Patent Application
20240116984
This application is filed with a Computer Readable Form of a Sequence Listing in accord with 37 C.F.R. § 1.821(c). The text file submitted by EFS, “092012-9146-WO01_sequence_listing_20-APR-2022_ST25.txt,” was created on Apr. 20, 2022, contains 22 sequences, has a file size of 27.3 Kbytes, and is hereby incorporated by reference in its entirety.
Described herein are N-amino peptides (NAPs) that inhibit disease-associated tau aggregation and prevent fibril formation. The NAPs are derived from the R2 and R3 domains of tau (VQIINK and VQIVYK, respectively) wherein the amide moiety is N-aminated. N-amination of the R2 and R3 domains of tau results in formation of soluble mimics of ordered β-strands that are aggregation resistant and can assemble into layered parallel β-sheets.
The higher-order assembly of proteins rich in β structure is correlated with poor prognosis in several neurodegenerative diseases. Intracellular accumulation of the tau protein into neurofibrillary tangles (NFTs) is linked to cognitive dysfunction in over 20 disorders collectively termed “tauopathies.” The normal function of tau is to stabilize microtubules (MTs), the support structures in axons. Pathogenic misfolding and aggregation of tau can be caused by mutations in the MAPT gene or by aberrant post-translational modifications. While toxicity has been associated with various forms of aggregated tau, current data supports oligomeric species as a primary driver of neuronal death. It is now accepted that tau pathology becomes self-perpetuating, with the capacity to spread from neuron to neuron and cause normal tau to become misfolded (
Tau is an intrinsically disordered protein harboring up to four MT-binding repeat domains (R1-R4) in the C-terminal half. See e.g., NCBI Reference Sequence No. NP_005901.2 and SEQ ID NO: 1-2 for the human tau isoform 2 (ON4R) wild type nucleotide and polypeptide sequences, respectively. Like many amyloidogenic proteins, tau fibrillization involves conformational reorganization into β-rich folds, followed by supramolecular assembly into layered parallel β-sheets (
Despite examples of peptidomimetic disruptors of β-sheet-mediated protein interactions, strategies to translate conformationally extended peptide leads into inhibitors remain limited. This is due in part to the inherent flexibility of short peptide sequences, coupled with the large surface areas and diverse modes of β-sheet interactions. The propensity for conformationally extended peptides to aggregate via exposed H-bonding edges presents another significant challenge in the design of soluble β-strand mimics. Presentation of a β-strand epitope for protein recognition typically relies on the templating effect of an auxiliary β-strand (as in linear and macrocyclic β-hairpins), intra-strand conformational restriction through covalent tethering, or backbone amide N-alkylation to preclude strand self-aggregation. While backbone amide substitution allows for retention of side chain information, N-methylation (or incorporation of Pro) can promote main chain torsions incompatible with β-sheet mimicry. An approach for β-strand stabilization was recently described based on peptide backbone N-amination (
What is needed is an approach to target β-rich amyloids by inhibiting disease-associated tau aggregation and preventing fibril formation.
One embodiment described herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof,
In another aspect, X1 is
In another aspect, R1 is —NHR7 and R2, R3, R4, R5, and R6 are each hydrogen. In another aspect, R3 is —NHR7 and R1, R2, R4, R5, and R6 are each hydrogen. In another aspect, R4 is —NHR7, and R1, R2, R3, R5, and R6 are each hydrogen. In another aspect, R5 is —NHR7, and R1, R2, R3, R4, and R6 are each hydrogen. In another aspect, R6 is —NHR7, and R1, R2, R3, R4, and R5 are each hydrogen. In another aspect, R1 and R3 are each —NHR7, and R2, R4, R5, and R6 are each hydrogen. In another aspect, R1 and R5 are each —NHR7, and R2, R3, R4, and R6 are each hydrogen. In another aspect, R3 and R5 are each —NHR7, and R1, R2, R4, and R6 are each hydrogen. In another aspect, R4 and R6 are each —NHR7, and R1, R2, R3, and R5 are each hydrogen. The compound of any one of clauses 1-7, or a pharmaceutically acceptable salt thereof, wherein R1, R3, and R5 are each —NHR7, and R2, R4, and R6 are each hydrogen. In another aspect, the compound is a compound of formula (I-a),
In another aspect, the compound is selected from:
In another aspect, the compound is stable in human blood, serum, plasma, or cerebrospinal fluid. In another aspect, the compound is non-toxic to human neuronal cells
Another embodiment described herein is a method for inhibiting tau protein fibrillization or aggregation, the method comprising contacting tau protein with one or more compounds described herein. In one aspect, the compounds comprise one or more of compounds 1-14 (SEQ ID NO: 7-20). In another aspect, the compounds comprise one or more of compounds 12 or 13 (SEQ ID NO: 18 or 19). In another aspect, the compounds have a concentration of at least 2-fold molar excess over the tau protein's concentration.
Another embodiment described herein is a method for preventing cellular transmission of neurofibrillary tangles (NFTs), the method comprising contacting cells containing NFTs with one or more compounds of the compounds described herein. In one aspect, the compounds comprise one or more of Compounds 1-14 (SEQ ID NO: 7-20). In another aspect, the compounds comprise one or more of Compounds 12 or 13 (SEQ ID NO: 18 or 19). In another aspect, the compounds have a concentration of about 2-5 μM.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.
As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “˜” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.
As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.
As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.
As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non-human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.
As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.
The spread of neurofibrillary tangles resulting from tau protein aggregation is a hallmark of Alzheimer's and related neurodegenerative diseases. Early oligomerization of tau involves conformational reorganization into parallel β-sheet structures and supramolecular assembly into toxic fibrils. Despite the need for selective inhibitors of tau propagation, β-rich protein assemblies are inherently difficult to target with small molecules.
Described herein is a minimalist approach to mimic the aggregation-prone modules within tau. A backbone residue scan was carried out and showed that amide N-amination completely abolishes the tendency of these peptides to self-aggregate, rendering them soluble mimics of ordered β-strands from the tau R2 and R3 domains. Several N-amino peptides (NAPs) inhibit disease-associated tau aggregation and prevent fibril formation in vitro. NAPs 12 and 13 are further demonstrated to be effective at blocking the cellular seeding of endogenous tau by both monomeric and fibrillar forms of extracellular tau. Peptidomimetic 12 is serum stable, non-toxic to neuronal cells, and selectivity inhibits the aggregation of tau over Aβ42. Structural analysis of lead NAPs shows considerable conformational constraint imposed by the N-amino groups. The enhanced rigidity and full complement of sidechains within NAPs thus enables tau fibril recognition. The described backbone N-amination approach thus provides a rational basis for the mimicry of other aggregation-prone peptides that drive pathogenic protein assembly.
One embodiment described herein is a compound of formula (I), or a pharmaceutically acceptable salt thereof,
In another aspect, X1 is
In another aspect, R1 is —NHR7 and R2, R3, R4, R5, and R6 are each hydrogen. In another aspect, R3 is —NHR7 and R1, R2, R4, R5, and R6 are each hydrogen. In another aspect, R4 is —NHR7, and R1, R2, R3, R5, and R6 are each hydrogen. In another aspect, R5 is —NHR7, and R1, R2, R3, R4, and R6 are each hydrogen. In another aspect, R6 is —NHR7, and R1, R2, R3, R4, and R5 are each hydrogen. In another aspect, R1 and R3 are each —NHR7, and R2, R4, R5, and R6 are each hydrogen. In another aspect, R1 and R5 are each —NHR7, and R2, R3, R4, and R6 are each hydrogen. In another aspect, R3 and R5 are each —NHR7, and R1, R2, R4, and R6 are each hydrogen. In another aspect, R4 and R6 are each —NHR7, and R1, R2, R3, and R5 are each hydrogen. The compound of any one of clauses 1-7, or a pharmaceutically acceptable salt thereof, wherein R1, R3, and R5 are each —NHR7, and R2, R4, and R6 are each hydrogen. In another aspect, the compound is a compound of formula (I-a),
In another aspect, the compound is selected from:
In another aspect, the compound is stable in human blood, serum, plasma, or cerebrospinal fluid. In another aspect, the compound is non-toxic to human neuronal cells
Another embodiment described herein is a method for inhibiting tau protein fibrillization or aggregation, the method comprising contacting tau protein with one or more compounds described herein. In one aspect, the compounds comprise one or more of compounds 1-14 (SEQ ID NO: 7-20). In another aspect, the compounds comprise one or more of compounds 12 or 13 (SEQ ID NO: 18 or 19). In another aspect, the compounds have a concentration of at least 2-fold molar excess over the tau protein's concentration.
Another embodiment described herein is a method for preventing cellular transmission of neurofibrillary tangles (NFTs), the method comprising contacting cells containing NFTs with one or more compounds of the compounds described herein. In one aspect, the compounds comprise one or more of Compounds 1-14 (SEQ ID NO: 7-20). In another aspect, the compounds comprise one or more of Compounds 12 or 13 (SEQ ID NO: 18 or 19). In another aspect, the compounds have a concentration of about 2-5 μM.
Fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 7-20 can be (i) ones in which one or more of the amino acid residues (e.g., 1, 2, 3, 4, 5, or 6 residues, or even more) are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). Such substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) ones in which one or more of the amino acid residues includes a substituent group (e.g., 1, 2, 3, 4, 5, or 6 residues or even more), or (iii) ones in which the mature polypeptide is fused with another polypeptide or compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) ones in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives, and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
In addition, fragments, derivatives, or analogs of the polypeptides of SEQ ID NO: 7-20 can be substituted with one or more conserved or non-conserved amino acid residue (preferably a conserved amino acid residue). In some cases these polypeptides, fragments, derivatives, or analogs thereof will have a polypeptide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the polypeptide sequence shown in SEQ ID NO: 7-20 and will comprise functional or non-functional proteins or enzymes. Similarly, additions or deletions to the polypeptides can be made either at the N- or C-termini or within non-conserved regions of the polypeptide (which are assumed to be non-critical because they have not been photogenically conserved).
As described herein, in many cases the amino acid substitutions, mutations, additions, or deletions are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein or additions or deletions to the N- or C-termini. Of course, the number of amino acid substitutions, additions, or deletions a skilled artisan would make depends on many factors, including those described herein. Generally, the number of substitutions, additions, or deletions for any given polypeptide will not be more than about 4, 3, 2, or 1.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
Various embodiments and aspects of the inventions described herein are summarized by the following clauses:
Unless stated otherwise, reactions were performed in flame-dried glassware under a positive pressure of argon or nitrogen gas using dry solvents. Commercial grade reagents and solvents were used without further purification except where noted. Anhydrous solvents were purchased directly from chemical suppliers. Thin-layer chromatography (TLC) was performed using silica gel 60 F254 pre-coated plates (0.25 mm). Flash chromatography was performed using silica gel (60 μm particle size). Reaction progress was judged by TLC analysis (single spot/two solvent systems) using a UV lamp, CAM (ceric ammonium molybdate), ninhydrin, or basic KMnO4 stain(s) for detection purposes. NMR spectra were recorded on a 500 or 800 MHz spectrometer. Proton chemical shifts are reported as δ values relative to residual signals from deuterated solvents (CDCl3, DMSO-d6, D2O).
A general scheme for the preparation of substituted oxaziridines is shown in Scheme 1.
To a solution of the amino benzyl ester (HCl salt, 1.0 equiv.) in a biphasic mixture of THF and sat. aq. NaHCO3 (1:1), 2-(tert-butyl)-3,3-diethyl-1,2-oxaziridine-2,3,3-tricarboxylate (1.1 equiv.) was added and the reaction mixture was allowed to stir at rt for 4 h. The reaction was diluted with EtOAc, and the aqueous layer drained. The organic layer was washed with additional water, then dried over anhydrous Na2SO4, filtered, and concentrated. Purification by flash chromatography over silica gel (15-50% EtOAc/hexanes) afforded the hydrazino ester as a clear oil (75-95% yield).
A general scheme for the preparation of moc N—NHR7 dipeptides from α-hydrazino ester intermediates is shown in Scheme 2.
A solution of Fmoc amino acid (1.2 equiv.) in DCM was treated with 1-chloro-N,N,2-trimethyl-1-propenylamine (1.6 equiv.) and stirred for 10 min. The solution was then transferred into a flask containing a mixture of the hydrazino ester above (1 equiv.) and NaHCO3 (3 equiv.) in DCM. The reaction was stirred for 6 h and quenched with water. The organic layer was collected, and the aq. phase extracted with additional DCM. The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. Purification by silica gel flash chromatography (15-75% EtOAc/hexanes) afforded the protected N-amino dipeptide as an off-white solid (73-82% yield).
To a solution of the protected N-amino dipeptide above (1.0 equiv.) in EtOAc was added 10% Pd/C (150 mg/mmol) and the mixture stirred under H2 atmosphere at rt for 6 h. The reaction was diluted with additional EtOAc, filtered through Celite, and concentrated. Purification by flash chromatography (25-100% EtOAc/hexanes) afforded the aminated carboxylic acid as a white solid (68-94%).
Exemplary schemes are shown in Schemes 3 and 4.
Obtained as a white solid (54% yield over 3 steps); 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 8.60 (s, 1H), 8.19 (s, 1H), 7.76 (d, J=7.5 Hz, 2H), 7.66-7.49 (m, 2H), 7.44-7.37 (m, 2H), 7.33-7.29 (m, 2H), 5.62-5.42 (m, 0.8H), 5.16 (m, 0.2H), 4.78-4.61 (m, 0.4H), 4.55-4.14 (m, 4.6H), 2.50-2.27 (m, 0.4H), 2.09-1.86 (m, 1.6H), 1.71-1.60 (m, 1H), 1.58-1.47 (m, 9H), 1.23-0.84 (m, 13H); 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 175.8, 175.7, 172.6, 171.5, 158.3, 157.5, 156.1, 155.7, 144.0, 143.6, 143.5, 141.3, 127.9, 127.8, 127.7, 127.1, 125.3, 125.2, 125.1, 125.0, 120.1, 120.0, 120.0, 84.7, 84.6, 77.4, 67.8, 67.6, 67.0, 64.1, 55.9, 54.8, 47.3, 46.9, 38.5, 38.0, 35.2, 28.3, 28.2, 27.9, 26.8, 24.6, 23.9, 23.6, 21.1, 20.1, 19.2, 19.1, 16.1, 15.8, 11.7, 10.5; HRMS (ESI-TOF) m/z [M+H]+ calculated for C31H42N3O7 569.3096, found 569.3121.
Obtained as a white solid (47% yield over 3 steps); 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 8.52 (s, 0.67H), 8.07 (s, 0.13H), 7.76 (d, J=7.5 Hz, 2H), 7.65-7.50 (m, 2H), 7.45-7.37 (m, 2H), 7.35-7.28 (m, 2H), 5.57-5.43 (m, 0.9H), 5.24 (s, 0.1H), 4.83-4.64 (m, 0.25H), 4.60 (d, J=8.0 Hz, 0.75H), 4.49-4.27 (m, 3H), 4.26-4.10 (m, 2H), 2.07 (bs, 0.25H), 1.99-1.86 (m, 0.75H), 1.79-1.61 (m, 2H), 1.58-1.37 (m, 10H), 1.28-1.15 (m, 2H), 1.09-0.84 (m, 13H); 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 175.6, 175.2, 173.9, 172.8, 171.3, 158.2, 157.4, 156.0, 155.5, 154.2, 143.8, 143.4, 143.3, 141.2, 127.8, 127.6, 127.6, 127.5, 127.1, 127.0, 125.2, 125.1, 125.0, 124.9, 124.9, 120.0, 119.9, 119.9, 119.8, 84.5, 82.5, 67.7, 66.9, 66.1, 62.9, 62.6, 55.8, 55.3, 54.6, 47.1, 46.8, 38.5, 37.9, 35.2, 35.1, 35.0, 33.0, 28.0, 27.7, 27.2, 26.8, 26.4, 24.5, 23.7, 23.5, 15.9, 15.6, 15.4, 12.1, 11.7, 11.5, 11.2, 10.3; HRMS (ESI-TOF) m/z [M+H]+ calculated for C32H44N3O7 583.3252, found 583.3255.
Obtained as a white solid (42% yield over 3 steps); 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 8.27 (bs, 0.2H), 8.00 (bs, 0.4H), 7.80-7.70 (m, 2H), 7.65-7.52 (m, 2H), 7.39 (q, J=7.5 Hz, 2H), 7.34-7.10 (m, 17H), 6.91 (bs, 0.5H), 6.73 (bs, 0.3H), 5.99-5.87 (m, 0.25H), 5.77 (d, J=8.0 Hz, 0.6H), 4.93-4.86 (m, 0.2H), 4.80-4.51 (m, 2H), 4.45-4.30 (m, 2H), 4.27-4.17 (m, 1H), 2.49-2.03 (m, 3H), 1.99-1.58 (m, 3H), 1.51-1.09 (m, 10H), 1.01-0.80 (m, 6H); 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 175.2, 174.5, 172.5, 172.0, 171.8, 156.1, 156.0, 154.9, 154.4, 144.2, 144.1, 143.7, 141.3, 141.2, 128.6, 127.9, 127.6, 127.0, 125.2, 125.1, 119.9, 83.3, 82.1, 77.2, 70.9, 66.8, 63.0, 51.1, 47.2, 34.5, 34.4, 33.2, 28.9, 28.6, 28.1, 27.8, 26.6, 15.8, 15.6, 11.8, 11.6; HRMS (ESI-TOF) m/z [M+H]+ calculated for C50H55N4O8 840.4093, found 840.4117.
Obtained as a white solid (45% yield over 3 steps); 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 7.76-7.65 (m, 3H), 7.53-7.46 (m, 2H), 7.38-7.22 (m, 5H), 7.06-7.00 (m, 0.5H), 6.91 (m, 1.5H), 6.72 (d, J=8.0 Hz, 1H), 6.04 (bs, 0.3H), 5.35-5.11 (m, 1.5H), 4.41-4.05 (m, 4H), 3.99-3.89 (m, 1H), 3.34-3.19 (m, 1.5H), 2.81 (t, J=13.8 Hz, 0.5H), 2.04-1.91 (m, 0.5H), 1.70 (s, 0.5H), 1.51-1.34 (m, 9H), 1.31-1.19 (m, 4H), 1.11 (s, 6H), 0.93-0.79 (m, 6H); 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 175.5, 174.9, 172.7, 171.6, 157.9, 156.8, 155.9, 155.7, 154.1, 153.8, 143.8, 143.6, 143.4, 141.1, 131.8, 130.6, 130.5, 129.3, 128.5, 127.6, 126.9, 125.1, 124.3, 124.1, 119.8, 84.4, 84.2, 78.3, 67.3, 66.9, 62.4, 58.7, 55.9, 55.7, 47.0, 46.9, 46.8, 33.5, 32.6, 31.0, 29.5, 29.4, 28.6, 28.0, 27.4, 19.5, 18.0, 16.5; HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H48N3O8 675.3514, found 675.3531.
Obtained as a white solid (53% yield over 3 steps); 1H NMR (500 MHz, CDCl3, mixture of rotamers) δ 8.63 (bs, 0.3H), 7.77 (d, J=7.5 Hz, 2H), 7.63-7.50 (m, 2H), 7.41 (t, J=7.5 Hz, 2H), 7.32 (t, J=7.4 Hz, 2H), 7.14-6.76 (m, 5H), 5.69-5.42 (m, 0.9H), 5.25 (m, 0.1H), 4.98-4.54 (m, 3H), 4.41-4.14 (m, 3H), 3.23-2.80 (m, 2H), 1.98 (bs, 0.6H), 1.81-1.65 (m, 1.3H), 1.56-1.24 (m, 35H); 13C NMR (126 MHz, CDCl3, mixture of rotamers) δ 174.5, 173.4, 173.0, 171.9, 156.2, 155.2, 154.0, 143.8, 143.5, 141.1, 130.9, 129.9, 127.5, 126.9, 125.1, 124.1, 124.0, 119.8, 84.3, 84.0, 82.8, 80.7, 79.1, 78.4, 78.3, 77.2, 67.4, 66.8, 62.3, 59.6, 59.0, 58.4, 52.6, 52.0, 47.0, 46.8, 41.2, 40.2, 39.7, 38.5, 37.6, 36.6, 33.9, 31.8, 29.6, 29.3, 28.7, 28.3, 28.0, 27.8, 23.7, 23.4; HRMS (ESI-TOF) m/z [M+H]+ calculated for C44H59N4O10 804.4304, found 804.4329.
Solid-phase peptide synthesis was carried out using CEM-Liberty Blue peptide synthesizer on Fmoc-capped polystyrene rink amide MBHA resin (100-200 mesh, 0.05-0.15 mmol scale). The following amino acid derivatives suitable for Fmoc SPPS were used: Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Ile-OH. Dry resin was washed with DMF 3× and allowed to swell in DMF for 5 min at elevated temperature prior to use. All reactions were carried out using gentle agitation. Fmoc deprotection steps were carried out by treating the resin with a solution of 20% piperidine/DMF (5 min×2). Coupling of Fmoc-protected amino acids as well as Fmoc-(N′-Boc)-hydrazino dipeptide acids was conducted using 5 equiv. HCTU (0.5 M in DMF), 10 equiv. NMM (1.0 M in DMF), and 5 equiv. of the carboxylic acid in DMF at 50° C. (10 min). After each reaction the resin was washed with DMF 2×, DCM 2×. Peptides were cleaved from the resin by incubating with gentle stirring in 2 mL of 95:2.5:2.5 TFA:H2O:TIPS at rt for 2 h. The cleavage mixture was filtered, and the resin was rinsed with an additional 1 mL of cleavage solution. The filtrate was treated with 8 mL of cold Et2O to induce precipitation. The mixture was centrifuged, and the supernatant was removed. The remaining solid was washed 2 more times with Et2O and dried under vacuum. Peptides were analyzed and purified on C12 RP-HPLC columns (preparative: 4 μm, 90 Å, 250×21.2 mm; analytical: 4μ, 90 Å, 150×4.6 mm) using linear gradients of MeCN/H2O (with 0.1% formic acid), then lyophilized to afford white powders. All peptides were characterized by LCMS (ESI), HRMS (ESI-TOF), and 1H NMR. Analytical HPLC samples for all purified peptides were prepared as 1 mM in MeCN. Linear gradients of MeCN in H2O (0.1% formic acid) were run over 20 or 12 minutes and spectra are provided for λ=220 nm.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 81% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C34H64N11O9 770.4883, found 770.4877.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 45% yield. HRMS (ESI-TOF) m/z [M+H]*+ calculated for C34H64N11O9 770.4883, found 770.4878.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 75% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C34H65N12O9 770.4883, found 770.4879.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 81% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 785.4992, found 785.4992.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 88% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 805.4931, found 805.4930.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 55% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 805.4931, found 805.4925.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 88% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 805.4931, found 805.4927.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 38% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 805.4931, found 805.4927.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 99% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H65N10O9 805.4931, found 805.4933.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 52% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H66N11O9 820.5040, found 820.5045.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 58% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H66N11O9 820.5040, found 820.5041.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 44% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H66N11O9 820.5040, found 820.5048.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 42% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H66N11O9 820.5040, found 820.5039.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 28% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H67N12O9 835.5149, found 835.5115.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 92% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C38H63N9O9 790.4822, found 790.4833.
The crude peptide was purified by preparative scale RP-HPLC using a 5-95% MeCN/H2O gradient (with 0.1% formic acid). The pure peptide was obtained in 50% yield. HRMS (ESI-TOF) m/z [M+H]+ calculated for C34H63N10O9 755.4774, found 755.4775.
Purified peptides were dissolved in CDCl3 or DMSO-d6. Final peptide concentration was 1 mM, determined by mass. Data were collected at 25° C. on a 500 MHz Bruker ASCEND 11.74 T, narrow bore 54 mm, BOSS-3 36 shim system, BSMS shim and digital lock control units with a 5 mm direct detect SMART probe (1H/13C/15N with Z-axis PFG), or an 800 MHz AVANCE II with UltraStabilized and UltraShield 18.79 T, 54 mm bore, BOSS-2 34 shim system and a 5 mm broadband (BBO) 15N-31P, 1H decoupling, Z-axis PFG. The TOCSY used a mixing time of 80 ms, and the ROESY had a mixing time of 200 ms. In the F2 direction, the TOCSY and ROESY had 2048 complex points collected, and in the F1 direction, 512 complex points were collected. Watergate 3-9-19 was used for solvent suppression where appropriate. Bruker TopSpin 4.0 or Mestrenova 10.0 software was used to process the data, and Gaussian functions were used before Fourier transformation.
The following is a list of inter-residue correlations: Val1 NH—Ac CH3 (s); Gln2 NH-Val1 α (s); Gln2 NH-Val1 β (s); Gln2 NH-Val1 γ (m); aIle3 NH2-Gln2 α (s); aIle3 NH2-Gln2 β (w); Val4 NH2-aIle3 α (s); Val4 NH2-aIle3 γ (m); aTyr5 NH2-Val4 α (s); Tyr5 NH2-Val4 β (s); aTyr5 ε-aIle3 β CH3 (w); aTyr5 δ-aIle3 γ CH3 (m); aTyr5 ε-aIle3 γ CH3 (s); Lys6 NH-aTyr5 α (s); Lys6 NH-aTyr5 β (s); C-term NH2-Lys6 NH (w) C-term NH2-Lys6 α (s); and C-term NH2-Lys6 β (m) C-term NH2-Lys6 δ (w).
Ac-Val-Gln-aIle-Val-aTyr-Lys-NH2 (12, EG09) in D2O
The following is a list of inter-residue correlations: Val1 NH—Ac CH3 (s); Gln2 NH-Val1 α (s); Gln2 NH-Val1 γ (m); aIle3 NH2-Gln2 β (m); Val4 NH2-aIle3 α (s); Val4 NH2-aIle3 γ (w); Val4 NH2-aIle3 δ (m); aTyr5 NH2-Val4 β (s); aTyr5 δ-aIle3 δ (w); aTyr5 ε-aIle3 δ (m); Lys6 NH-aTyr5 α (s); Lys6 NH-aTyr5 β (m); C-term NH2-Lys6 α (s); and C-term NH2-Lys6 β (w).
Ac-Val-Gln-Ile-aVal-Tyr-aLys-NH2 (13, EG08) in DMSO-d6.
The following is a list of inter-residue correlations: Val1 NH—Ac CH3 (s); Gln2 NH-Val1 α (s); Gln2 NH-Val1 β (m); Gln2 NH-Val1 γ (w); Ile3 NH-Gln 2 α (s); aVal4 NH2-Ile3 α (s); aVal4 NH2-Ile3 β (w); aVal4 NH2-Ile3 γ (w); Tyr5 NH-aVal4 α (s); Tyr5 NH-aVal4 β (m); Tyr5 NH-aVal4 γ (m); Tyr5 δ-Ile3 γ (w); Tyr5 ε-Ile3 γ (w); Tyr5 ε-Ile3 δ (w); Tyr5 ε-aLys6 β (w); aLys6 NH2-Tyr5 α (s); aLys6 NH2-Tyr5 β (m); aLys6 NH2-Tyr5 δ (w); C-term NH2-aLys6 α (s); and C-term NH2-aLys6 β (w).
Ac-Val-Gln-Ile-aVal-Tyr-aLys-NH2 (13, EG08) in D2O
The following is a list of inter-residue correlations: Val1 NH—Ac CH3 (s); Gln2 NH-Val1 α (s); Ile3 NH-Gln2 α (s); Ile3 NH-Gln2 β (m); Ile3 NH-Gln2 γ (w); aVal4 NH2-Ile3 α (s); aVal4 NH2-Ile3 β (m); aVal4 NH2-Ile3 γ (w); aVal4 NH2-Ile3 δ (w); Tyr5 NH-aVal4 α (s); Tyr5 NH-aVal4 γ (m); Tyr5 δ-Ile3 γ CH3 (w); Tyr5 ε-Ile3 γ CH3 (w); Tyr5 δ-Ile3 δ (m); Tyr5 ε-Ile3 δ (m); aLys6 NH2-Tyr5 α (s); aLys6 NH2-Tyr5 β (m); and aLys6 NH2-Tyr5 δ (w).
1H NMR data for Compounds12 (EG09) and 13 (EG08) (δ values in ppm)
Human tauP301L (0N4R) (SEQ ID NO: 3-4) was cloned into pET28b with an N-terminal Hise tag. Briefly, transformed BL21(DE3) cells were grown in LB+kanamycin media at 37° C. until OD600 reached 0.8 and was then induced with 1 mM IPTG overnight at 16° C. Cells were then harvested, resuspended, and lysed by probe sonication in the lysis buffer containing 20 mM Tris, 500 mM NaCl, 10 mM imidazole, Roche cOmplete™ protease inhibitor cocktail, adjusted to pH 8.0. The lysate was then boiled for 20 minutes in a water bath and the debris was pelleted by centrifugation at 20,000×g for about 40 minutes 4° C. The supernatant obtained was then injected onto a 5 mL IMAC Ni-charged affinity column (Profinity™) and eluted over a gradient of 10-200 mM imidazole. Eluted tau-containing fractions were further purified and using GE HiPrep™ 16/60 Sephacryl™ S-200 high-resolution size exclusion chromatography into a storage buffer containing 20 mM Tris·HCl, 150 mM NaCl, and 1 mM DTT, adjusted to pH 7.6. The purity of the protein was confirmed was SDS-PAGE analysis (
Recombinant tauP301L (10 μM final concentration) and NAP inhibitors 20 μM final concentration) were mixed in an aggregation buffer (100 mM sodium acetate, 10 μM ThT, 10 μM heparin, 2 mM DTT, 0.5% DMSO, pH 7.4) in a 96-well clear bottom black plate with a final reaction volume of 200 μL. The plate was then sealed with a clear sealing film and allowed to incubate at 37° C. with continuous shaking in a Biotek Synergy H1 microplate reader. An automated method was used to carry out ThT fluorescence measurements at an excitation wavelength of 444 nm and an emission wavelength of 485 nm at an interval of every 5 minutes for 48 hours. Experiments were carried out in technical replicates on at least two different days. Every experiment included control wells that lacked tau301L, heparin, or NAPs. The average of tau-only (no heparin) wells was used to subtract background fluorescence and the average of the first and last 10 data points of tau+heparin wells, after blank subtraction, was used to normalize the data. All data plots were generated with SigmaPlot.
For analyzing fibrils by transmission electron microscopy (TEM), aggregation was carried out under conditions similar to the assay above but with ThT excluded and a final reaction volume of 100 μL Samples were incubated in a microcentrifuge tube for 4 days at 37° C. with a mixing speed of 100 rpm. A 10 μL aliquot of the sample was then applied to 400-meshed formvar/carbon-coated copper grids and negative stained with 2% uranyl-acetate. Micrographs were taken on a JEOL 2011 TEM at 200 kV.
TauP301L was diluted to a final concentration of 10 μM in an aggregation buffer containing 100 mM sodium acetate, 10 μM heparin, 2 mM DTT, pH 7.4. The protein was incubated in a microcentrifuge tube for 4 days at 37° C. with a shaking speed of 100 rpm. Control vials included those wherein (1) buffer was added in place of tauP301L, and (2) buffer was added in place of heparin.
HEK293 cells stably expressing tau-RD (LM)-YFP were cultured in DMEM media containing 10% FBS, 1% penicillin/streptomycin, and 1% Glutamax™ (Gibco) in a 75 cm2 cell culture flask under 5% CO2 at 37° C. For each experiment, cells were plated at a density of 15000 cells/well into 96 well tissue culture plates.
Monomeric tauP301L was co-incubated with NAPs for 4 days in an aggregation buffer at 37° C. (see above section). Following incubation, the reaction mixture was diluted in low serum Opti-MEM® media (Gibco), mixed with lipofectamine 2000 in 20:1 ratio (complex:lipofectamine) and allowed to incubate for an additional 20 minutes at RT. A mixture of 0.19 μM of Tau+1.9 μM or 0.009 μM of inhibitors (final concentrations) was added to the cells. Cells were incubated for additional 48 h before taking measurements on a BioTek Cytation 5 cell imager and microplate reader. 10×10 pictures/well were taken at 20× magnification under FITC channel and the punctate counting was carried out using built-in software. Each data set was collected from technical replicates on at least two different days. Every experiment included control wells (no tau, no heparin, and no NAP). All data plots were generated with SigmaPlot. Error bars shown are standard deviation from technical replicates.
TauP301L fibrils were prepared as described above (see section on fibril formation) and sonicated for 3 minutes prior to use in this assay. In a reaction volume of 40 μL, 8 μL of fibrils was diluted with 31 μL of low-serum Opti-MEM® (Gibco) media and then mixed with 1 μL of NAPs (DMSO concentration was constant across various concentration of inhibitors). The reaction mixture was then allowed to incubate at 37° C. for 36 h, then mixed with 2 μL of lipofectamine 2000 and further incubated for 20 minutes at R.T. A 10 μL aliquot of this mixture was then added into 90 μL of cells (15000 cells/well). Cells were incubated for additional 48 h before taking measurements on BioTek Cytation 5 cell imager and microplate reader. 10×10 pictures/well were taken at 20× magnification under FITC channel and the punctate counting were carried out using built-in software. Each data set were collected from technical replicates on at least two different days. Every experiment had Tau control well (no Tau but rest all), heparin control well (no heparin but rest all), and Tau alone well (no inhibitors but rest all). Every experiment included control wells (no tau, no heparin, and no NAP). All data plots were generated with Sigma plot. IC50 values were calculated by fitting the data set using sigmoidal logistic 4 parameter equation. Error bars represent standard deviation from technical replicates. % Tau infection was calculated using following formula:
The stability of NAPs in 25% human serum (Millipore Sigma) was assessed by HPLC. The reaction was started by adding NAPs at a final concentration of 500 μM in pre-warmed serum. The mixture was incubated at 37° C. for 24 h. A 100 μL aliquot of the reaction mixture was taken out at 0 h, 1 h, 4 h, and 24 h and was mixed with an equal volume of 20% TCA and incubated at 4° C. for 15 minutes to precipitate serum proteins. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and mixed with an internal standard (1 mg/mL of Cbz-Tyr-OH dissolved in MeCN) and stored at −20° C. Samples were then analyzed by LC-MS and the percentage of peptide remaining was calculated by integrating peaks.
MTT cell viability assays were carried out on both HEK293 cells stably expressing tau-RD (LM)-YFP and SH-SY5Y cells. Cells were cultured in DMEM/F12 complete media containing 10% FBS, 1% penicillin/streptomycin and 1% Glutamax™ (Gibco) in a 75 cm2 cell culture flask under 5% CO2 at 37° C. Cell viability was determined using MTT reduction assay. Briefly, 15,000 cells/well were plated in a 96 well tissue culture plate and were allowed to incubate overnight in a CO2 incubator. The media was aspirated, and the NAP inhibitor prepared in complete media was added at a given final concentration. The plate was then allowed to incubate for additional 48 h in a CO2 incubator and the media was aspirated again and replaced with 0.5 mg/mL of MTT prepared in complete media and incubated for additional 3 h. Media was then replaced with DMSO to dissolve formazan crystals and the absorbance was measured at 570 nm using Synergy H1 micro plate reader. Each data set were collected from technical replicates on at least two different days.
Simulated Annealing with NOE Distance Restraints
The simulated annealing protocol includes the following steps: (1) Structures of 12 (EG09; SEQ ID NO: 18) and 13 (EG08; SEQ ID NO: 19) were prepared using Maestro. (2) Each initial structure was first energy minimized in vacuum. (3) Next, beginning with the minimized structure, 100 replicas were generated with different initial velocities and each replica was heated from 300 K to 800 K in 100 ps and simulated at 800 K for another 100 ps. (4) After annealing, each replica was solvated. The dimensions of the box were chosen such that the distance between the walls of the box and any atom of the compound was at least 1.0 nm. Minimal counter ions were added to neutralize the net charge of the system. The entire system was then energy minimized using the steepest descent algorithm to remove any bad contacts. (5) Next, the system underwent a 500 ps NVT equilibration at 300 K. (6) Lastly, the system was annealed from 300 K to 500 K and then subsequently down to 5 K over 1 ns in an NPT ensemble (the temperature was increased from 300 K to 500 K in the first 100 ps, maintained at 500 K for 100 ps, decreased to 300 K in the following 500 ps, maintained at 300 K for 100 ps, and then decreased to 5 K in the last 200 ps). (7) After all the simulation steps, the final frames from each of the 100 trajectories were used for the analysis.
GROMACS 4.6.7 suite with the OPLS2005 force field with TIP4P water model was used for simulations. Throughout the simulated annealing protocol, NOE-derived distance restraints were applied to the compound with a force constant of 10,000 kJ·mol−1·nm−2. The temperature was regulated using a v-rescale thermostat, with a coupling time constant of 0.1 ps. The pressure was regulated using a Berendsen barostat, with a time coupling constant of 2.0 ps and isothermal compressibility of 4.5×10−5 bar−1. The leapfrog algorithm with an integration time step of 2 fs was used to evolve the dynamics of the system. The LINCS algorithm was used to constrain all bonds containing hydrogens to the equilibrium bond lengths. For simulations in vacuum, the cutoffs of all non-bonded (electrostatics and van der Waals) interaction were set to 999.0 nm and the neighbor list was only constructed once and never updated. For simulations in solvent, all non-bonded interactions as well as neighbor searching were truncated at 1.0 nm. Long-range electrostatics beyond the 1.0 nm were calculated using the particle mesh Ewald method with a Fourier spacing of 0.12 nm and an interpolation order of 4. To account for truncation of the Lennard-Jones interactions, a long-range analytic dispersion correction was applied to both energy and pressure.
Dihedral principal component analysis (dPCA) was performed on the backbone (ø, ψ) angles of residues V, Q, I, V, Y, K of 12 (EG09; SEQ ID NO: 18) and 13 (EG08; SEQ ID NO: 19). The first three principal components were used for further cluster analysis. The population for each cluster was calculated and the conformational entropy for each system was computed via the relation: S=−RΣipi ln pi, where pi is the population of cluster i, and R is the ideal gas constant. For 12 (EG09; SEQ ID NO: 18), 99 structures were grouped into 18 clusters. For 13 (EG08; SEQ ID NO: 19), 87 structures were grouped into 16 clusters.
Conventional molecular dynamics (MD) simulations were performed for AcPHF6 (EE02; SEQ ID NO: 22), 12 (EG09; SEQ ID NO: 18), and 13 (EG08; SEQ ID NO: 19). Initial structures were built using Maestro. The topology file for each compound was generated using the Schrödinger utility ffId_server and converted to the GROMACS format using the ffconv.py script. All MD simulations in this study were performed using the GROMACS 4.6.7 suite with the OPLS 2005 force field and the TIP4P water model. The initial structure was first energy minimized for 10,000 steps and then solvated in a cubic box of water molecules. The box size was chosen such that the distance between the compound and the box wall was at least 1.0 nm. Minimal explicit counter ions were also added to neutralize the net charge of the system. With all heavy atoms restrained, the solvated system was further energy minimized for 5,000 steps. With all the heavy atoms remained restrained to their initial coordinates, a 50-ps NVT equilibration at 300 K was performed, followed by a 50-ps NPT equilibration at 300 K and 1 bar to adjust the solvent density. Then, the position restraints on heavy atoms were removed. The system underwent a further equilibration process in the NVT ensemble for 100 ps, and in the NPT ensemble for 100 ps. The equilibrated system then underwent a 500 ns production run in the NPT ensemble at 300 K and 1 bar. In all the simulations, the temperature was regulated using the v-rescale thermostat with a coupling time constant of 0.1 ps. To avoid the “hot solvent/cold solute” artifacts, two separated thermostats were applied to the solvent (water and ions) and the compound. For the NPT simulations, the pressure was maintained using the isotropic Parrinello-Rahman barostat with a coupling time of 2.0 ps and compressibility of 4.5×10−5 bar−1. Bonds involving hydrogen were constrained using the LINCS algorithm. A 2-fs time step was used with the leapfrog integrator. The nonbonded interactions (Lennard-Jones and electrostatic) were truncated at 1.0 nm. Long-range electrostatic interactions were treated using the Particle Mesh Ewald summation method. A long-range analytic dispersion correction was applied to both the energy and pressure to account for the truncation of Lennard-Jones interactions. The last 400 ns of each production run was used for further analysis.
The 306VQIVYK311 hexapeptide motif (SEQ ID NO: 6) is widely accepted as the key amyloidogenic core of tau because filaments formed from this motif closely resemble those observed from Alzheimer's disease (AD) tau. However, recent crystal structures of the 275VQIINK280 motif (SEQ ID NO: 5) show tighter side chain packing and strand interdigitation relative to the R3 hexapeptide, suggesting it to be a more powerful driver of tau aggregation. Since the specific contribution of individual residues in these sequences have not yet been studied, a backbone N-amino scan was performed along the length of each hexapeptide. The NAP-based library included mono-, di-, and tri-N-aminated analogues. Poly-N-amino peptides were limited to those harboring amide substitutions on a single H-bonding edge, thus retaining a fully intact edge for interaction with tau.
Fourteen NAP β-strand mimics were synthesized on solid support as shown in
Thioflavin T (ThT), an amyloid specific fluorescent dye that binds to β-sheet assemblies, was chosen to first evaluate the effect of NAPs on recombinant tau aggregation. For these studies, full-length tau featuring a P301L mutation (
To confirm the effect of NAPs on tau fibril growth, transmission electron microscopy (TEM) was used to visualize the morphology and maturity of fibrillar species. Heparin-induced tauP301L fibrils were allowed to grow over 96 h in the presence or absence of inhibitors. Untreated tauP301L afforded large, helical, amyloid-like filamentous fibrils. In contrast, no elongated or mature fibrils were observed in the presence of a two-fold molar excess of the 6 NAP inhibitors above. Di-N-aminated peptides 4 (EG05; SEQ ID NO: 10), 12 (EG09; SEQ ID NO: 18), and 13 (EG08; SEQ ID NO: 19) were particularly effective at blocking fibrillization, resulting in non-fibrillary amorphous aggregates similar to control wells containing tauP301L without heparin (
Recent studies show that extracellular tau fibrils spread in a prion-like fashion from one cell to the next. This mode of propagation is important for the spread of NFTs, neuropil threads, and plaque-associated neurites-all of which contribute to the progression of AD. To test whether NAP inhibitors are able to block the seeding activity of recombinant tauP301L, HEK293 biosensor cells were employed that stably express a tau-yellow fluorescent protein fusion (tau-RD(LM)-YFP). When these cells were treated with preformed heparin-induced fibrils of tauP301L. a large number of intracellular tau aggregates were observed, as indicated by punctate fluorescence after 48 h. These wells exhibited a mean of 38% aggregate-containing cells over 3 separate experiments, demonstrating the ability for fibrillar tauP301L to enter cells and seed the aggregation of the endogenous tau-RD(LM)-YFP (
Given that pathogenic tau can be secreted from cells in various forms (as oligomers, aggregates, or mature fibrils), the ability of NAPs to cap pre-formed tau fibrils to block cellular transmission was tested. In this experiment NAPs were incubated with mature tauP301L fibrils for 36 h prior to treatment of cells expressing tau-RD(LM)-YFP. Indeed, compounds 12 (EG09; SEQ ID NO: 18) and 13 (EG08; SEQ ID NO: 19) were found to be able to effectively inhibit propagation in a dose-dependent manner. A fibril capping IC50 in cells in the 5 μM range across 3 repeated experiments was determined (
Consistent with the seeding experiments above using monomeric tauP301L, di-NAP 4 was generally ineffective at capping pre-formed fibrils and blocking propagation (
Compounds 12 (EG09; SEQ ID NO: 18) and 13 (EG08; SEQ ID NO: 19) feature two hydrazide bonds within the peptidomimetic backbone. Their utility as tau ligands in cell-based experiments would benefit from resistance to proteolytic degradation. Stability studies were carried out in human serum and degradation was monitored by RP-HPLC (
Cellular seeding experiments with tauP301L in the presence or absence of di-NAPs did not result in detectable toxicity to HEK293 biosensor cells (
Di-NAPs that cap mature tau fibrils are expected to adopt parallel-sheet-like conformations. The X-ray crystallographic structure of a model di-N-aminated tripeptide previously demonstrated its self-association as a dimeric species with extended backbone geometries. To gain insight into the solution structure of the lead tau ligands, 2D-NMR spectroscopy was carried out followed by simulated annealing. While AcPHF6 was insoluble in water, gCOSY, TOCSY, and ROESY NMR spectra in 9:1 H2O:D2O for compounds 12 (EG09; SEQ ID NO: 18) and 13 (EG08; SEQ ID NO: 19) were able to be obtained. NMR spectra in D2O were remarkably well resolved and devoid of significant minor rotamers despite the presence of two N-substituted amide bonds. Moreover, inter-residue NOEs were limited to correlations consistent with an extended solution conformation (iα→i+1NH). Though short linear peptides are expected to be highly flexible in solution, the absence of characteristic turn correlations suggests conformational restriction imparted by the N-amino groups. Distance-restrained simulated annealing and clustering based on backbone dihedral angles afforded ensembles of the three most populated conformers of compound 12 (EG09; SEQ ID NO: 18) (
Di-NAP 12 does not Inhibit Aβ42 Aggregation In Vitro
Many small molecule protein aggregation inhibitors exhibit undesired promiscuity. A peptidomimetic approach to tau inhibition offers prospects for achieving selectivity over other amyloids rich in β structure. As a preliminary test, the best-performing tau mimic, compound 12 (EG09; SEQ ID NO: 18), was selected and its effect on Aβ42 aggregation in vitro was determined (
Described herein is the design, synthesis, and biological evaluation of a novel class of β-strand mimics that block tau aggregation and propagation. Using an amide-to-hydrazide replacement strategy, a positional scan of aggregation-prone peptide sequences derived from the R2 and R3 domain of tau was carried out. Several NAP analogues inhibited the fibrillization of recombinant full-length tau as well as its seeding capacity in an in-cell aggregation assay. Key features of the described NAP inhibitors include increased conformational rigidity, resistance toward self-aggregation, and remarkable stability toward serum proteases. The most effective inhibitor of tau fibrillization and seeding showed no effect on the in vitro aggregation of Aβ42. Discrimination between structurally related β-rich assemblies is potentially enabled by NAPs, which exhibit a full complement of side chains in a minimalist single-strand design. In using the structure of tau to guide the design of its own inhibitors, this work sets the stage for the development of selective ligands of other pathogenic amyloids. Given that disease-associated conformational strains of tau are known to propagate in vivo with high fidelity, it is also expected that a NAP-based strategy can be used to target unique structural motifs within such polymorphs. The current study thus provides a rational basis for the design of soluble β-strand mimics with high levels of specificity.
This application claims priority to U.S. Provisional Patent Application No. 63/179,350, filed on Apr. 25, 2021, which is incorporated by reference herein in its entirety.
This invention was made with government support under grant number CHE 2021265 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/026110 | 4/25/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63179350 | Apr 2021 | US |
