Patent Application
20140154180
The instant application contains a Sequence Listing which has been submitted via paper and CD-R format and is hereby incorporated by reference in its entirety. Hexamer fiber-forming segments of Aβ and tau having the sequences KLVFFA (SEQ ID NO:1) and VQIVYK (SEQ ID NO:2), respectively, are referred to throughout this application. The sequence of the Aβ used in the present experiments is represented by SEQ ID NO:21. The sequence of tau is represented by SEQ ID NO:22.
The devastating and incurable dementia known as Alzheimer's disease affects the thinking, memory, and behavior of dozens of millions of people worldwide. The challenge of developing chemical interventions for Alzheimer's disease has proceeded in a virtual vacuum of information about the three-dimensional structures of the two proteins most widely accepted as being involved in the etiology. These are amyloid-beta (Aβ, sometimes referred to herein as Abeta) and tau [1,2]. Both convert from largely natively disordered, soluble forms to toxic oligomers and fibers [2,3] that may be related in structure [4]. Indeed, analogs of the well-established ligands to amyloid fibers, congo-red and thioflavin T, also bind Aβ oligomers labeling them in vitro and in vivo [5]. Screens of chemical libraries have uncovered dozens of small molecules that interact with amyloid [6-8]. Curcumin and various antibiotics are a few of many fiber inhibitors that also inhibit oligomer formation [7,9,10], supporting a common underlying structure in fibers and oligomers. Despite this progress, until now there have been no atomic-level structures showing how small molecules bind to amyloid and, consequently, no means for structure-based design of specific binders.
More is known about the molecular structure of amyloid fibers, both those associated with Alzheimer's disease and with the numerous other amyloid conditions [11-15]. Common to all amyloid fibers is their X-ray fiber-diffraction pattern, with two orthogonal reflections at about 4.8 Å and 10 Å spacing suggesting a “cross-β structure” [16,17]. The determination of the first amyloid-like atomic structures revealed a motif consisting of a pair of tightly mated β-sheets, called a “steric zipper,” which is formed from a short self-complementary segment of the amyloid-forming protein [12,18,19]. The steric zipper structures elucidate the atomic features that give rise to the common cross-β diffraction pattern, corresponding to the 4.8 Å spacing between strands forming β-sheets and the ˜10 Å spacing between two mating β-sheets. The structures imply that stacks of identical short segments form the “cross-β spine” of the protofilament, the basic unit of the mature fiber, while the rest of the protein adopts either native-like or unfolded conformations peripheral to the spine [12,20].
The short segments forming steric zippers, when isolated from the rest of the protein, form well-ordered fibers on their own, with essentially all properties of the fibers of their full-length parent proteins [21,22]. These properties include similar fiber diameters and helical pitch, similar cross-β diffraction patterns, similar fiber-seeding capacities, similar stability, and similar dye binding. That stacked short amyloidogenic segments can constitute the entire spine of an amyloid-like fiber has been demonstrated with the enzyme RNase A, containing an insert of a short amyloidogenic segment [20,23]. These RNase A fibers retain enzymatic activity, showing that native-like structure remains intact with only the stacked segments forming the spine. Thus while short amyloidogenic segments cannot recapitulate the entire complexity of their parent proteins, they nonetheless serve as good models for full amyloid fibers [24] and offer the informational advantage that they often grow into microcrystals whose atomic structures can be determined [12]. To date, structures for over 90 such steric zippers have been determined from a variety of disease-associated proteins ([18,19,25-27] and Colletier et al. (2011) Molecular basis for amyloid-beta polymorphism, Proc. Natl. Acad. Sci. USA 108, 16938-43).
The patent or application file contains at least one drawing executed in color. It is noted that many of these color drawings are present in the publication, Landau et al. (2011), Towards a Pharmacophore for Amyloid, PLoS Biol 9(6): e1001080. doi:10.1371.
This application relates, e.g., to computer-based methods for designing and/or selecting (screening for) small molecule compounds which bind to amyloid fibers and/or which inhibit a biological function of the amyloid (e.g, inhibit amyloid-mediated cellular toxicity). The present inventors discovered that by co-crystallizing fiber-forming segments of amyloid proteins (e.g. Aβ and tau) with small molecule binders and by determining the structures of the resulting microcrystals by X-ray microcrystallography, they were able to characterize features of the drug binding environment (e.g. binding surfaces and/or binding pockets) of the amyloid binders, which allow for computer-based identification of additional small molecules that exhibit improved binding and/or inhibitory properties compared to the small molecules used to generate the co-crystals.
This represents the first time that, by using the adhesive segments of amyloid-forming proteins (such as Aβ), which on their own, isolated from the rest of the protein, form amyloid-like fibers, and growing co-crystals of such segments complexed with amyloid-binding ligands (to form microcrystals of about 1 micrometer in cross section), recording useful diffraction data from them, and determining the structures, it was possible to perform structure-based computational design of improved small molecule diagnostic and therapeutic agents. An improved docking program is also disclosed, which allows one to apply a docking program to identify compounds by targeting the amyloid fibril structure, and to successfully identify active compounds by docking a large compound database. (about 18,000 compounds) to amyloid fibril structures.
Compounds identified by methods of the invention, pharmaceutical compositions comprising the compounds, methods of using the compounds for diagnosis and/or treatment of amyloid-mediated diseases or conditions, and computer-related embodiments, such as a computer-readable medium providing the structural representation of a co-crystal of a protofilament of an amyloid protein with a small molecule that is known to bind to the amyloid protein, are also described.
An advantage of the small molecule compounds of the invention is that they are expected to readily cross the blood brain barrier. This property enhances their ability to visualize, for example, amyloid plaques and/or tau tangles in the brains of subjects having amyloid-mediated diseases, and to be effective as therapeutic agents for the treatment of such subjects.
One aspect of the invention is a method for determining on a computer the relevant criteria for designing or selecting (screening for) on a computer a small molecule amyloid binder or inhibitor (e.g., for creating a computer-based replica or a pharmacophore representing the criteria), comprising
In one embodiment of the invention, test compounds are selected which exhibit an energy below an empirically determined threshold value based on the comparative values of energy found for co-crystals made with many candidate amyloid binders. The threshold values will differ depending on which co-crystal is being analyzed and which docking program used in the analysis. For example, when using the energy score obtained with the ROSETTA program, the calculated binding energy of Orange-G/Aβ co-crystals is about −8 kcal/mol, so compounds with an energy of below −8 kcal/mol are selected. For other co-crystals, using the ROSETTA energy score, the energy values can range from about −5 kcal/mol to about −15 kcal/mol. When using other programs, such as AutoDock or DOCK, the energy values may be considerably different. In embodiments of the invention, a structural representation of the co-crystal is provided in a storage medium on a computer; and a computer is used to apply structure-based drug design techniques to the structural representation.
In one embodiment of this method, the amyloid protein is Aβ, and the small molecule is a polar (e.g., charged) molecule comprising one or more flat aromatic rings (e.g., a polar molecule), such as Orange-G. The atomic coordinates of the three-dimensional structure are shown in Table 3, and the amino acid residues of the amyloid molecule which contact the amyloid binder are selected from one or more of Lys16, Leu17, Val18, Phe19, or Phe20, or combinations thereof (e.g., the binding site or pocket comprises one or more of these amino acid residues).
In another embodiment, the amyloid protein is tau, and the small molecule is a polar (e.g. charged) molecule comprising one or more flat aromatic rings (e.g., a polar molecule), such as Orange-G. The atomic coordinates of the three-dimensional structure are shown in Table 4, and the amino acid residues of the amyloid molecule which contact the amyloid binder are selected from one or more of Gln2, Val4, or Lys6, or combinations thereof.
In another embodiment, the amyloid protein is tau, and the small molecule is an elongated (having a ratio of length to width of greater than 2:1) apolar molecule, such as curcumin or DDNP. The atomic coordinates of the three-dimensional structure are shown in Table 5 or 6, respectively, and the amino acid residues of the amyloid molecule which contact the amyloid binder are selected from one or more of Val1, Gln2, Ile3, Val4, Tyr5 or Lys6, or combinations thereof.
Another aspect of the invention is a method for designing or selecting (screening for) on a computer a candidate small molecule amyloid binder or inhibitor, comprising
In one embodiment of the invention, the docking in a method as above is accomplished by a docking program in which the test molecule and protein side chain tortion angles and small molecule rotamers are sampled in a near native perturbation fashion. Many of the currently available docking programs are high resolution and are designed to fit test molecules into deep binding pockets of whole proteins. For the 3-D structures of the present invention, in which the binding surfaces or binding pockets are much shallower, it is desirable to use a docking program at lower resolution, allowing for more rapid screening. In many currently available docking programs, all possible side chain angles and revolutions are tested. For docking test molecules to the present 3-D structures, it is desirable to sample ligand and protein side-chain torsion angles and ligand rotamers in a near “native” perturbation fashion. By near “native” is meant limiting the possible side chain torsion angles to deviations (+/−0.33, 0.67, 1 sd) around each input torsion, based on the standard deviation value of the same torsion bin from the backbone-dependent Dunbrack rotamer library. See Example III for details.
Any of the preceding methods can further comprise (a) testing the candidate amyloid binders for their ability to inhibit amyloid-mediated cell toxicity, and identifying and selecting candidate amyloid inhibitors which inhibit amyloid-mediated cell toxicity to a greater degree than the small molecule which was co-crystallized with the amyloid; (b) characterizing and validating the candidate binders by X-ray crystallography, NMR spectroscopy (titration), ITC (isothermal titration calorimetry), thermal denaturation, mass spectrography, SPR (surface plasmon resonance), to measure the binding affinity to the amyloid fibers and also to oligomers, and/or an activity assay; (c) deriving on a computer a refined pharmacophore based on the identified candidate amyloid inhibitors (e.g. using methods as discussed herein).
Starting with the refined pharmacophore derived above, one can test a new set of candidate amyloid binders by repeating the docking and selecting steps, and testing the candidate amyloid binders for their ability to inhibit amyloid-mediated cell toxicity, in order to identify a further refined pharmacophore. Then, starting with this further refined pharmacophore, one can repeat the docking and screening steps, and test the candidate amyloid binders for their ability to inhibit amyloid-mediated cell toxicity in order to identify a yet further refined pharmacophore. This series of steps can be reiterated (repeated) as many times as desired.
Another aspect of the invention is a pharmaceutical composition comprising one or more of the compounds BAF4, BAF8, BAF11, BAF12, BAF14, BAF30 or BAF31, as shown in
Another aspect of the invention is a method for determining the presence of Aβ or tau oligomers or fibers (particularly fibers) in a sample, comprising contacting a sample suspected of comprising such oligomers or fibers with an effective amount of one or more of the 35 BAF compounds listed in Table 9, or suitable derivatives thereof. In one embodiment, the compounds are selected from one or more of the first set of (twelve) active compounds of the invention. The compounds may be detectably labeled. The contacting step is followed by measuring the amount of (bound) label in the sample, wherein a statistically significantly higher amount of label than that in a control sample lacking fibers indicates the presence of the fibers in the sample. In embodiments of this method, the determination is carried out on an in vitro sample (e.g. a tissue culture sample) or is carried out on a subject (e.g. the sample is removed from the subject, and can be, for example, blood or cerebral spinal fluid (CSF)). When the determination of Aβ or tau oligomers or fibers is in a sample from a subject, the method can be a method for diagnosing the presence of an amyloid-mediated disease or condition, such as Alzheimer's disease. Compounds that are found by a method of the invention to diagnose one disease or condition may also be useful for diagnosing a different amyloid-mediated disease or condition; and compounds found to reduce amyloid-mediated toxicity or to be useful for treating one amyloid-mediated disease or condition may also be useful for reducing amyloid-mediated toxicity or for treating a different amyloid-mediated disease or condition.
Compounds of the invention can also be used to detect the presence of Aβ or tau oligomers or fibers in a subject, in vivo, comprising introducing into the subject an effective amount of one or more of the 35 BAF compounds listed in Table 9, or suitable derivatives thereof. In one embodiment, the compounds are selected from one or more of the first set of (twelve) active compounds of the invention. In this method, the compound is labeled with a nuclide that can be detected by PET. The amount of bound label in the brain is them measured by PET (imaging the brain by PET). A statistically significantly higher signal than that in a control sample lacking the oligomers or fibers indicates the presence of the oligomers or fibrils in the brain of the subject.
Another aspect of the invention is a method for reducing or inhibiting amyloid-based (cellular) toxicity, comprising contacting amyloid protofilaments with an effective amount of one or more of the first set of (twelve) active compounds of the invention. This method can be carried out in vitro (e.g. in tissue culture) or in vivo (in a subject).
When carried out in a subject, the method can be a method for treating an amyloid-mediated disease or condition (e.g. a disease or condition mediated by Aβ or tau), comprising administering to a subject having or likely to have the disease or condition an effective amount of one or more of the set of twelve amyloid-inhibiting compounds. In one embodiment of the invention, a cocktail of more than one of these amyloid-inhibiting compounds is administered. As is described elsewhere herein, the inventors observed that different amyloid polymorphs bind different small molecules, suggesting that a cocktail of compounds directed against more than one of the polymorphs may provide improved therapies by binding to the several amyloid polymorphs present.
Another aspect of the invention is a computer readable medium providing the structural representation of a co-crystal of a protofilament of an amyloid protein with a small molecule that is known to bind to the amyloid protein.
Another aspect of the invention is a kit for carrying out any of the methods described herein (e.g., for identifying new compounds which bind to amyloid and/or inhibit amyloid toxicity, for diagnostic assays, for therapeutic applications, etc).
In the Examples shown herein, the present inventors first used a core fiber-forming hexamer segment from Aβ [KLVFFA (SEQ ID NO:1)] and one from tau [VQIVYK (SEQ ID NO:2)] to form co-crystals with low molecular weight compounds that were reported to bind to and/or to inhibit fibrillation of the amyloid fibers—the dye orange-G, the natural compound curcumin, and the Alzheimer/s diagnostic compound DDNP; and they then determined the atomic structures of the fiber-like complexes by X-ray microcrystallography. The atomic coordinates of the crystal structures of Orange-G/Aβ, Orange-G/tau, curcumin/tau and DDNP/tau are shown in Tables 3-6, respectively. The first two crystal structures are deposited in the Protein Data Bank (PDB) with accession codes 3OVJ and 3OVL. The rest of the structures and crystallographic tables are accessible at the world wide web site people.mcbi.ucla.edu/meytal/CoCrystalPaper.
The atomic structures of the fiber-like complexes reveal that they consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. Cylindrical cavities run along the β-spines of the fibers. Negatively charged orange-G wedges into a specific binding site between two sheets of the fiber, combining apolar binding with electrostatic interactions with lysine side chains of adjacent sheets, whereas uncharged compounds slide along the cavity. The three dimensional (3-D) structures thus determined allow for a structure-based design of improved small molecule diagnostics and therapeutics. The structural characteristics which allow for such design are sometimes referred to herein as pharmacophores.
Having obtained the co-crystals and the 3-D structures, the inventors developed a computer-based method to identify new candidates for small molecule amyloid binders. As proof of principle, the inventors employed a 3-D structure determined from a co-crystal of the Aβ fiber-forming segment, KLVFFA (SEQ ID NO: 1), and the negatively charged small molecule, Orange-G. They first assembled a database of test compounds containing a total of about 20,000 small molecules, which met certain initial criteria as described in Example III. The test molecules were docked on the computer to the crystal structure to determine if they fit, and to determine the energy of the fit. By determining for each test molecule the position and orientation having minimal energy, and using a threshold cut-off value that is below the calculated binding energy of the Orange-G molecule in the co-crystal (e.g., when using the Rosetta program exemplified herein, about 8 kcal/mol), 35 candidate amyloid binding molecules were identified for further study. See Table 9.
These 35 candidate molecules were then further characterized and validated by other criteria, including NMR titration, electron microscopy, and cell viability studies. Nine compounds were shown to inhibit amyloid cellular toxicity to a greater degree than the Orange-G used to form the original co-crystals. Of these, 7 compounds have not, to our knowledge, been reported to reduce amyloid toxicity and are particularly good candidates for therapeutic and/or diagnostic agents for amyloid diseases.
In subsequent steps, the inventors expanded the set of test compounds to include derivatives (homologs) of the active molecules described above. 25 such derivatives were selected, based on the crystal structure described above. Viability assays revealed that 7 of these derivatives can reduce amyloid toxicity, 5 of which have not, to our knowledge, been reported to inhibit amyloid toxicity, giving a total of 12 new small molecule amyloid inhibitors.
Using the identified amyloid inhibitors, the inventors designed a more refined general set of rules for identifying compounds which bind to Aβ fibers (a more refined pharmacophore), which can then be used, e.g. in a method as described above, to identify additional and/or improved amyloid inhibitors. This process can be reiterated for as many rounds as desired, to obtain additional, improved agents for use as diagnostic or therapeutic agents.
Flow charts shown in
As used herein, the term pharmacophore” refers to a specific, three-dimensional map of chemical and biological structures, properties, and features common to a set of ligands that exhibit a particular activity. A pharmacophore can be used as a model for the design of specific molecules that exhibit the same structural and functional features as the ligand(s) from which the pharmacophore was derived. Examples of pharmacophores according to the invention are displayed throughout this application.
Features of pharmacophores that relate to functional, structural, chemical or biological descriptors that describe a substituent and interaction of ligands with their receptors or binding sites include, e.g, hydrogen bond donors, hydrogen bond acceptors, hydrophobic regions, hydrophilic regions, ionizable regions, or aromatic rings. The features may further be described by the distances separating the features. For example, a feature may be a hydrogen bond donor that is 3 Å from a hydrogen bond acceptor. Pharmacophore features may be arranged in three-dimensional space and define points of interaction with the residues lining a binding site. In addition, features may further be described by torsional degrees of freedom of an atom or groups of atoms that define distinct, low energy conformations.
As used herein, the following terms have the meanings as indicated:
The term “small molecule” refers to a low molecular weight organic compound, e.g. having a molecular weight of less than about 800 Daltons (e.g. <700, 600, 500, 400, 300 Daltons). Small peptides (e.g. about 6 amino acids) are not included. As used herein, “about” means plus or minus 5% of the value.
An “amyloid” protein refers to one of a class of proteins having the structural and functional characteristics described in the Background Information section of this application and in the references cited therein. Inappropriately folded (misfolded) versions of the proteins interact with one another or other cell components to form insoluble fibrils (e.g. plaques or tangles). A skilled worker will recognize a wide variety of amyloid proteins that can be used in a method of the invention to design or select small molecule binders or inhibitors. These amyloid proteins have been implicated in the etiology of a variety of diseases or conditions, including neurodegenerative ones, and include, e.g., beta amyloid (Alzheimer's disease, cerebral amyloid angiopathy), tau (Alzheimer's disease and a large number of tauopathies, including frontotemporal dementia and progressive supranuclear palsy), amylin (diabetes type 2), PrP (Creutzfeldt-Jacob Disease, fatal familial insomnia, other prior-based conditions), SOD1, TDP-43, FUS (ALS), alpha-synuclein (Parkinson's disease), p53 (many cancers), and beta 2 microglobulin (dialysis related amyloidosis).
Such conditions are sometimes referred to herein as “amyloid-mediated” conditions or diseases. A disease or condition that is “mediated” by an amyloid is one in which the amyloid plays a biological role. The role may be direct or indirect, and may be necessary and/or sufficient for the manifestation of the symptoms of the disease or condition. It need not necessarily be the proximal cause of the disease or condition
A “protofilament” refers to the basic unit of a mature amyloid fiber. For the Abeta and tau structures described herein, each protofilament generally contains two of the beta sheets. Each sheet is formed from stacks of identical fiber-forming segments, as represented by the hexamers described herein. A pair of sheets forms a “cross-β spine” of the protofilament.
An “oligomer” amyloid structure contains between about 20 and 1,000 amyloid molecules.
The terms amyloid “fiber” and “fibril” are used interchangeably herein, and refer to structures with thousands of amyloid molecules.
“Fibrillation” refers to the aggregation of amyloid molecules to form fibers.
Without wishing to be bound by any particular mechanism, it is suggested, particularly with regard to Alzheimer's disease, that soluble aggregation intermediates such as amyloid oligomers are more toxic than amyloid fibers, while fibrils may serve as reservoirs of toxic oligomers. In this suggested model, fiber-binding molecules can inhibit amyloid toxicity by shifting the equilibrium from toxic oligomers toward end-stage fibers. See, e.g.,
An “amyloid binder” is a molecule which binds to an amyloid, preferably to the degree required to detect the presence of the amyloid (e.g., in a diagnostic assay). In some, but not all, cases, an amyloid binder can also elicit a biological effect (such as the inhibition of amyloid-induced cellular toxicity), in which case it is referred to herein as an “amyloid inhibitor.”
Any of a variety of fiber-forming segments of amyloid proteins can be used to generate co-crystals with small molecule amyloid binders, in addition to the hexamers described herein. These include, e.g., for Abeta, NKGAII (two polymorphic crystal forms) (SEQ ID NO:23), GAIIGL (SEQ ID NO:24), AIIGLM (SEQ ID NO:25), MVGGVVIA (2 POLYMOPRHIC CRYSTAL FORMS) (SEQ ID NO:26); MVGGVV (2 FORMS) (SEQ ID NO:27), GGVVIA (SEQ ID NO:28); for tau (Alzheimer's disease), VQIINK (SEQ ID NO:29); for Alpha synuclein (Parkinson's disease), GVTTVA (SEQ ID NO:30), GVATVA (SEQ ID NO:31), VVTGVTA (SEQ ID NO:32), TGVTAVA (SEQ ID NO:33); for insulin (Injection amyloidosis, and keeping insulin from forming fibers while stored), VEALYL (SEQ ID NO:34), LYQLEN (SEQ ID NO:35); for lysozyme (lysozyme amyloidosis), IFQINS (SEQ ID NO:36), TFQINS (SEQ ID NO:37), for Islet amyloid polypeptide (aka IAPP or amylin)—Diabetes type 2, NNFGAIL (SEQ ID NO:38), SSTNVG (SEQ ID NO:39); for p53—Cancer, TITTLE (SEQ ID NO:40), LTITTLE (SEQ ID NO:41); for Beta-2-microglobulin—Dialysis amyloidosis, NHVTLS (SEQ ID NO:42), NHVTLSQ (SEQ ID NO:43), KDWSFY (SEQ ID NO:44); for Transthyretin—several different amyloidosis, TIAALLS (SEQ ID NO:45), AADTWE (SEQ ID NO:46), YTIAAL (SEQ ID NO:47), SOD1 (SEQ ID NO:48), GVIGIAQ (SEQ ID NO:49), GVTGIAQ (SEQ ID NO:50), DSVISLS (SEQ ID NO:51), VQGIINFE (SEQ ID NO:52), for Prion protein (aka PrP)—prion diseases CJD etc., GTHSQW (SEQ ID NO:53), GTHSQWN (SEQ ID NO:54), AGAAAA (SEQ ID NO:55), GAVVGG (SEQ ID NO:56), GYMLGS (SEQ ID NO:57), GYVLGS (SEQ ID NO:58), IIHFGS (SEQ ID NO:59), NQVYYR (SEQ ID NO:60), PMDEYS (SEQ ID NO:61), SNQNNF (SEQ ID NO:62), NQNNFV (SEQ ID NO:63); QHTVTT (SEQ ID NO:64).
Aspects of a method of the invention for designing and/or selecting candidate amyloid-binding compounds comprise determining on a computer the 3-D structure of the co-crystal, thereby determining the atomic coordinates of the binding pocket or binding surface (pharmacophore).
Techniques for determining the three-dimensional (3-D) structure of such a co-crystal are conventional and well-known in the art. See, e.g., the Examples herein. Such a determination can comprise providing a structural representation of the co-crystal in a storage medium on a computer.
The storage medium (computer readable medium) in which the co-crystal structural representation is provided may be, e.g., random-access memory (RAM), read-only memory (ROM e.g. CDROM), a diskette, magnetic storage media, hybrids of these categories, etc. The storage medium may be local to the computer, or may be remote (e.g. a networked storage medium, including the Internet). The present invention also provides methods of producing computer readable databases containing coordinates of 3-D co-crystal structures of the invention; computer readable media embedded with or containing information regarding the 3-D structure of a co-crystal of the invention; a computer programmed to carry out a method of the invention (e.g. for designing and/or selecting small molecule amyloid binders or inhibitors), and data carriers having a program saved thereon for carrying out a method as described herein.
Any suitable computer can be used in the present invention.
A “binding surface” or “binding pocket” refers to a site or region in a co-crystal of the invention that, because of its shape, likely associates with a substrate or ligand. Atomic coordinates of the co-crystals of the invention define the binding surface or pocket. The amino acid residues of the Aβ or tau hexamer segments used to form the co-crystals described herein, which bind to the ligands and where are therefore important for binding small molecules designed or selected by a method of the invention, include one of more of the following amino acid residues, or combinations thereof: for the Orange-G/Aβ co-crystals, Lys16, Leu17, Val18, Phe 19, and Phe20; for the Orange-G/tau co-crystals, Gln2, Val4, and Lys6; and for the DDNP or curcumin/tau co-crystals, Val1, Gln2, Ile3, Val4, Tyr5 or Lys6. The numbering of the amino acid residues is as described elsewhere herein.
In aspects of a method of the invention for designing and/or selecting candidate amyloid-binding compounds, test molecules (for small molecule amyloid binders) are “docked” in a computer to determine if they fit well and bind tightly. Docking aligns the 3-D structures of two or more molecules to predict the conformation of a complex formed from the molecules. According to the present invention, test molecules are docked with a co-crystal 3-D structure of the invention to assess their ability to interact with the amyloid. Docking can be accomplished by either geometric matching of the ligand and its receptor or by minimizing the energy of interaction. This generally requires rotation and translation of a compound to achieve the best alignment with the 3-D structure (pharmacophore), i.e., the lowest energy conformation or interaction.
Suitable docking algorithms are well-known to those of skill in the art and include, e.g., DOCK [Kuntz et al. (1982) J. Mol. Biol. 161:269-288; available from UCSF]; AUTODOCK [Goodsell & Olson (1990) Proteins: Structure, Function and Genetics 8:195-202; Available from Oxford Molecular (<http://www.oxmol.co.uk/>]; MOE-DOCK [Available from Chemical Computing Group Inc. (<http://www.chemcomp.com/>); FLExX [Available from Tripos Inc (<http://www.tripos.com)]; GOLD [Jones et al. (1997) J. Mol. Biol. 267:727-748]; and AFFINITY [Available from Molecular Simulations Inc (<http://www.msi.com/>)]. The docking method described in the Examples herein is a modified version of the RosettaLigand program.
The test compounds may be known compounds or based on known compounds. Suitable libraries of compounds will be evident to a skilled worker. Several such compound libraries are discussed in the Examples herein.
Alternatively, the test compounds may be designed and made de novo. The binding surface or pharmacophore of a co-crystal 3-D structure of the invention can be used to map favorable interaction positions for functional groups (e.g. protons, hydroxyl groups, amine groups, hydrophobic groups and/or divalent cations) or small molecule fragments. Compounds can then be designed de novo in which the relevant functional groups are located in the correct spatial relationship to interact with CD81.
Once functional groups or small molecule fragments which can interact with specific sites on the binding surface or in the binding pocket of a co-crystal of the invention have been identified, they can be linked in a single compound using either bridging fragments with the correct size and geometry or frameworks which can support the functional groups at favorable orientations, thereby providing a compound according to the invention. While linking of functional groups in this way can be done manually, perhaps with the help of software such as QUANTA or SYBYL, automated or semi-automated de novo design approaches are also available. These include, e.g., MCDLNG [Gehlhaar et al. (1995) J. Med. Chem. 38:466-72]; MCSS/HOOK [Caflish et al. (1993) J. Med. Chem. 36:2142-67; Eisen et al. (1994) Proteins: Str. Funct. Genet. 19:199-221]; LUDI [2 Bohm (1992) J. Comp. Aided Molec. Design 6:61-780); GROW [Moon & Howe (1991) Proteins: Str. Funct. Genet. 11:314-328]; GROUPBUILD [Rotstein et al. (1993) J. Med. Client. 36:1700]; CAVEAT Lauri & Bartlett (1994) Comp. Aided Mol. Design. 8:51-66]; RASSE [Lai (1996) J. Chem. Inf. Comput. Sci. 36:1187-1194]; and others.
An amyloid inhibitor of the invention inhibits a measurable amount of one or more functions of an amyloid (e.g. it can inhibit or reduce amyloid-mediated or induced cellular toxicity; disrupt the structure of an amyloid oligomer; bind to an amyloid oligomer or fiber; stabilize amyloid fibers, thereby shifting the equilibrium to favor the formation of fibers rather than oligomers; etc.) Methods for assaying such amyloid-mediated effects are conventional and well-known to those of skill in the art. Some such methods, e.g., for measuring amyloid-mediated cellular toxicity, are described in the Examples.
In aspects of the invention, candidate inhibitors are further characterized and/or validated by any of a variety of methods, including X-ray crystallography, NMR spectroscopy (titration), ITC (isothermal titration calorimetry), thermal denaturation, mass spectroscopy, SPR (surface plasmon resonance), to measure the binding affinity to the amyloid fibers and also to oligomers, and/or an activity assay. In one embodiment of the invention, amyloid-mediated cell toxicity is monitored by assaying for cell viability, using an assay such as the MIT assay. Such methods are conventional and well-known in the art; some of them are described in the Examples herein.
A compound of the invention can be in the form of a pharmaceutically acceptable salt, solvate or salt. Suitable acids and bases that are capable of forming salts with the compounds of the present invention are well known to those of skill in the art, and include inorganic and organic acids and bases. “Solvates” refers to solvent additions forms that contain either stoichiometric or non stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate. Hydrates are formed by the combination of one or more molecules of water with one of the substances in which the water retains its molecular state as H2O, such combination being able to form one or more hydrate.
A “pharmaceutical composition” comprises a compound of the invention plus a pharmaceutically acceptable carrier or diluent. In some embodiments, the compound is present in an effective amount for the desired purpose.
“Pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use. For example, “pharmaceutically acceptable salts” of a compound means salts that are pharmaceutically acceptable, as defined herein, and that possess the desired pharmacological activity of the parent compound.
One aspect of the invention is a method for reducing or inhibiting amyloid-based cellular toxicity, or for treating an amyloid-mediated disease or condition, comprising contacting amyloid protofilaments with an effective amount of a compound of the invention, or, if the method is conducted in vivo (in a subject), administering an effective amount of the compound to the subject.
An “effective amount” of a compound or pharmaceutical composition of the invention is an amount that can elicit a measurable amount of a desired outcome, e.g. for a diagnostic assay, an amount that can detect a target of interest, such as an amyloid oligomer or fiber, or in a method of treatment, an amount that can reduce or ameliorate, by a measurable amount, a symptom of the disease or condition that is being treated.
A “subject” can be any subject (patient) in which amyloid molecules associated with an amyloid-mediated disease or condition can be detected, or in which the disease or condition can be treated by a compound of the invention. Typical subjects include vertebrates, such as mammals, including laboratory animals, dogs, cats, non-human primates and humans.
The compounds of the invention can be formulated as pharmaceutical compositions in a variety of forms adapted to the chosen route of administration, for example, orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.
Suitable oral forms for administering the compounds include lozenges, troches, tablets, capsules, effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
The compounds of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They may be enclosed in coated or uncoated hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the compounds may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. For compositions suitable for administration to humans, the term “excipient” is meant to include, but is not limited to, those ingredients described in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins, 21st ed. (2006) (hereinafter Remington's).
The compounds may be combined with a fine inert powdered carrier and inhaled by the subject or insufflated. Such compositions and preparations should contain at least 0.1% compounds. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of a given unit dosage form.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
In addition, the compounds may be incorporated into sustained-release preparations and devices. For example, the compounds may be incorporated into time release capsules, time release tablets, and time release pills. In some embodiments, the composition is administered using a dosage form selected from the group consisting of effervescent tablets, orally disintegrating tablets, floating tablets designed to increase gastric retention times, buccal patches, and sublingual tablets.
The compounds may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the compounds can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations can contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the compounds which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
Sterile injectable solutions are prepared by incorporating the compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the compounds may be applied in pure form. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water/alcohol/glycol blends, in which the compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art.
For example, the concentration of the compounds in a liquid composition, such as a lotion, can be from about 0.1-25% by weight, or from about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder can be about 0.1-5% by weight, or about 0.5-2.5% by weight.
Effective dosages and routes of administration of agents of the invention are conventional. The exact amount (effective dose) of the agent will vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See, e.g, The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an, effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy to dosages for comparable therapeutic agents.
The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.
In general, however, a suitable dose will be in the range of from about 0.001 to about 100 mg/kg, e.g., from about 0.01 to about 100 mg/kg of body weight per day, such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose may be about 1 mg/kg, 10 mg/kg, or 50 mg/kg of body weight per day.
The compounds are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form. In some embodiments, the dosage unit contains about 1 mg, about 10 mg, about 25 mg, about 50 mg, about 75 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 750 mg, or about 1000 mg of active ingredient.
One aspect of the invention is a method for detecting the presence of amyloid (e.g., Abeta or tau) oligomers or fibers in a sample, comprising contacting a sample suspected of containing such oligomers or fibers with an effective amount of a detectably labeled compound of the invention and measuring the amount of (bound) label in the sample. Phrases such as “detecting an oligomer or fiber in a sample” are not meant to exclude samples or determinations (detection attempts) where no oligomer or fiber is contained or detected. In a general sense, this invention involves assays to determine whether the target is present in a sample, irrespective of whether or not it is detected.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, “a” compound of the present invention, as used above, can be two or more compounds.
The contacting step can comprise, e.g., (1) taking a sample of body fluid or tissue (e.g., a suitable blood sample likely to contain amyloid molecules; (2) contacting the sample with a detectably labeled compound of the invention, under conditions effective for the compound to bind to the oligomers or fibers, e.g., reacting or incubating the sample and the compound; and (3) assaying the contacted sample for the presence of labeled compound which has bound to the amyloid oligomer or fiber.
Suitable labels which enable detection (e.g., provide a detectable signal, or can be detected), and methods for labeling compounds of the invention with the labels, are conventional and well-known to those of skill in the art. Suitable detectable labels include, e.g., radioactive active agents, fluorescent labels, and the like. Assays for detecting such labels are conventional
Conditions for binding a compound of the invention to an amyloid oligomer or fiber, and treating the sample as necessary to detect the targets to which the compound has bound, are conventional and well-known to those of skill in the art.
Suitable samples include tissues and bodily fluids, such as blood, cerebral spinal fluid (CSF), saliva, gastric secretions, mucus, or the like, which will be evident to a skilled worker;
In one aspect of the invention, amyloid oligomers or fibers are detected in a subject, e.g. in the brain of a subject. In one embodiment of the invention, a compound of the invention is labeled with a radionuclide which can be detected in a non-invasive manner. For example, if the compound is used in diagnosis according to single photon emission computed tomography (SPECT), examples of a radionuclide that can be used may include gamma-ray-emitting radionuclides such as 99mTc, 111In, 67Ga, 201Tl, 123I, or 133Xe. When the compound is used in diagnosis according to Positron Emission Tomography (PET), examples of a radionuclide that can be used may include positron-emitting radionuclides such as 11C, 13N, 15O, 18F, 62Cu, 68Ga, or 76Br. When the compound is administered to animals other than human, radionuclides having a longer half-life, such as 125I, may also be used. Methods for formulating the labeled compounds, administering them to a subject, and imaging them with a suitable apparatus are conventional. Other labeled agents are currently being used to detect amyloid fibers in brain, and methods similar to those can be used with compounds of the present invention.
Another aspect of the invention is a kit for performing a method of the invention (e.g. for detecting amyloid in a sample or in a subject, or for inhibiting or reducing amyloid toxicity, in vitro or in a subject). The kit may comprise a suitable amount of a compound or pharmaceutical composition of the invention. Kits of the invention may comprise instructions for performing a method, such as a diagnostic method. Other optional elements of a kit of the invention include suitable buffers, media components, or the like; a computer or computer-readable medium for storing and/or evaluating the assay results; containers; or packaging materials. Reagents for performing suitable controls may also be included. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stabilized liquids. The reagents may also be in single use form, e.g., in single reaction form for diagnostic use.
In the foregoing and in the following examples, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.
Peptide segments (custom synthesis) were purchased from CS Bio. Orange-G and curcumin were purchased from Sigma-Aldrich. DDNP was synthesized as described in [38,58].
All crystals were grown at 18° C. via hanging-drop vapor diffusion. All crystals appeared within 1 wk, except the negative control crystals of VQIVYK (SEQ ID NO: 2)+DDNP that took 8 mo to grow.
VQIVYK (SEQ ID NO: 2)+orange-G. The drop was a mixture of 10 mM VQIVYK (SEQ ID NO: 2) and 1 mM orange-G in water, and reservoir solution (0.1 M zinc acetate dehydrate, 18% polyethylene glycol 335.0). The structure was solved to 1.8 Å resolution and contained one segment, one orange-G, two water molecules, two zinc atoms, and one acetate molecule in the asymmetric unit.
VQIVYK (SEQ ID NO: 2)+DDNP. The drop was a mixture of 6 mM VQIVYK (SEQ ID NO: 2) and 1 mM DDNP in 60% ethanol, and reservoir solution (0.52 M potassium sodium tartrate, 0.065 M HEPES-Na pH 7.5, 35% glycerol). The structure was solved to 1.2 Å resolution and contained one segment and three water molecules in the asymmetric unit.
VQIVYK (SEQ ID NO: 2)+DDNP from second crystallization conditions. The drop was a mixture of 6 mM VQIVYK (SEQ ID NO: 2) and 1 mM DDNP in 60% ethanol, and reservoir solution (1.2 M DL-malic acid pH 7.0, 0.1 M BIS-TRIS propane pH 7.0). The structure was solved to 1.65 Å resolution and contained one segment, and three water molecules in the asymmetric unit.
Negative control crystals to VQIVYK (SEQ ID NO: 2)+DDNP. The drop was a mixture of 6 mM VQIVYK (SEQ ID NO: 2) in 60% ethanol and reservoir solution (0.52 M potassium sodium tartrate, 0.065 M HEPES-Na pH 7.5, 35% glycerol). The structure was solved to 1.2 Å resolution and contained one segment, and one water molecule in the asymmetric unit.
VQIVYK (SEQ ID NO: 2)+curcumin. The drop was a mixture of 10 mM VQIVYK (SEQ ID NO: 2) and 1 mM curcumin in 80% dimethyl sulfoxide (DMSO), and reservoir solution (0.1 M Tris.HCl pH 8.5, 70% (v/v) MPD (2-methyl-2,4-pentanediol)). The structure was solved to 1.3 Å resolution and contained one segment, and two water molecules in the asymmetric unit.
Negative control crystals to VQIVYK (SEQ ID NO: 2)+curcumin. The drop was a mixture of 10 mM VQIVYK (SEQ ID NO: 2) in 80% DMSO and reservoir solution (0.1 M Tris.HCl pH 8.5, 70% (v/v) MPD (2-methyl-2,4-pentanediol)). The structure was solved to 1.3 Å resolution and contained one segment, and one water molecule in the asymmetric unit.
KLVFFA (SEQ ID NO: 1)+orange-G. The drop was a mixture of 10 mM KLVFFA (SEQ ID NO: 1) and 1 mM orange-G in water, and reservoir solution (10% w/v polyethylene glycol 1,500, 30% v/v glycerol). Another drop was a mixture of 5 mM KLVFFA (SEQ ID NO: 1) and 1 mM orange-G in water, and reservoir solution (30% w/v polyethylene glycol 1,500, 20% v/v glycerol). The structure was solved to 1.8 Å resolution and contained four segments, two orange-G molecules, and 11 water molecules in the asymmetric unit.
Negative control crystals to KLVFFA (SEQ ID NO: 1)+orange-G. The drop was a mixture of 10 mM KLVFFA (SEQ ID NO: 1) in water, and reservoir solution (10% w/v polyethylene glycol 1,500, 30% v/v glycerol). Another drop was a mixture of 5 mM KLVFFA (SEQ ID NO: 1) in water, and reservoir solution (30% w/v polyethylene glycol 1,500, 20% v/v glycerol). The structure was solved to 2.1 Å resolution and contained one segment and three water molecules in the asymmetric unit.
X-ray diffraction data were collected at beamline 24-ID-E of the Advanced Photon Source (APS), Argonne National Laboratory; wavelength of data collection was 0.9792 Å. Data were collected at 100 K. Molecular replacement solutions for all segments were obtained using the program Phaser [59]. The search models consisted of available structures of the same segment or geometrically idealized n-strands. Crystallographic refinements were performed with the program Refmac5 [60]. Model building was performed with Coot [61] and illustrated with PyMOL [62]. There were no residues that fell in the disallowed region of the Ramachandran plot. Simulated annealing composite omit map was generated using CNS [63,64]; 10% was omitted.
Three-dimensional (3-D) structures of the small molecules were generated using Corina (Molecular Networks; http://www.molecular-networks.com/online_demos/corina_demo) and Chemical Identifier Resolver (http://cactus.nci.nih.gov/translate/). Additional 3-D conformations were generated using OpenEye Omega [65]. The small molecule was placed in approximate location according to the electron density map. The small molecule was docked to the peptide fibrillar structure using RosettaLigand [66,67]. The protein side chains were fixed. The generated docked structures (1,000 for KLVFFA (SEQ ID NO: 1)-orange-G and 500 for the rest of the structures) were further refined using Refmac5 [60] and the 10 best structures (based on lowest free-R [68]) were analyzed and showed to be very similar to each other. The best structures were further optimized and refined and the one with the lowest free-R was chosen as the final structure.
The area buried of the small molecules within the fiber structure was calculated using Areaimol [69,70] with a probe radius of 1.4 Å. The difference between the accessible surface areas of the fiber structure alone and with the small molecule constitutes the reported area buried. The Areaimol [69,70] calculations were also used to report the segment atoms that are in contact with the small molecules (shown in
Binding energy and corresponding dissociation constant of one orange-G molecule to the KLVFFA (SEQ ID NO: 1) fiber were estimated from the apolar surface area (contributed by carbon atoms) that is covered by the interaction and was calculated using Areaimol [69,70]. The difference between the apolar accessible surface areas of the fiber structure atone and with the small molecule was added to the difference between the apolar accessible surface areas of the small molecule alone and with the fiber. These calculations resulted in 500 Å2 of apolar surface area covered. The binding energy was calculated from the formula [71] ΔG0=18 cal×Å−2×mol−1=18×500 cal/mol=9 kcal/mol. The dissociation constant was calculated from ΔG0−RT ln K. Thus, K=exp(−ΔG/RT)=3×10−7 M=0.3 μM.
Liquid chromatography tandem mass spectrometry (LC-MSMS) was used to measure the molar ratios of the peptide segments and the small molecules within the crystals. Authentic samples of the peptides and each of the small molecules were used to prepare standard response curves. Crystals from each of the four mixtures of peptides and small compounds were individually picked (using a sharpened glass capillary) and re-dissolved in 5%-10% acetonitrile. The samples were divided into two aliquots, one for the peptide analyses and the other for the small molecule analyses, and the amount of each component in the samples was interpolated using the standard curves.
Peptide standards (dry powder of VQIVYK (SEQ ID NO: 2) and KLVFFA (SEQ ID NO: 1)) were dissolved in water and prepared in concentrations ranging from 0.05 μM to 0.01 mM in 0.1% TFA. Aliquots of the standards and the re-dissolved crystals were separately injected (50 μL) onto a polymeric reverse phase column (PLRP/S, 2×150 mm, 5 μm, 300 Å; Varian) equilibrated in Buffer A (0.1% formic acid in water) and eluted (0.25 mL/min) with an increasing concentration of Buffer B (0.1% formic acid in acetonitrile). The effluent from the column was directed to an Ionspray source attached to a triple quadrupole mass spectrometer (Perkin Elmer/Sciex API III+) operating under previously optimized positive ion mode conditions. Data were collected in the positive ion multiple reaction monitoring (MRM) mode in which the intensity of specific parent→fragment ion transitions were recorded (VQIVYK (SEQ ID NO: 2), m/z 749.5→341.3, 749.5→409.4, 749.5→440.3, 749.5→522.5; KLVFFA (SEQ ID NO: 1), 724.4→84, 724.4→488.3, 362.7→84, 362.7→120.1).
Similar procedures were used for the analyses of the small molecules. Orange-G was dissolved in water and diluted with 10% ammonium acetate to concentrations ranging from 2 nM to 20 μM. Solutions of the standard and the re-dissolved crystals were separately injected (50 μL) onto a silica based reverse phase column (Supelco Ascentis Express C18, 150×2.1 mm, 2.7 μm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.2 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1:1 containing 10 mM ammonium acetate). The negative ion MRM transitions were m/z 407.1→302.1 and 407.1→222.1.
DDNP was dissolved in 95% ethanol and diluted with 10% ammonium acetate to concentrations ranging from 2 nM to 20 μM. Solutions of the standards and the re-dissolved crystals (further diluted with acetonitrile:methanol:water:acetic-acid (41:23:36:1, v/v/v/v) to ensure dissolution) were separately injected (50 μL) onto a silica based reverse phase column (Supelco Ascentis Express C18, 150×2.1 mm, 2.7 μm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.2 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1:1 containing 10 mM ammonium acetate). The positive ion MRM transition was: DDNP—m/z 262.1→247.1.
Curcumin was dissolved and diluted in acetonitrile:methanol:water:acetic-acid (41:23:36:1, v/v/v/v) to concentrations ranging from 2 nM to 2 μM. Aliquots of the standards and the re-dissolved crystals (further diluted with acetonitrile:methanol:water:acetic-acid (41:23:36:1, v/v/v/v) to ensure dissolution) were injected (100 μL) onto a silica based reverse phase column (Waters Symmetry Shield RP18 5 μM, 3.9×150 mm) equilibrated in Buffer A (10 mM ammonium acetate) and eluted (0.5 mL/min) with an increasing concentration of Buffer B (acetonitrile/Isopropanol 1:1 containing 10 mM ammonium acetate). The negative ion MRM transitions were m/z 367.1→173.1, 367.1→149.
B. Screening for Co-Crystals of Amyloid-Like Segments with Small Molecules
In our attempts to obtain complexes of small molecules with amyloid-like segments from disease-related proteins, we screened for co-crystals grown from dozens of mixtures (Table 1). The majority of the resulting crystals yielded X-ray diffraction too poor for structure determination. Others led to structure determinations of the small molecule or amyloid-like segment alone. Out of hundreds of co-crystallization trials (Table 1), four mixtures, described below, yielded co-crystals with suitable X-ray diffraction from segments of Aβ and tau with amyloid binders.
C. Crystal Structure of the KLVFFA (SEQ ID NO: 1) Segment from Aβ Complexed with Orange-G
The KLVFFA (SEQ ID NO: 1) segment (residues 16-21) from Aβ contains apolar residues that participate in a hydrophobic spine in Aβ fibers and itself acts as an inhibitor of Aβ fibrillation [28,29]. We previously determined the atomic structure of the KLVFFA (SEQ ID NO: 1) segment in three crystal forms; all show the common steric zipper motif associated with amyloid fibers (Colletier et al. unpublished results). Orange-G (
All four crystal forms of KLVFFA (SEQ ID NO: 1), including the complex with orange G, show an anti-parallel β-strand stacking in the steric zipper (Colletier et al. (supra) and
D. Crystal Structures of the VQIVYK (SEQ ID NO: 2) Segment from the Tau Protein with Orange-G
The VQIVYK (SEQ ID NO: 2) segment of tau was suggested as the minimal interaction motif for fiber formation [37]. We previously determined the crystal structure of VQIVYK (SEQ ID NO: 2) in two crystal forms; both show the common steric zipper motif of amyloid fiber-like structures [18,25]. Co-crystallization of VQIVYK (SEQ ID NO: 2) with orange-G resulted in deep orange crystals (
Crystallization of VQIVYK (SEQ ID NO: 2) alone, under identical conditions to the co-crystallization of the VQIVYK (SEQ ID NO: 2)-orange-G mixture, resulted in the formation of colorless fibrous crystals (
E. Crystal Structures of the VQIVYK (SEQ ID NO: 2) Segment from the Tau Protein with Curcumin and DDNP
Curcumin (
Despite the lack of differentiated electron density for curcumin and DDNP in VQIVYK (SEQ ID NO: 2), there is strong evidence for the presence of the small molecules in the crystals. The crystals show a distinctive color, whereas the control crystals (grown under identical condition without the small molecule) are colorless (
The common feature of the structures of four amyloid/small-molecule complexes is that the small molecules bind to fibers in a similar orientation, along the β-sheets, with their long axes parallel to the fiber axis. This orientation was previously proposed for the binding of thioflavin T to bovine insulin and bovine β-lactoglobulin amyloid fibrils using polarized laser confocal microscopy [42]. A similar mode of binding was seen in co-crystals of oligomer-like β-2-microglubulin with thioflavin T, showing that thioflavin T is bound between β-sheets, orthogonal to the β-strands [43]. The orientation of congo-red was also suggested to be parallel to the amyloid long axis based on electron diffraction, linear dichroism [44], and a recent NMR-based model of congo-red bound to the fungal prion domain HET-s (218-289) [45].
Our crystal structures of small molecules bound within amyloid-like steric zippers define molecular frameworks, or pharmacophores, for the design of diagnostics and drugs for Alzheimer's and other aggregation diseases. The amyloid components in our structures are steric zippers formed by stacks of six-residue segments from Alzheimer-related proteins. Although these steric zippers cannot represent all aspects of the full-length amyloid parent proteins, they share many properties and are commonly used as models of the amyloid β-spine and of aggregation [22,24]. The small molecules in our structures bind along the 3-spine, and because the parent amyloids contain the same segments, we expect a similar mode of binding along the spine of the full-length parent amyloid fibers. Moreover, we expect the steric zipper spine of the parent fibers to be flanked with the rest of the protein residues in a native-like or unfolded conformation [12,20] and therefore to contain more solvent channels, or accessible sites for the binding of the small molecules, compared to the very compact packing of the steric zipper segments. Consistently, orange-G, curcumin, and DDNP all bind to, or affect fibrillation of, full-length fibers [7,9,39].
Overall, the complexes presented here define two molecular frameworks for the binding of small molecules to amyloid fibers. The first molecular framework pertains to site-specific binders, such as charged compounds that form networks of interactions with sequence motifs, and is relatively well defined. The second molecular framework, far less well defined at this point, pertains to broad-spectrum binders, such as uncharged aromatic compounds that bind to tube-like cavities between β-sheets. Without wishing to be bound by any particular mechanism, it is suggested that for binding amyloid deposits in the brain, uncharged molecules may be more effective because of superior blood-brain-barrier penetrability. The same frameworks, offering cavities along β-sheets, are also expected to exist in amyloid oligomers known to be rich in β-sheets and possibly fiber-like [46], similar to the observed binding of amyloid markers to β-sheets in non-fibrillar structures [43,47]. Consistent with this, both oligomers and fibers are inhibited by similar compounds, including curcumin [7,9].
The specific binding of orange-G allows definition of the chemical properties of a specific molecular framework. The prominent feature of amyloid structures is the separation of β-strands (forming a (β-sheet) by ˜4.8 Å. In structures with strands packed in an antiparallel orientation, as observed for the KLVFFA (SEQ ID NO: 1) fibers and for a rare mutation in Aβ that is associated with massive depositions of the mutant protein and early onset of the disease [34,48], the separation of repeating units (2 strands) is twice as great, ˜9.6 Å. Orange-G contains two negatively charged sulfonic acid groups facing the same direction, with the sulfur atoms spaced ˜5 Å apart and the oxygen atoms separated by 4.5-7.5 Å. This framework allows the formation of salt links between the sulfonic acid groups and lysine ammonium ions from every repeating strand in both KLVFFA (SEQ ID NO: 1) (anti-parallel orientation) and VQIVYK (SEQ ID NO: 2) (parallel orientation) fibers (
Within our framework, an apolar aromatic spine is another essential moiety [22]. The largely apolar KLVFFA (SEQ ID NO: 1) segment attracts the apolar surface of orange-G, stabilizing the binding (
Despite the lack of atomized electron density for the binding of curcumin and DDNP in VQIVYK (SEQ ID NO: 2) fibers, the location of the binding cavity is clear. It is narrow, restricting rotation of the small molecule (
Our structures show that different small molecules bind along β-spine of amyloid-like fibers. In case fibers contain more than a single spine, the molecules might bind to multiple sites. This is more likely for the broad-spectrum hydrophobic compounds but can also apply for charged compounds. For example, we observed orange-G to bind to two different steric zippers, of KLVFFA (SEQ ID NO: 1) and VQIVYK (SEQ ID NO: 2), with the commonality of binding to lysine side chains protruding from the β-sheets.
Congo-red, a known amyloid marker, contains two sulfonic acid groups, similar to orange-G, but they are spaced ˜19 Å apart, which might account for its lack of specificity [44]. In a recent model, built using NMR constrains, congo-red was computationally docked to the fungal prion domain HET-s (218-289), suggesting that the sulfonic acid groups interact with lysine residues protruding from the sheets [45], similar to orange-G in our structures. However, in the model, the strands of HET-s are arranged in an anti-parallel orientation and the sulfonic acid groups of congo-red interact with every other lysine along the fiber [45], while orange-G interacts with every single lysine in both the KLVFFA (SEQ ID NO: 1) and VQIVYK (SEQ ID NO: 2) complexes (
Defining these two molecular frameworks illuminates functional attributes of specific and broad-spectrum amyloid binders. This distinction is consistent with competitive kinetic experiments demonstrating that the binding of FDDNP (the fluoridated analog of DDNP) to Aβ fibrils is displaceable by the uncharged non-steroidal anti-inflammatory naproxen, but not by the common charged dyes congo-red and thioflavin T [53]. Moreover, in vitro FDDNP labels amyloid-like structures in a fashion similar to congo-red and thioflavin T, providing further evidence for the broad-spectrum type of binding [54]. Knowledge of both frameworks can lead to the design of more potent and specific compounds. Without wishing to be bound by any particular mechanism, it is suggested that these molecules can act as binders and be used as diagnostics, or serve as inhibitors of aggregation by either destabilizing steric zippers by wedge action (
In the case of the complexed curcumin and DDNP structures, we expect that the tube-like cavity along the β-sheets provides an adequate site for the binding of many compounds of similar properties. However, the lack of specific interactions allows the small molecule to drift along the fiber axis, leading to lower occupancy and a degree of fluidity in the structure. Extrapolating from our structures, we expect that various aromatic compounds, such as polyphenols [6], would bind to a variety of amyloid-forming sequences because of a cylindrical, partially apolar cavity that forms between the pairs of β-sheets forming the fibers. These cavities are also expected to provide binding sites for various kinds of apolar drugs, such as benzodiazepines and anesthetics, explaining some of the altered pharmacokinetic properties and increased sensitivity detected in elderly [55].
One implication of our structures for the design of effective therapeutic treatments is the specificity they reveal of ligand binding to particular fiber polymorphs (
Four crystal structures of small molecules bound to fiber-forming segments of the two main Alzheimer's disease proteins show common features. The small molecules bind with their long axes parallel to the fiber axis. The structures reveal a sequence-specific binder which forms salt links with side-chains of the steric zipper spines of the fibers and non-specific binders which lie in cylindrical cavities formed at the edges of several steric zippers. Small-molecule binding is specific to particular steric-zipper polymorphs, suggesting that for effective Alzheimer's diagnostics and therapeutics, it may be advantageous to have to be mixtures of various compounds to bind to all polymorphs present. The complexes presented here providet routes for structure-based design of combinations of compounds that can bind to a spectrum of polymorphic aggregates, to be used as markers of fibers and as inhibitors of aggregation.
In all four structures reported in Example I, the electron density attributed to the small molecule was undifferentiated (to different extents), which hindered the determination of the structures in atomic detail. After assignment of the peptide segment into the electron density 2Fo-Fc map, the difference Fo-Fc map showed positive density that resembled a narrow and long tube running along the fiber (see e.g. in
The generated docked structures were refined and evaluated based on their free-R value [3] (Methods). In the case of KLVFFA (SEQ ID NO: 1) or VQIVYK (SEQ ID NO: 2) with orange-G, the crystallographic refinement in the presence of the small molecule significantly decreased the free-R value (by 5% and 2%, respectively). In the case of VQIVYK (SEQ ID NO: 2) with DDNP or curcumin, the refinement with the small molecule did not improve the free-R value and we concluded that the x-ray diffraction does not allow the determination of the position of the small molecule in atomic detail.
In the three structures with VQIVYK (SEQ ID NO: 2) complexed with orange-G, DDNP and curcumin, the lengths of the small molecules (DDNP ˜12×5 Å, curcumin ˜19×5 Å and orange-G ˜9.5×8 Å) span multiple unit cells of the fibril (4.8-4.9 Å along the fiber axis;
Based on our structures we extrapolate that apolar compounds, such as DDNP and curcumin, bind to cylindrical cavities formed between pairs of β-sheets in amyloid structures. These cavities are frequently surrounded by hydrophobic and aromatic side chains [6,7], forming a binding motif for poly-aromatic compounds often reported to affect fibrillation [7-14]. Nevertheless, the binding is insufficiently specific, such that the molecules can be situated with different spacing along the fiber. Moreover, since the main constraint on binding is the width of the cylindrical cavity, the small molecule can not only drift along the fiber, but also rotate along its long dimension, and flip 180° perpendicular to its long dimension. In the crystalline form, these degrees of freedom will lead to crystal disorder along the fiber axis, as we see here for the DDNP and curcumin complexes.
The binding of orange-G to the fibers is more specific than the binding of apolar compounds, via salt links between the negatively charged sulfonic acid groups of orange-G and the lysine side chains (
The high abundance of orange-G in the KLVFFA (SEQ ID NO: 1) fiber corresponds to the detailed electron density for orange-G obtained following the computational docking (
We choose small molecules that were reported to affect fibrillation of different amyloid-forming proteins [10,11,13], including natural compounds [15], a Thioflavin derivative: Pittsburgh compound B (PIB) [16], as well as a molecule that constitutes half of the curcumin molecule: (−)-2-Methoxy-4-methylphenol (Creosol). We also screened for complexes with biological marker that detect amyloid fibers in-vivo, developed and synthesized by Jorge R. Barrio and co-workers [17-20].
We used 34 different small-molecules combined with different amyloid-like segments to generate an overall of 89 different mixtures. We note that several different molecular ratios (ranging between 1:1 and 1:10 small-molecule:segment) were tested (details are not specified in the table) resulting in >100 different co-crystallization trials. Each mixture was screened for the formation of co-crystals with 768 different crystallization conditions. In many cases, crystals grown from various conditions were tested (details are not specified in the table). We note that soaking experiments (adding the small molecule after growing crystals from the amyloid-like segment alone), tested for several of the different combinations, failed to show the presence of the small molecule. This is expected due to the lack of solvent channels in the crystal packing of the amyloid-like segments.
From the 89 mixtures detailed in the Table, 4 structures of complexes were determined. 14 mixtures did not show formation of crystals in the conditions tested over several months. 47 mixtures resulted in fibrous or colorless crystals that were not tested, or crystals with too poor x-ray diffraction to be determined. Crystals grown from 21 mixtures showed the presence of only the amyloid-like segment, while 3 showed the presence of only the small molecule.
VQIVYK from tau (SEQ ID NO: 2)
KLVFFAEN (residues 16-23) -
GGVVIA (residues 37-42) from Aβ (SEQ
GVVEVD (residues 734-739) from Aβ A4
VQIVYK from tau (SEQ ID NO: 2)
GVVEVD (residues 734-739) from Aβ A4
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
KLVFFA (residues 16-21) from Aβ (SEQ
GGVVIA (residues 37-42) from Aβ (SEQ
GVVEVD (residues 734-739) from Aβ A4
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
VQIVYK from tau (SEQ ID NO: 2)
(*)Values in brackets are for the highest resolution shells.
(a)Rmerge(square) = Σ(I − I(mean))2/ΣI2, where I is the observed intensity of the reflection HKL and the sum is taken over all reflections HKL.
(b)The incompleteness (<80%) of the highest resolution shell is likely due to the rapid decay of the peptide crystals that are naturally susceptible to radiation decay due to their small size.
(c)Rwork = Σ||Fo| − |Fc||/Σ|Fo|.
(d)Rfree as defined by [3].
(e)The statistical significance of Rfree is limited in the case of peptide structures, where the small size of the unit cell leads to a paucity of reflections. In these cases, the Rfree statistic has proven to be a useful, but less sensitive tool compared with Rfree statistics reported from macromolecular structure.
In order to identify additional compounds that can act as amyloid binders and/or inhibitors, we started with the crystal structure described above of a fiber-forming segment of Aβ in complex with the small molecule binder Orange-G. We then computationally identified candidate compounds from very large databases of compounds which interact favorably with amyloid fibers. The top-ranking compounds were then experimentally characterized by, e.g., NMR titration, Electron Microscope (EM), and MIT cell viability experiments.
Flow charts summarizing the approach used in this Example are shown in
Step I. Determination of the Co-Crystal Structure of a Fiber-Forming Segment of Aβ in Complex with the Small Molecule Binder Orange-G
Amyloid beta was chosen as a target for inhibitor design. Amyloid beta (Aβ) is a peptide of 39-42 amino acids processed from the Amyloid precursor protein (APP), and it is most commonly known in association with Alzheimer's disease (AD). The segment 16KLVFFA21 (SEQ ID NO: 1) has been well studied and identified as an amyloid-forming peptide involved in the fiber core structure. As shown in Examples I and II above, we have determined the crystal structure of this fiber-forming segment in complex with the small molecule binder Orange-G (see, e.g.,
Step II. Select Compounds from Compound Databases
We selected compounds for docking from two choices of purchasable compound libraries of compounds:
102,236 organic compounds having crystal structures with R-factor of better than 0.1 were extracted from the Cambridge Structure Database (version 5.32, November 2010) using ConQuest. The SMILES string of each structure was then used to locate its purchasing information among the ZINC purchasable set (http://zinc.docking.org/) by OpenBabel package (http://openbabel.org/). The fast index table of all SMILES strings of the ZINC purchasable set was generated to allow the fast search of each CSD structure against ZINC purchasable set. CSD structures failed in locating their purchasing information (that is, without any hit in searching against ZINC purchasable set) were omitted. A library of 11,057 compounds was finally compiled. A total of 13,918 structures from CSD representing 11,057 compounds were compiled, whose purchasing information is annotated by ZINC purchasable database. The information of CSD code and ZINC entry can be downloaded from the world wide web site people.mbi.ucla.edu/jiangl/AmyloidInhibitor Paper.
A library of 6,589 compounds containing phenol and less than 3 freely rotatable bonds were extracted from the ZINC database (http://zinc.docking.org/). Those compounds have a common feature of planar aromatic ring, a so called “flat” compound. The flat compound library includes those compounds which have similar chemical structures to naturally fibril-binding molecules, for instance, Thioflavin-T (ThT), Congo Red, Green tea epigallocatechin-3-gallate (EGCG) and Curcumin. And it also includes many natural phenols, such as gallic acid, ferulic acid, coumaric acid, propyl gallate, epicatechin, epigallocatechin, epigallocatechin gallate, and etc. The complete list of ZINC entries of these compounds can be downloaded from the world wide web site people.mbi.ucla.edu/jiangl/AmyloidInhibitor Paper.
From these two compound libraries, each molecule was then prepared. Hydrogens of each molecule were added if there is any missing hydrogen by using the program Omega (v. 2.3.2, OpenEye). Ligand atoms were represented by the most similar Rosetta atom type, their coordinates were re-centered to the origin, and their partial charges were assigned by OpenEye's AMI-BCC implementation. The ligand perturbation ensemble near the crystal conformation (CSD set) or starting conformation (FC set) of each was then generated. For each rotatable bond of the ligand, small degree torsion angle deviation) (+/−5° was applied. K-mean clustering method was used to generate the ligand perturbation ensemble and similar/redundant conformation (rmsd to the selected conformation is less than 0.5 Å) was omitted. Finally, up to 100 conformations for each ligand were generated, and ready for Rosetta LigandDock.
Step III. Rosetta LigandDock with Additional Near “Native” Perturbation Sampling
We developed a general approach for docking a large library of commercial compounds onto the flat surface of the amyloid fiber. Starting from the template of the 16KLVFFA21 (SEQ ID NO: 1)/Orange-G structure described above, we computationally identified small molecule inhibitors that bind the side of the 16KLVFFA21 (SEQ ID NO: 1) fiber.
The docking algorithm is similar to the method previously described in the RosettaLigand docking paper (J Mol. Biol. 2009 Jan. 16; 385(2):381-92. Epub 2008 Nov. 18.), following the same three stages: coarse-grained stage, Monte Carlo minimization (MCM) stage and gradient-based minimization stage. The original RosettaLigand method performed a full sampling of the ligand internal and protein side-chain degrees of freedom in. In order to enable the fast run time required by any screening method, we sampled the ligand and protein side-chain torsion angles in near-“native” perturbation fashion, where only the near-“native” conformation of side-chain and ligand rotamers were allowed and any conformation far away from the starting conformation were omitted. For each protein side-chain, the deviations (+/−0.33, 0.67, 1 sd) around each input torsion was applied based on the standard deviation value of the same torsion bin from the backbone-dependent Dunbrack rotamer library. For each internal torsion of the ligand, the deviations) (+−5° around the input torsion was applied as described above. This near-“native” perturbation sampling makes count for both high-resolution finer sampling around the starting conformation and fast speed required by screening a large library.
A summary of the method of structure-based selection/identification of small compound inhibitors of Aβ is shown in
We tested candidate compounds by the MTT-based cell proliferation/viability assay, as described on the ATCC web site. Briefly, the ATCC MTT Cell Proliferation Assay quantitates the reduction of the yellow tetrazolium salt (MTT) in response to an external factor, such as treatment with a compound of the present invention, as a measure of a cell population's response to the external factor. The assay measures the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. In our tests, Hela and PC12 cell lines were used to assess the toxic effect of Abeta protein. Abeta at 0.5 μM was a positive control. The small molecule inhibitors were added to samples with different concentrations (such as 2.5 μM). After 12 h incubation at room temperature, the absorbance of reduced MIT was measured at 570 nm. Each of the experiments was repeated 3 times with 4 replicates per sample per concentration. Our MTT cell viability assay quantified the percentage of survival cells upon the treatment of the mixture of Abeta and compound inhibitors. The rescuing percentage of each compound was calculated by normalizing the survival percentage using the buffer-treated cell as 100% viability and Abeta-treated cell as 0% viability.
These studies support the proposed model shown in
Nine compounds were shown to inhibit Aβ toxicity. These compounds are listed in Table 7. Of these, seven compounds have never been reported to inhibit Aβ toxicity. The structures of these seven compounds are shown in
We studied derivatives/homologs of the compounds we determined to be active. These derivatives are listed in Table 8.
The results of activity studies of some representative compounds, BAF11 and BAF30, and the active derivatives thereof are shown in
The geometries defined in this amyloid pharmacophore are highlighted in
The defined interactions and geometries are:
1) H-bond acceptor (or negative charge) of the inhibitor should make either a hydrogen bond or a salt bridge to the sidechain nitrogen atoms (NZ) of at least two Lysine 16 redidues from adjacent Abeta sheets. Our data suggest that the active inhibitors should bind across 2 to 4 adjacent Abeta strands.
2) The hydrogen bond or salt bridge described in 1) should follow the general rule of H-bond geometry. They are:
As a validation of our computational approach, we used nuclear magnetic resonance (NMR) to characterize the interactions between our BAF compounds and KLVFFA (SEQ ID NO: 1) and Aβ fibers. First the 1H NMR spectra for two representative compounds (BAF1 & BAF8) and the binder molecule Orange-G were collected in the presence of increasing concentrations of KLVFFA (SEQ ID NO: 1) fibers. By monitoring the BAF1 compound peak area over a range of KLVFFA (SEQ ID NO: 1) fiber concentrations, we estimate the apparent dissociation constant (Kd) value of the interaction of BAF1 with KLVFFA (SEQ ID NO: 1) fibers to be ˜12 μM. We also performed an NMR titration experiment for BAF8 and obtained a binding affinity Kd of ˜24 μM. Since our computational approach identified candidate molecules that have a stronger predicted binding affinity than that of our template molecule Orange-G, we then measured the apparent Kd of Orange-G (˜43 μM). The weak binding affinity of Orange-G confirmed the success of our computational approach.
NMR samples contained 550 μL of designed compounds were added from 1 mM stocks in H2O to a final concentration of 100 μM. Fibrillar KLVFFA (SEQ ID NO: 1) and Abeta were added at the indicated concentrations. 500 MHz 1H NMR spectra were collected on a Bruker DRX500 at 283 K. H2O resonance was suppressed through presaturation. Spectra were processed with XWINNMR 3.6.
We used NMR spectroscopy to validate the direct binding of designed compounds to Abeta fibers. Moreover, Electron Microscope (EM) studies showed those designed compounds cannot inhibit fibrillation of Abeta. Those compounds showed inhibition of the Abeta toxicity in mu cell viability assays. The ability of their derivative/variant molecules to reduce Abeta toxicity correlated well with our structural models (crystal structure and docked models), and those data of cell viability allow us to derive the precise model of an “amyloid pharmocophore”. Supporting our hypothesis, our results showed that the designed compounds bind to amyloid proteins and greatly inhibit amyloid toxicity, indicating that these agents will likely be effective therapeutic and/or diagnostic agents for amyloid disease.
b
b
c
amolecule weight (anhydrous basis) excluding the salt and water molecules
b with the standard of NCI free compound library
c analytical data for AldrichCPR products are not available
drescue percentage is a scaled cell survival rate
eentry code for the ZINC database (http: //zinc.docking.org)
indicates data missing or illegible when filed
aNational Cancer Institute (NCI) free compound library (http://dtp.nci.nih.gov/)
b The toxicity results of BAF34 were not consistent for several independent replica experiments, which can be due to the possible impurity and the large molecule weight of the compound.
cZINC entry of the compound is not applicable, and the catalog number from Sigma-Aldrich is provided.
Compounds of the invention that are shown to protect human cells in vitro from the toxic effects of Abeta and/or tau will be tested in art-recognized animal models, including in D. melanogaster, C. elegans, and mice. Examples of transgenic animals constructed to exhibit amyloid disease include the Drosophila flies; mice produced by Jackson et al. (A Genomic Screen for Modifiers of Tauopathy Identifies Puromycin-Sensitive Aminopeptidase as an Inhibitor of Tau-Induced Neurodegeneration (2006) Neuron 51, 549-560); and the Abeta expressing mice of G. Cole et al. (Science, 274, 99-102 (1996)). Other suitable models will also be evident to skilled workers.
Compounds will be administered to the test animals by conventional methods, depending on the nature of the compounds, e.g. by adding them to the animals' food, injecting intravenously, or administering (pumping) directly into the spinal column or brain. The animals will be monitored for the effect of the compounds on suitable characteristics of the amyloid disease, compared to suitable controls. Details of such protocols are standard in the art, and will be well-known to those of skill in the art.
It is expected that the compounds being tested will elicit a reduction of symptoms or manifestations of the disease or condition.
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications (including U.S. provisional application 61/507,810, filed Jul. 14, 2010), particularly with regard to the specific disclosure for which they are referenced herein cited above, and in the figures, are hereby incorporated in their entirety by reference.
Abeta is a cleavage product of the precursor protein APP with UniProt accession code P05067 (A4_HUMAN). Among the best-studied peptide products are Abeta-40 and Abeta-42. A skilled worker will know the sequence of a variety of forms of Abeta that are suitable for use in the present invention. A representative sequence, of Abeta-42, is referred to herein as SEQ ID NO:21:
The sequence of the human tau protein (SEQ ID NO:22), is according to the document isoform of tau with the UniProt accession code P10636 (TAU_HUMAN). This sequence is referred to herein as SEQ ID NO:22:
This application claims the benefit of the filing date of U.S. Provisional Application 61/507,810, filed Jul. 14, 2011, which is incorporated by reference in its entirety herein
This invention was made with Government support under Grants No. AG029430 and AG016570, awarded by the National Institutes of Health. The Government has certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2012/046945 | 7/16/2012 | WO | 00 | 1/14/2014 |
| Number | Date | Country | |
|---|---|---|---|
| 61507810 | Jul 2011 | US |
