 3rznA; 3rzvA; 3s06A; 3sOaA; 3s1xA; 3s25A; 3s2jA; 3s2rA; 3s3tA; 3s3zA; 3s44A; 3s4eA; 3s4yA; 3s5bA; 3s5 mA; 3s5qA; 3s5wA; 3s6bA; 3s6eA; 3s83A; 3s8gA; 3s8gB; 3s8gC; 3s8iA; 3s8 mA; 3s8sA; 3s90C; 3s9fA; 3s9jA; 3s9xA; 3sb1A; 3sb4A; 3sbmA; 3sbtA; 3sbtB; 3sc7X; 3scyA; 3sd7A; 3sdbA; 3sdeA; 3se5A; 3seeA; 3sfvB; 3sfxB; 3sg0A; 3sggA; 3sh4A; 3shgB; 3shgA; 3shoA; 3shqA; 3sibA; 3si1A; 3sjhB; 3sj1D; 3sj1A; 3sjqC; 3sk2A; 3sk7A; 3sk9A; 3skxA; 3s11A; 3s12A; 3s1rA; 3s1zA; 3smaA; 3smdA; 3smvA; 3smzA; 3snoA; 3so5A; 3so6A; 3soeA; 3sojA; 3sonA; 3soyA; 3sp7A; 3sp8A; 3sq7A; 3sqfA; 3sqzA; 3sreA; 3sriA; 3sriB; 3ss7X; 3ssbC; 3ssbI; 3ssoA; 3su6A; 3sukA; 3sumA; 3suvA; 3sw0X; 3swoA; 3swyA; 3sxmA; 3sxuA; 3sxuB; 3sxyA; 3sy1A; 3sy6A; 3sz3A; 3sz7A; 3szaA; 3szvA; 3szyA; 3t0hA; 3t0A; 3t31A; 3t3wA; 3t4lA; 3t43A; 3t47A; 3t49A; 3t4lA; 3t64A; 3t6 pA; 3t6sA; 3t7dA; 3t7hA; 3t71A; 3t8kA; 3t8wA; 3t92A; 3t94A; 3t9yA; 3tbjA; 3tbmA; 3tbnA; 3tboA; 3tc3A; 3tc5A; 3tc8A; 3tcqA; 3tcvA; 3tdsA; 3te8A; 3teeA; 3tejA; 3tekA; 3tewA; 3tg0A; 3tg2A; 3tghA; 3thgA; 3thkA; 3ti2A; 3tiaA; 3tjeF; 3tjmA; 3tjyA; 3tk8A; 3tm4A; 3tm8A; 3tosA; 3towA; 3tp4A; 3tr9A; 3trbA; 3trdA; 3trgA; 3trqA; 3ts3A; 3ts9A; 3tt9A; 3ttcA; 3ttgA; 3tu3B; 3tu3A; 3tu8A; 3tuoA; 3tvjA; 3tvqA; 3txsA; 3ty1A; 3typA; 3tysA; 3u0bA; 3u1dA; 3u1jA; 3u11A; 3u1uA; 3u1wA; 3u2aA; 3u2sC; 3u2uA; 3u31C; 3u3zA; 3u4gA; 3u4vA; 3u5vA; 3u65A; 3u6gA; 3u7qA; 3u7qB; 3u7zA; 3u8vA; 3u97A; 3u99A; 3u9gA; 3u9rB; 3u9wA; 3uanA; 3ub1A; 3ub6A; 3uc9A; 3ucjA; 3ucpA; 3ucsC; 3ucsA; 3ud1A; 3udfA; 3ue2A; 3uejA; 3uekA; 3uenA; 3uf6A; 3uf8A; 3ufbA; 3ufeA; 3ugfA; 3uguA; 3uidA; 3ujcA; 3u1bA; 3u1jA; 3u1tA; 3umhA; 3umoA; 3unnA; 3uoaB; 3up3A; 3up1A; 3upsA; 3upvA; 3uq8A; 3ur8A; 3urgA; 3urrA; 3ushA; 3utkA; 3utmA; 3utmC; 3uueA; 3uu1A; 3uuwA; 3uv0A; 3uv4A; 3uv9A; 3ux2A; 3uxfA; 3uxjA; 3v0dA; 3v0rA; 3v1aA; 3v1vA; 3v34A; 3v31A; 3v46A; 3v4cA; 3v4gA; 3v4kA; 3v5cA; 3v68A; 3v6oA; 3v75A; 3v7bA; 3v7 nA; 3v7qA; 3v9oA; 3vaaA; 3vbcA; 3vc1A; 3vcaA; 3vcxA; 3vdjA; 3vejA; 3venA; 3vfzA; 3vgiA; 3vgpA; 3vgzA; 3vh8G; 3vhjA; 3vi6A; 3viiA; 3vj9A; 3vk0A; 3vk5A; 3vk6A; 3v1aA; 3vmnA; 3vmvA; 3vn0A; 3vn3A; 3vn5A; 3vocA; 3vorA; 3votA; 3vp5A; 3vpbA; 3vpbE; 3vpyA; 3vpzA; 3vgjA; 3vqtA; 3vrdB; 3vrdA; 3vsnA; 3vthA; 3vtoA; 3vtxA; 3vubA; 3vupA; 3vv1A; 3vvvA; 3vvyA; 3vwnX; 3vxcA; 3vypA; 3vz9B; 3vz9D; 3w06A; 3w08A; 3w0fA; 3w0kA; 3w0oA; 3w0tA; 3w15A; 3w15B; 3w1oA; 3w20A; 3w36A; 3w42A; 3w4rA; 3w4sA; 3w57A; 3w5fA; 3w5jA; 3w5 nA; 3w5sA; 3w5xA; 3w63A; 3w6bA; 3w6sA; 3w6wA; 3w7tA; 3w9kA; 3wa1A; 3wa2X; 3wa5B; 3waiA; 3warA; 3wasA; 3wdhA; 3wdqA; 3we5A; 3we9A; 3weoA; 3weuA; 3wfvA; 3wg3A; 3wgxA; 3wgxC; 3wh1A; 3wh2A; 3whjA; 3wiaA; 3wihA; 3wisA; 3witA; 3wiwA; 3wjpA; 3wkgA; 3wkyA; 3w1iA; 3wmiA; 3wmiB; 3wmqA; 3wmtA; 3wmvA; 3wmyA; 3wndA; 3wnoA; 3wnzA; 3wpaA; 3wppA; 3wpuA; 3wqbA; 3wqbB; 3wqcA; 3wsgA; 3wt0A; 3wu4A; 3wurA; 3wuzA; 3wv7A; 3wvaA; 3wvqA; 3wvtA; 3wvzA; 3ww8A; 3ww9A; 3wwaA; 3wwcA; 3wwhA; 3wwIA; 3wwnA; 3wwqC; 3wx4A; 3wx7A; 3wxyA; 3wydA; 3wz3A; 3wzsA; 3x0iA; 3x0tA; 3x0uA; 3x0vA; 3x2 mA; 3x30A; 3x34A; 3x38A; 3zbdA; 3zbqA; 3zdmA; 3zdsA; 3zeuA; 3zeuB; 3zg1A; 3zg9B; 3zg9A; 3zh0A; 3zhiA; 3zhnA; 3zi1A; 3zibA; 3zidA; 3zitA; 3zj0A; 3zjaA; 3zjbA; 3zl8A; 3zmdA; 3zmrA; 3zn4A; 3zn6A; 3znvA; 3zojA; 3zoqB; 3zpxA; 3zpyA; 3zqkA; 3zqoA; 3zrdA; 3zrgA; 3zriA; 3zrqA; 3zrxA; 3zscA; 3zsjA; 3zsuA; 3zt9A; 3ztvA; 3zucA; 3zuiA; 3zuzA; 3zv1A; 3zvqA; 3zvqB; 3zvsA; 3zw5A; 3zx1A; 3zx4A; 3zxcA; 3zxkA; 3zxnA; 3zxoA; 3zxyA; 3zy7A; 3zy1A; 3zypA; 3zyqA; 3zywA; 3zzoA; 3zzpA; 3zzsA; 4a02A; 4a0dA; 4a0pA; 4a0tA; 4a0zA; 4a1iA; 4a29A; 4a2vA; 4a35A; 4a37A; 4a3 pA; 4a3xA; 4a3zA; 4a42A; 4a4jA; 4a56A; 4a57A; 4a5 nA; 4a5sA; 4a5uB; 4a5xA; 4a6hA; 4a6qA; 4a7kA; 4a7wA; 4a8uA; 4a8xA; 4a8xB; 4a94C; 4aajA; 4aanA; 4ab1A; 4ac1X; 4ac7C; 4ac7A; 4ac7B; 4aciA; 4acjA; 4adiA; 4admA; 4adnA; 4adzA; 4ae2A; 4ae5A; 4ae7A; 4aeqA; 4afA 4affA; 4afkA; 4afmA; 4aghA; 4agkA; 4aivA; 4ajyC; 4ajyV; 4ajyB; 4ak1A; 4ak2A; 4ak5A; 4a10A; 4a1zA; 4ammA; 4ap9A; 4apoA; 4apxB; 4aq1A; 4aqoA; 4aqrD; 4ar9A; 4armA; 4ascA; 4asmB; 4at0A; 4at7B; 4at7A; 4ateA; 4atgA; 4athA; 4atmA; 4aumA; 4avaA; 4avrA; 4avsA; 4aw2A; 4aw7A; 4ax2A; 4axiA; 4ay0A; 4ay7A; 4aycA; 4ay1A; 4az6A; 4b0aA; 4b0mA; 4b1mA; 4b29A; 4b2fA; 4b2hA; 4b46A; 4b4cA; 4b4sA; 4b4uA; 4b60A; 4b62A; 4b6gA; 4b6iA; 4b6 mA; 4b89A; 4b8eA; 4b8vA; 4b93B; 4b93A; 4b9dA; 4b9gA; 4b9iA; 4b9kA; 4ba0A; 4bb9A; 4bc3A; 4bdxA; 4be3A; 4begA; 4beuA; 4bfaA; 4bfcA; 4bfoA; 4bg7A; 4bgbA; 4bgcA; 4bgoA; 4bgpA; 4bh5A; 4bhuA; 4bi3A; 4bi8A; 4bixA; 4bj0A; 4bjaA; 4bjiA; 4bjsA; 4bjtA; 4bjtD; 4bjzA; 4bk0A; 4bk8A; 4b10B; 4b1pA; 4bmhA; 4bnrI; 4bo1A; 4boqA; 4bouA; 4bpfA; 4bpsA; 4bpyA; 4bq2A; 4bqhA; 4bqnA; 4bt7A; 4bt9A; 4bu0A; 4bv1A; 4bvaA; 4bvwA; 4bvxB; 4bvxA; 4bwcA; 4bwrA; 4byzA; 4bz4A; 4bz7A; 4bzaA; 4bzpA; 4c08A; 4c0nA; 4c12A; 4c18A; 4c1oA; 4c1uA; 4c1wA; 4c24A; 4c2eA; 4c3sA; 4c4aA; 4c4pB; 4c5cA; 4c5eA; 4c5eE; 4c5wA; 4c6aA; 4c6eA; 4c6sA; 4c76A; 4c97A; 4c9bB; 4c9sA; 4cahB; 4cayA; 4cayB; 4cayC; 4cbeA; 4cbuA; 4cbuG; 4ccdA; 4ccsA; 4ccvA; 4ccwA; 4cd8A; 4cdjA; 4cdpA; 4ce7A; 4cfiA; 4cfqQ; 4cfsA; 4cgoA; 4cgqA; 4cgsA; 4ch7A; 4cheA; 4ci7A; 4ci9A; 4cicA; 4ci1A; 4citA; 4cj0B; 4cj0A; 4cj2A; 4ck4A; 4c11A; 4cmrA; 4cn0A; 4cn9A; 4cndA; 4cngA; 4cn1A; 4co8A; 4cogA; 4cosA; 4cpyA; 4cq4A; 4cqbA; 4cqiA; 4crhA; 4cruB; 4cruA; 4crwA; 4cs4A; 4csrA; 4csrB; 4ct3A; 4cu9A; 4cuaA; 4cv4A; 4cv7A; 4cvbA; 4cvdA; 4cvoA; 4cvrA; 4cvuA; 4cw4A; 4cxfA; 4cxfB; 4cybA; 4czgA; 4czuA; 4czxA; 4czxB; 4d02A; 4d05A; 4d0pA; 4d0qA; 4d11A; 4d3xA; 4d40A; 4d4zA; 4d53A; 4d5aA; 4d5bA; 4d5rA; 4d6gA; 4d73A; 4d77A; 4d7jA; 4d7 pA; 4d86A; 4d8bA; 4d9bA; 4d9iA; 4da2A; 4damA; 4db5A; 4dbbA; 4dd5A; 4ddpA; 4de9A; 4devA; 4deyB; 4df0A; 4dgfA; 4dh2B; 4dhiB; 4di9A; 4dipA; 4dixA; 4djaA; 4djgA; 4dk2A; 4dkaC; 4dkcA; 4d1hA; 4d1qA; 4dm1A; 4dm5A; 4dm7A; 4dmgA; 4dmiA; 4dmvA; 4dn7A; 4dnyA; 4do4A; 4do7A; 4dpbX; 4dpzX; 4dq6A; 4dq9A; 4dqaA; 4dqjA; 4driB; 4dt4A; 4dt5A; 4dthA; 4dv8A; 4dvcA; 4dwsA; 4dyqA; 4dz1A; 4dz4A; 4dziA; 4dzoA; 4dzzA; 4e01A; 4e0aA; 4e15A; 4e19A; 4e1bA; 4e1oA; 4e1pA; 4e29A; 4e2aA; 4e2gA; 4e2uA; 4e2zA; 4e3eA; 4e3yA; 4e40A; 4e45A; 4e45B; 4e45E; 4e4rA; 4e57A; 4e5rA; 4e5vA; 4e6fA; 4e6kG; 4e6uA; 4e74A; 4e8dA; 4e97A; 4e91A; 4e9sA; 4e9xA; 4ea9A; 4eadA; 4eaeA; 4ebbA; 4ebgA; 4ebjA; 4ebyA; 4ecfA; 4ed9A; 4edhA; 4edpA; 4edqA; 4ee6A; 4eetB; 4efoA; 4efpA; 4egcB; 4egcA; 4egdA; 4eguA; 4ehcA; 4ehsA; 4ehuA; 4ehxA; 4ei0A; 4eibA; 4eicA; 4eiuA; 4eivA; 4ekfA; 4ekxA; 4e16A; 4e11A; 4emdA; 4emtA; 4eo0A; 4eo1A; 4eo3A; 4ep4A; 4ep8B; 4epiA; 4epsA; 4epzA; 4eq8A; 4eqaC; 4eqaA; 4eqbA; 4eqpA; 4eqsA; 4ercA; 4errA; 4eryA; 4es1A; 4es8A; 4esmA; 4esqA; 4esrA; 4esuA; 4eswA; 4etyA; 4eu9A; 4eukA; 4eunA; 4euoA; 4euuA; 4ev8A; 4evfA; 4evmA; 4evqA; 4evuA; 4evwA; 4evxA; 4evyA; 4ew5A; 4ew7A; 4eweA; 4ewfA; 4ex6A; 4exkA; 4exoA; 4exrA; 4eyzA; 4ezgA; 4eziA; 4fD1A; 4fD3A; 4fD6A; 4fDjA; 4fDwA; 4fDzC; 4f1jA; 4f1vA; 4f27A; 4f2eA; 4f21A; 4f2 nA; 4f3 mA; 4f3 nA; 4f3vA; 4f4hA; 4f54A; 4f55A; 4f67A; 4f6 pA; 4f6tA; 4f7uB; 4f7uG; 4f7uA; 4f7uE; 4f7uF; 4f7uP; 4f80A; 4f87A; 4f8cA; 4f8kA; 4f98A; 4f9dA; 4faiA; 4fayA; 4fb2A; 4fb7A; 4fbcA; 4fbjA; 4fbrA; 4fbsA; 4fc9A; 4fcgA; 4fchA; 4fcjA; 4fd4A; 4fd7A; 4fdbA; 4fe3A; 4fekA; 4fetA; 4fevA; 4ff5A; 4ff1A; 4ffuA; 4fg1A; 4fgqA; 4fh0A; 4fhgA; 4fhrA; 4fhrB; 4fibA; 4fk9A; 4fkbA; 4fkeA; 4f13A; 4f1bA; 4fm1A; 4fmzA; 4fn7A; 4fnvA; 4fojA; 4fp5D; 4fqgA; 4fqnA; 4fr9A; 4fs7A; 4fs8A; 4ftdA; 4ftfA; 4fvdA; 4fvgA; 4fw1A; 4fx5A; 4fxiA; 4fxqA; 4fypA; 4fzoA; 4fzvA; 4fzvB; 4g0xA; 4g1iA; 4g1qA; 4g1qB; 4g22A; 4g26A; 4g29A; 4g2uA; 4g38A; 4g3aA; 4g3fA; 4g3 nA; 4g3oA; 4g3vA; 4g4gA; 4g4kA; 4g5aA; 4g68A; 4g6tA; 4g6tB; 4g6xA; 4g75A; 4g78A; 4g79A; 4g71A; 4g7xB; 4g7xA; 4g94B; 4g94A; 4g9eA; 4g9 mA; 4g9qA; 4g9sA; 4g9sB; 4ga2A; 4gakA; 4gb5A; 4gb7A; 4gbfA; 4gbmA; 4gc3A; 4gc5A; 4gcoA; 4gd5A; 4gdaA; 4gdoA; 4gdzA; 4gehA; 4gehB; 4gekA; 4ge1A; 4gf3A; 4gf3B; 4gftA; 4ggcA; 4ggfC; 4gggA; 4ggjA; 4gh9A; 4ghnA; 4gi3C; 4gi5A; 4gimA; 4gj4A; 4gjzA; 4gk6A; 4gkgA; 4g1qA; 4gm6A; 4gmqA; 4gmuA; 4gn0A; 4gn4B; 4gneA; 4gnrA; 4gofA; 4goqA; 4gosA; 4gpvA; 4gq4A; 4gqmA; 4gqnA; 4gqzA; 4gr2A; 4gr6A; 4grdA; 4gt6A; 4gt8A; 4gt9A; 4gucA; 4gudA; 4gvbB; 4gvfA; 4gvqA; 4gwbA; 4gwgA; 4gwmA; 4gx8A; 4gxbA; 4gxbB; 4gxtA; 4gxwA; 4gy7A; 4gymA; 4gywA; 4gzcA; 4gzkA; 4h08A; 4h0aA; 4h0cA; 4h0sA; 4h14A; 4h18A; 4h1xA; 4h27A; 4h2gA; 4h2wA; 4h3uA; 4h3vA; 4h3wA; 4h4lA; 4h4dA; 4h4 nA; 4h59A; 4h5bA; 4h5iA; 4h5sA; 4h6cA; 4h6qA; 4h6xA; 4h79A; 4h7uA; 4h7wA; 4h7yA; 4h87A; 4h89A; 4h8eA; 4h9nC; 4hadA; 4haeA; 4hamA; 4hatC; 4hatB; 4hb9A; 4hbqA; 4hbzA; 4hc5A; 4hchA; 4hcjA; 4hcsA; 4hczA; 4hddA; 4hdeA; 4hdoA; 4he6A; 4heiA; 4hemA; 4hesA; 4hf0A; 4hfqA; 4hfsA; 4hfvA; 4hg2A; 4hguA; 4hh3A; 4hh3C; 4hh5A; 4hhjA; 4hhrA; 4hhvA; 4hi8A; 4hi8B; 4hiaA; 4hjfA; 4hjgE; 4hjiA; 4hjzA; 4hkgA; 4hkhA; 4h12A; 4h1bA; 4hmsA; 4hn9A; 4hnoA; 4hpmA; 4hpmB; 4hq1A; 4hqzA; 4hroA; 4hs1A; 4hs2A; 4hsqA; 4hstB; 4hstA; 4htfA; 4ht1A; 4htrA; 4hu2A; 4hu8A; 4hujA; 4hvkA; 4hvtA; 4hvyA; 4hw0A; 4hw6A; 4hwhA; 4hwmA; 4hwvA; 4hxfB; 4hxyA; 4hy4A; 4hyeA; 4hziA; 4hzoA; 4i05A; 4i0oA; 4i0wB; 4i0wA; 4i0xB; 4i0xA; 4i17A; 4i1fA; 4i3gA; 4i4cA; 4i4kA; 4i4oA; 4i66A; 4i68A; 4i6 nA; 4i6rA; 4i6xA; 4i6yA; 4i71A; 4i79A; 4i84A; 4i8iA; 4i90A; 4i93A; 4i9oA; 4ia6A; 4iabA; 4iajA; 4iauA; 4ibgA; 4ibnA; 4ic3A; 4ic9A; 4ichA; 4iciA; 4icvA; 4id9A; 4idhA; 4idiA; 4iejA; 4ienA; 4ifaA; 4igiA; 4igkA; 4igvA; 4iibA; 4iikA; 4ii1A; 4iiyA; 4ij5A; 4ijnA; 4ijrA; 4ik8A; 4ikbA; 4ikdA; 4iknA; 4ikvA; 4i17A; 4i1fA; 4indA; 4ineA; 4inkA; 4inwA; 4inzA; 4ipcA; 4ipiA; 4ipuA; 4iq0A; 4ignA; 4iqyA; 4irgA; 4it6A; 4iu2A; 4iu6A; 4iujA; 4iumA; 4iupA; 4iusA; 4iuwA; 4ivgA; 4ivkA; 4ivnA; 4iwbA; 4ix3A; 4ixjA; 4iyaA; 4iyjA; 4iykA; 4iysA; 4iz7B; 4izxA; 4j0dA; 4j0wA; 4j25A; 4j27A; 4j32A; 4j32B; 4j33A; 4j37A; 4j3hA; 4j3vA; 4j42A; 4j44A; 4j4hA; 4j4zA; 4j6oA; 4j78A; 4j7hA; 4j7 nA; 4j7qA; 4j7zA; 4j87A; 4j8cA; 4j81A; 4j8sA; 4j9tA; 4j9yB; 4jaqA; 4jb3A; 4jb7A; 4jb8A; 4jbbA; 4jbdA; 4jbeA; 4jbuA; 4jccA; 4jdeA; 4jdnA; 4jduA; 4je5A; 4jeaA; 4jejA; 4jemA; 4jf3A; 4jf8A; 4jg2A; 4jgiA; 4jg1A; 4jhnA; 4jhyA; 4jifA; 4jifB; 4jiuA; 4jivD; 4jj7A; 4jjaA; 4jjoA; 4jk8A; 4jm1A; 4jmdA; 4jmgA; 4jmpA; 4jmqA; 4jn7A; 4jndA; 4jnuA; 4jo5A; 4jo7A; 4joqA; 4jp0A; 4jp6A; 4jqfA; 4jqpA; 4jquB; 4jr6A; 4jrfA; 4jtmA; 4jvoA; 4jvuA; 4jwjA; 4jwoA; 4jxeA; 4jxhA; 4jxrA; 4jykA; 4jysA; 4jz5A; 4jzzA; 4jzzR; 4k00A; 4k02A; 4k05A; 4k0nA; 4k12B; 4k12A; 4k22A; 4k2 mA; 4k2 pA; 4k35A; 4k3zA; 4k4kA; 4k6 nA; 4k70A; 4k73A; 4k7bA; 4k7cA; 4k7jA; 4k82A; 4k84A; 4k8wA; 4k90A; 4k90B; 4k9zA; 4ka1A; 4kcaA; 4kdrA; 4kdwA; 4ke2A; 4kefA; 4kemA; 4kfuA; 4kg3A; 4kg7A; 4kgdA; 4kh8A; 4kh9A; 4kiaA; 4kjmA; 4kk7A; 4k10A; 4k1kA; 4k1xA; 4km6A; 4kmdA; 4km1A; 4kmnA; 4kmrA; 4kn8A; 4knkA; 4ko8A; 4kopA; 4kq7A; 4kq9A; 4kqdA; 4kqiA; 4kqpA; 4krdB; 4krgA; 4kruA; 4ksnA; 4ksyA; 4kt3A; 4kt3B; 4kt6A; 4kt6B; 4ktwA; 4ku0A; 4ku0D; 4kuiA; 4kukA; 4kunA; 4kv2A; 4kv7A; 4kv9A; 4kvgB; 4kwaA; 4kxvA; 4kyqA; 4kyxA; 4kzkA; 4kzpA; 4l0cA; 4l0jA; 4l0 nA; 4l1jA; 4l2hA; 4l2iB; 4l2iA; 4l3uA; 4l51A; 4l57A; 4l58A; 4l5eA; 4l63A; 4l68A; 4l6hA; 4l7gA; 4l7 mA; 4l7 nA; 4l8aA; 4l8 pA; 4l9aA; 4l9bA; 4l9eA; 4l9hA; 4l9 nA; 4l9oA; 4l9 pA; 4l9pB; 4l9uA; 4la2A; 4lanA; 4layA; 4lbaA; 4lbhA; 4lc1A; 4ld1A; 4ld6A; 4ld8A; 4ldfA; 4ldmA; 41dvA; 4e1A; 4lebA; 4lerA; 4lDA; 4lftA; 4lg1A; 4lg8A; 4lgjA; 4lgoA; 4lgxA; 4lgyA; 4lhdA; 4lhsA; 4lijA; 4limA; 4lixA; 4lizA; 4lj1A; 4lj9A; 4ljiA; 4ljoA; 41ldA; 41ldB; 41loB; 41loA; 41lqB; 41lyA; 4lm6A; 4lm8A; 4lmiA; 4lmsA; 4lmyA; 4ln7A; 4losA; 4lowA; 4lpqA; 4lpsA; 4lq6A; 4lqbA; 4lqkA; 4lqzA; 4lrdA; 4lrjA; 4lrtA; 4lrtB; 4ls3A; 4lsbA; 4ltnA; 4lttA; 4ltyA; 4luaA; 4lukA; 4lunU; 4lv5B; 4lvfA; 4lw1A; 4lx2A; 4lx2B; 4lx3A; 4lx3B; 4lxoA; 4lxqA; 4ly7A; 4lypA; 4lzhA; 4lzkA; 4lz1A; 4lzxB; 4m02A; 4m0nA; 4m0wA; 4m1bA; 4m1gA; 4m1uA; 4m1xA; 4m20A; 4m2 mA; 4m37A; 4m3 pA; 4m51A; 4m5bA; 4m5dA; 4m5 dB; 4m5eA; 4m5rA; 4m6bA; 4m6bC; 4m7tA; 4m82A; 4m85A; 4m88A; 4m8aA; 4m8dA; 4m8iA; 4m8kA; 4m91A; 4m9 pA; 4maaA; 4maiA; 4makA; 4mamA; 4maqA; 4maxA; 4mb4A; 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4nkpA; 4n19A; 4n19C; 4n1mA; 4nm9A; 4nmuA; 4nmwA; 4nmyA; 4nn2A; 4nn5B; 4nn5C; 4nn5A; 4nnbA; 4nnoA; 4nnrA; 4noaA; 4nobA; 4nohA; 4npdA; 4npfX; 4np1A; 4npnA; 4npsA; 4npxA; 4nrpA; 4ns5A; 4nsvA; 4nt1A; 4ntcA; 4ntdA; 4ntkA; 4nu3A; 4nuaA; 4nurA; 4nutB; 4nuuA; 4nuuC; 4nuzA; 4nv0A; 4nv4A; 4nwbA; 4nwkA; 4nwyA; 4nx8A; 4nxyA; 4nyhA; 4nyqA; 4nzjA; 4nzkA; 4nz1B; 4nznA; 4nzrM; 4nzvA; 4o06A; 4o0cA; 4o1eA; 4o1rA; 4o1wA; 4o36B; 4o3vA; 4o4fA; 4o4oA; 4o590; 4o5fA; 4o5qA; 4o65A; 4o66A; 4o6 mA; 4o6uA; 4o6yA; 4o7kA; 4o7qA; 4o87A; 4o8aA; 4o8oA; 4o8vA; 4o8yA; 4o9dA; 4o91A; 4oa3A; 4oahA; 4ob0A; 4ob1A; 4ocvA; 4od6A; 4od8A; 4od8C; 4od9B; 4od9A; 4odkA; 4odpA; 4oe9A; 4oebA; 4oe1B; 4oe1A; 4oevA; 4of8A; 4offA; 4ofkA; 4oh7A; 4ohjA; 4ohxA; 4oi3A; 4oieA; 4ojuA; 4ojxA; 4ok4A; 4okeA; 4okiA; 4okvE; 4okzA; 4o19A; 4o1tA; 4om8A; 4ombA; 4omfA; 4omfB; 4omfG; 4omgA; 4omjA; 4oncA; 4onmA; 4onrA; 4onwA; 4ooaA; 4oopA; 4ooxA; 4opcA; 4opmA; 4opwA; 4oq1A; 4oqjA; 4oqpA; 4oqvA; 4ou0A; 4ou9A; 4ouhA; 4oujA; 4ounA; 4ousA; 4ov4A; 4ovjA; 4ovkA; 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4q5eA; 4q5wA; 4q62A; 4q63A; 4q68A; 4q6kA; 4q6uA; 4q6vA; 4q7fA; 4q7oA; 4q7qA; 4q82A; 4q8kA; 4q8rA; 4q8wA; 4q98A; 4q9bA; 4qa8A; 4qasA; 4qb0A; 4qbbA; 4qb1A; 4qbnA; 4qboA; 4qbsA; 4qbuA; 4qc6A; 4qcjA; 4qdcA; 4qdjA; 4qdnA; 4qe0A; 4qekA; 4qfuA; 4qgoA; 4qgpA; 4qgsA; 4qhjA; 4qhqA; 4qhwA; 4qi3A; 4qiuA; 4qjkA; 4qjvA; 4qjvB; 4qkdA; 4qkwA; 4q1pA; 4q1pB; 4qmaA; 4qmdA; 4qmhA; 4qn8A; 4qndA; 4qo5A; 4qp5A; 4qpkA; 4qp1A; 4qpnA; 4qptA; 4qpvA; 4qpwA; 4qq0A; 4qq4A; 4qrhA; 4qrkA; 4qr1A; 4qmA; 4qrvA; 4qt6A; 4qtcA; 4qtpA; 4qtqA; 4qttB; 4qucA; 4qusA; 4qvhA; 4qwoA; 4qx5A; 4qxbA; 4qxbB; 4qx1A; 4qy7A; 4r03A; 4r0jA; 4r12A; 4r1dB; 4r1dA; 4r1hA; 4r1jA; 4r2fA; 4r2xA; 4r33A; 4r38A; 4r31A; 4r3 nA; 4r3qA; 4r42A; 4r4kA; 4r4 mA; 4r4xA; 4r52A; 4r5rA; 4r6fA; 4r6hA; 4r6kA; 4r6yA; 4r75A; 4r78A; 4r7kA; 4r7qA; 4r81A; 4r82A; 4r84A; 4r8xA; 4r9fA; 4r9iA; 4r9kA; 4r9 pA; 4rajA; 4raxA; 4rayA; 4rbrA; 4rcoA; 4rd4A; 4rd8A; 4rdbA; 4re5A; 4rekA; 4re1A; 4reoA; 4repA; 4rexA; 4rf6A; 4rfuA; 4rg1A; 4rgdA; 4rgiA; 4rguA; 4rgyA; 4rhaA; 4rhsA; 4ri5A; 4jwA; 4rjzA; 4rk2A; 4rk4A; 4rk6A; 4rkqA; 4rksA; 4r11A; 4r13A; 4r1cA; 4r1jB; 4r1jA; 4rm6A; 4rm8A; 4rmkA; 4rmxA; 4mcA; 4mwA; 4rnxA; 4rnzA; 4ro3A; 4rojA; 4rp3A; 4rp9A; 4rpmA; 4rptA; 4rriA; 4rs7A; 4rscA; 4rswA; 4rt1A; 4rthA; 4ru1A; 4ru3A; 4ru5A; 4rugA; 4ruqA; 4ruwA; 4rv0B; 4rv5A; 4rvcA; 4rvqA; 4rw0A; 4rwfA; 4rwfB; 4rwhA; 4rwuA; 4rxiA; 4rxvA; 4ry1A; 4ry8A; 4ryaA; 4ryeA; 4ryoA; 4rz3A; 4rz9A; 4rzaA; 4rzyA; 4s12A; 4s1aA; 4s1hA; 4s1pA; 4s28A; 4s36A; 4s39A; 4s3iA; 4s3jA; 4s3oB; 4tjvA; 4tkbA; 4tkcA; 4tkxL; 4t16A; 4t1vA; 4tmdA; 4tmeA; 4tmxA; 4tndA; 4tnsA; 4tpsA; 4tpsB; 4tpvA; 4tq1A; 4tq1B; 4tqxA; 4tr3A; 4tr6A; 4trkA; 4trtA; 4tsdA; 4tshB; 4tshA; 4ttwA; 4tv5A; 4tvcA; 4tveA; 4tvvA; 4tx5A; 4txdA; 4txgA; 4txrA; 4txrB; 4txrC; 4txvB; 4txwA; 4tyzA; 4tzhA; 4u09A; 4u0pB; 4u12A; 4u1eI; 4u1eB; 4u1eG; 4u31A; 4u3eA; 4u3sB; 4u3vA; 4u4pB; 4u4 pA; 4u5hA; 4u5qA; 4u5rA; 4u5wA; 4u5wB; 4u5yD; 4u63A; 4u68A; 4u72A; 4u7aA; 4u7iA; 4u89A; 4u8fA; 4u98A; 4u9cA; 4u9hL; 4u9hS; 4u9oA; 4u9 pA; 4u9uA; 4u9vB; 4ua3A; 4uabA; 4uafB; 4uafE; 4uapA; 4uasA; 4uavA; 4uc1A; 4uc8A; 4ud4A; 4udgA; 4udqA; 4udsA; 4udxX; 4ue0A; 4ue8A; 4ue8B; 4uf0A; 4uf7C; 4uf7A; 4ufqA; 4ug1A; 4uhcA; 4uhoA; 4uhqA; 4uhtA; 4uiqA; 4uj7A; 4uj8A; 4um7A; 4umgA; 4umiA; 4um1A; 4un2B; 4unuA; 4uobA; 4up0A; 4up3A; 4upiA; 4uqxA; 4uqzB; 4usaA; 4usiA; 4uskA; 4usoA; 4ut1A; 4utuA; 4uu3A; 4uuuA; 4uuxA; 4uvqA; 4uwxA; 4uwxC; 4uxeA; 4uybA; 4uydA; 4uyiA; 4uyrA; 4uytA; 4uz1A; 4uz3A; 4v00A; 4v0hA; 4v0wB; 4v0xB; 4v12A; 4v17A; 4v1gA; 4v1kA; 4v1sA; 4v24A; 4v2xA; 4v33A; 4v3iA; 4v3 1C; 4w1tA; 4w4 kB; 4w4kA; 4w4oC; 4w4tA; 4w5xA; 4w5zA; 4w64A; 4w6yA; 4w78A; 4w78B; 4w79A; 4w71A; 4w7wA; 4w82A; 4w8bA; 4w8hA; 4w8 pA; 4w8pB; 4w8qA; 4w9wA; 4wa0A; 4wb7A; 4wbdA; 4wbjA; 4wbsA; 4wbtA; 4wbyA; 4wcjA; 4wckA; 4wctA; 4wcxA; 4wd1A; 4wdcA; 4we2A; 4weeA; 4wesB; 4wesA; 4wfoA; 4wftA; 4wh5A; 4wh9A; 4whiA; 4whsB; 4whsA; 4wi1A; 4wi1A; 4wiqA; 4wjiA; 4wjsA; 4wjtA; 4wk0B; 4wk0A; 4wkaA; 4wksC; 4wksA; 4wkyA; 4wkzB; 4wkzA; 4w1hA; 4w1iA; 4w1rA; 4wIrB; 4wmaA; 4wmaD; 4wmuA; 4wmyA; 4wn5A; 4wndA; 4wndB; 4wp2A; 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5do6A; 5docA; 5dofA; 5domA; 5dp2A; 5dpoA; 5dqvA; 5dtcA; 5dthA; 5du9A; 5dutA; 5dv4A; 5dviA; 5dvwA; 5dwdA; 5dx6A; 5dx1A; 5dymA; 5dyqA; 5dzeA; 5dzoA; 5e0uD; 5e0yA; 5e0zA; 5e10A; 5e13A; 5e16A; 5e1qA; 5e1wA; 5e2cA; 5e37A; 5e3bA; 5e3eB; 5e3eA; 5e3qA; 5e4bA; 5e4gA; 5e50A; 5e56A; 5e57A; 5e5uB; 5e5yA; 5e68A; 5e6vA; 5e6xA; 5e6zA; 5e72A; 5e75A; 5e7hA; 5e71A; 5e8sA; 5e9 nA; 5e9 pA; 5ec6A; 5ecuA; 5edfA; 5ed1A; 5ee2A; 5eehA; 5efrA; 5efzA; 5eh1A; 5ehaA; 5ehrA; 5eipA; 5eiuA; 5ej3A; 5ej8A; 5ejrA; 5ejyA; 5ekiA; 5ekzA; 5e13A; 5e19A; 5e1bA; 5e1nA; 5embA; 5emiA; 5emxA; 5enfA; 5enqA; 5enuA; 5eovA; 5ep0A; 5ep2A; 5epeA; 5epwA; 5eq0A; 5eq7A; 5eqzA; 5er9A; 5ereA; 5erqA; 5erxA; 5escA; 5eu0A; 5eu0B; 5eurA; 5evcA; 5evfA; 5evhA; 5ewoA; 5ewpA; 5ewuA; 5ewyA; 5ex2A; 5exeA; 5exeB; 5exeC; 5exjA; 5expA; 5ey0A; 5eynA; 5ezqA; 5ezuA; 5fUeB; 5f18A; 5f1sA; 5f2kA; 5f30A; 5f3kA; 5f3 pA; 5f47A; 5f4cA; 5f4wA; 5f5 nA; 5f67A; 5f61A; 5f61B; 5f61J; 5f6rA; 5f7rA; 5f7uA; 5f7vA; 5f86A; 5f8cA; 5fa8A; 5faaA; 5faiA; 5favA; 5fbfA; 5fc2B; 5fc9A; 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6b9xC; 6b9xD; 6b9xE; 6b9xA; 6bcbA; 6bevA; 6bgdA; 6bhdA; 6bk0A; 6b1kA; 6b1mA; 6bmeA; 6bo0A; 6bphA; 6bu6A; 6bus1; 6bvcA; 6bweA; 6bxgA; 6c0cA; 6c4qA; 6c4vA; 6ehiA; 6ekbA; 6ektA; 6e1mA; 6ensA; 6eofA; 6eonA; 6eroA; 6es9A; 6euwA; 6fDpA; 6f72A; 6f8 pA; 6ff1A; 6fg8A; 7fd1A;
This application claims priority to U.S. Provisional Application No. 63/054,585, entitled “DESIGNED PROTEINS FOR LIGAND BINDING” and filed on Jul. 21, 2020, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with Government support under grant numbers R35 GM122603, awarded by The National Institutes of Health, and 1709506, awarded by the National Science Foundation, and FA9550-19-1-0331, awarded by the Air Force Office of Scientific Research. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/042647 | 7/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63054585 | Jul 2020 | US |
