WO2010054127A1

WO2010054127A1 - Methods and agents for stabilizing non-pathological amyloidogenic polypeptides

Info

Publication number: WO2010054127A1
Application number: PCT/US2009/063457
Authority: WO
Inventors: Gergely Toth; Christofer Lendel; Carlos W. Bertoncini; Nunilo Cremades; Michele Vendruscolo; Christopher M. Dobson; Dale B. Schenk; John Christodoulou
Original assignee: Elan Pharmaceuticals, Inc.; Cambridge Enterprise Limited
Priority date: 2008-11-05
Filing date: 2009-11-05
Publication date: 2010-05-14

Abstract

The disclosure relates to non-pathological forms of amyloidogenic polypeptides. The disclosure further relates to agents that preferentially bind and stabilize monomeric or prefibrillar non-pathological forms of amyloidogenic polypeptides. Further disclosed are biophysical methods, and particularly NMR- and fluorescence-based methods for screening for agents that bind and stabilize non-pathological forms of amyloidogenic polypeptides.

Description

Methods and Agents for Stabilizing Non-Pathological Amyloidogenic Polypeptides

PRIORITY CLAIM

Priority is claimed to U.S. Provisional Application No. 61/111,625, filed November 5, 2008, the entire contents of which are hereby incorporated by reference for all purposes.

TECHNICAL FIELD

The invention resides in the technical fields of drug discovery and medicine.

BACKGROUND OF THE INVENTION

The information provided below is not admitted to be prior art to the present invention, but is provided solely to assist the understanding of the reader.

A number of severe disorders, including Alzheimers's (AD), Parkinson's (PD), Huntington's (HD) and prion disease are linked to protein misfolding and aggregation (Chiti & Dobson 2006 Annu Rev Biochem 75:333-66). The mechanisms by which proteins of wide structural diversity are transformed into morphologically similar aggregates seem to be a generic property of the peptide backbone. Structural transformations into fibrillar assemblies have been observed for a range of globular proteins and in particular from intrinsically unstructured poly-peptides, like the Amyloid β (Aβ) peptide, protein Tau and α-synuclein (Uversky & Fink 2004 Biochim. Biophys. Acta. 1698:131-153). The characteristic cross β-sheet structure of the inclusions found in patients suffering from the disorders associated with these proteins is indeed similar to the structures of the aggregates induced by in vitro assays, which offers opportunities to characterize the details of the self-assembly processes at a molecular level.

Formation of intracellular aggregates containing the pre-synaptic protein α-synuclein is the hallmark of a group of neurodegenerative disorders called α-synucleinopathies, with the most well-known variant being PD (Uversky 2007 J. Neurochemistry 103:17; Lee & Trojanowski 2006 Neuron 52: 33-38). α-synculein is a 14 kDa intrinsically unstructured protein whose normal function is not yet well understood, but it's overexpression and aggregation are closely associated with the development of PD (Spillantini et al 1997 Nature, 388:839-840; Voiles & Lansbury 2003 Biochemistry 42:7871-7878). Upon interaction with phospholipid membranes it folds into a highly helical state (Chandra et al 2003 JBC 278: 15313-15318; Ulmer et al 2005 JBC 280: 9595-9603) and characterization of free unstructured protein, using NMR spectroscopy, has revealed the presence of local and long range interactions within the conformational ensemble (Eliezer et al 2001 JMB 307:1061-1073; Dedmond et. al. 2005 JACS 127:476; Bertoncini et al. 2005 PNAS 102:1430-1435). Furthermore, many aggregation promoting conditions, such as high ionic strength, low pH or elevated temperature, seem to induce structural changes in the protein and the aggregation pathway has been proposed to proceed via a partially folded intermediate (Uversky et al. 2001 JBC 276:10737-10744, Fink 2006 Ace. Chem. Res. 39:628-634).

The growing class of mis-folding diseases provide an extremely challenging task for drug discovery and even though a large number of small molecules have been shown to inhibit fibril formation in vitro, no approved therapeutic that blocks the formation of amyloid structures exists. Different inhibitors seem to affect different steps along the pathway of fibril formation, as illustrated for Aβ by the Necula and co-workers (Necula et al. 2007 J. Biol. Chem. 282: 10311). There is a lack of understanding of the degree of toxicity of the protein species along the aggregation pathways (Cookson et al 2006 Experimental Neurology, 199:238-242).

Applicants believe that an effective therapeutic strategy would keep the proteins in their native states and thus hinder the formation of all oligomeric species. In the case of globular proteins, partial unfolding has been found to be necessary as an initial step for aggregation in many systems and therefore stabilization of the globular fold would provide a possible avenue for aggregation inhibition. The situation is different in the case where the amyloidogenic protein is intrinsically unstructured, such as, for example, non-globular amyloidogenic polypeptides. The use of conventional techniques to identify a stabilizer of the native state is not as straight forward, particularly for small molecule stabilizers.

Amyloidosis is a general term that describes a number of diseases characterized by the existence of pathological forms of amyloid proteins, often involving multimeric aggregates of the proteins, and frequently extracellular deposition of protein fibrils, which form numerous "amyloid deposits" or "amyloid plaques," which may occur in local sites or systematically. These deposits or plaques are composed primarily of a naturally occurring soluble protein or peptide, assembled into extensive insoluble deposits 10-100 μm in diameter in a variety of tissue sites. The deposits are composed of generally lateral aggregates of fibrils that are approximately 10- 15 nm in diameter. Amyloid fibrils produce a characteristic apple green birefringence in polarized light, when stained with Congo Red dye. Generally, the fibrillar composition of these deposits is an identifying characteristic for the various forms of amyloid disease.

The peptides or proteins forming the plaque deposits are often produced from a larger precursor protein. More specifically, the pathogenesis of amyloid aggregates such as fibril deposits generally involves proteolytic cleavage of an "abnormal" precursor protein into fragments that aggregate into anti-parallel β pleated sheets.

The fibrillar composition of these deposits is an identifying characteristic for the various forms of amyloid disease. For example, intracerebral and cerebrovascular deposits composed primarily of fibrils of beta amyloid peptide (β-AP) are characteristic of Alzheimer's disease (both familial and sporadic forms), islet amyloid protein peptide (IAPP; amylin) is characteristic of the fibrils in pancreatic islet cell amyloid deposits associated with type II diabetes, and β2-microglobulin is a major component of amyloid deposits which form as a consequence of long term hemodialysis treatment. More recently, prion-associated diseases, such as Creutzfeld-Jacob disease, have also been recognized as amyloid diseases.

In general, primary amyloidoses of the disease are characterized by the presence of "amyloid light chain-type" (AL-type) protein fibrils, so named for the homology of the N-terminal region of the AL fibrils to the variable fragment of immunoglobulin light chain (kappa or lambda).

The various forms of disease have been divided into classes, mostly on the basis of whether the amyloidosis is associated with an underlying systematic illness. Thus, certain disorders are considered to be primary amyloidoses, in which there is no evidence for preexisting or coexisiting disease. In secondary or reactive (AA type) amyloidosis characterized by the presence deposition of amyloid protein A (AA) fibrils, there is an underlying or associated chronic inflammatory or infectious disease state. Heredofamilial amyloidoses may have associated neuropathic, renal, or cardiovascular deposits of the ATTR transthyretin type. Other heredofamilial amyloidoses include other syndromes and may have different amyloid components (e.g. , familial Mediterranean fever which is characterized by AA fibrils). Other forms of amyloidosis include local forms, characterized by focal, often tumor-like deposits that occur in isolated organs. Other amyloidoses are associated with aging, and are commonly characterized by plaque formation in the heart or brain. Also common are amyloid deposits associated with long term hemodialysis. These and other forms of amyloid disease are summarized in Table 1 (Tan, S.Y. and Pepys, Histopathology 25:403-414, 1994; Harrison's Handbook of Internal Medicine, 13^th Ed., Isselbacher, K. J., et al, eds, McGraw- Hill, San Francisco, 1995) and are described in U.S. Patent Nos. 6,875,434, 6,890,535, 6,913,745, 6,923,964, and 6,936,246, which are incorporated by reference herein in their entirety. Especially incorporated are disclosures relating to amyloidogenic polypeptides and proteins.

SUMMARY OF THE INVENTION

The present disclosure provides a method to identify an agent which stabilizes a non- pathological form of an amyloidogenic polypeptide, comprising: comparing an NMR spectrum of the amyloidogenic polypeptide in the presence and absence of at least one test agent; selecting one or more test agents for which, in their presence, the amyloidogenic polypeptide has an altered NMR spectrum; and comparing aggregation of the amyloidogenic polypeptide in the presence and absence of the selected test agent, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide.

The present disclosure provides a method to identify an agent which stabilizes a non- pathological form of an amyloidogenic polypeptide, comprising: providing an amyloidogenic polypeptide having a predetermined NMR spectrum; contacting the amyloidogenic polypeptide with at least one test agent; determining the resultant NMR spectrum of the contacted amyloidogenic polypeptide; selecting at least one test agent wherein the resultant NMR spectrum is different compared to the predetermined NMR spectrum; and evaluating the aggregation of the amyloidogenic polypeptide in the presence of the selected test agent, wherein a decrease in aggregation relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide.

In some methods, the amyloidogenic polypeptide is labeled with N. In some methods, each amide nitrogen of the amyloidogenic polypeptide is labeled with ¹⁵N.

In some methods, the amyloidogenic polypeptide is labeled with ¹³C. In some methods, each carboxyl carbon of the amyloidogenic polypeptide is labeled with ¹³C.

In some methods, the NMR spectrum is a 2D ¹H-¹⁵N heteronuclear single quantum correlation spectrum (HSQC).

In some methods, the NMR spectrum is a protonless 2D ¹³CO-¹⁵N heteronuclear NMR spectrum.

In some methods, the NMR spectrum is a pulsed field gradient NMR spectrum.

Some methods include the detection or measurement of the fluorescence of the sample. For example, a stabilization of a non-pathological form of the amyloidogenic polypeptide may perturb or quench the fluorescence observed with a pathological form of the amyloidogenic polypeptide. For example, the fluorescence may be the fibril-dependent fluorescence of thioflavin-T.

Some methods include the detection or measurement of the circular dichroism of the sample in the presence and/or absence of a test agent.

Some methods include the detection or measurement of optical rotory dispersion of the sample in the presence and/or absence of a test agent.

Some methods include the detection or measurement of dynamic light scattering of the sample in the presence and/or absence of a test agent.

Some methods include the detection or measurement of isothermal calorimetry of the sample in the presence and/or absence of a test agent. Some methods include the detection or measurement of an amyloidogenic protein to a test agent by surface plasmon resonance (SPR).

The amyloidogenic polypeptide may have at least one non-pathological form, which may, depending on the particular amyloidogenic polypeptide, be a monomeric, dimeric, trimeric, tetrameric or alternative non-pathological form. For example, non-pathological tetrameric forms of transthyretin, non-pathological monomeric forms of α-synuclein and non-pathological monomeric forms amyloid beta peptide exist. The at least one non-pathological form may be an intrinsically unstructured globular form. The non-pathological form may undergo a structural transformation into a pathological form, such as, for example, an oligomeric assembly or a fibrillar assembly. Some fibrillar forms may possess a cross β-sheet structure.

Non- limiting examples of amyloidogenic polypeptides include, but are not limited to, serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, apolipoprotein Al (ApoAl), transthyretin (TTR), lysozyme, fϊbrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β-APP), β₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, a peptide hormone, microtubule associated protein tau, huntingtin, and α-synuclein. Such amyloidogenic polypeptides also include mutant proteins, protein fragments and proteolytic peptide products of such proteins, such as, for example, those listed below in Table 1.

The present disclosure also provides a pharmaceutical composition comprising a pharmaceutically effective amount of at least one agent which stabilizes a non-pathological form of an amyloidogenic polypeptide, for example, a pharmaceutically effective amount of an agent that alters the NMR spectrum of the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide. Some pharmaceutical compositions comprise a pharmaceutically effective amount of an agent identified according to any of the methods described herein.

The present disclosure also provides a method of stabilizing a non-pathological form of an amyloidogenic polypeptide in a mammal, comprising administering a pharmaceutically effective amount of at least one agent to the mammal; wherein, the at least one agent alters the NMR spectrum of the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide. The present disclosure comprehends a mammal as including a human. In such embodiments, the pharmaceutical composition may be a drug for administration to a human as defined by the FDA and may require FDA approval.

The present disclosure comprehends a mammal as including a non-human animal. In such embodiments, the pharmaceutical composition may be a veterinary drug for administration to a non-human mammal.

Some methods of stabilizing the non-pathological form of an amyloidogenic polypeptide, comprise contacting the amyloidogenic polypeptide with an agent that alters the NMR spectrum characteristic for the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide relative to the NMR spectrum and aggregation observed for the amyloidogenic polypeptide in the absence of the agent.

The present disclosure also provides methods of treating or preventing or delaying the onset of diseases characterized by pathological oligomerization or deposition of amyloidogenic polypeptides. The disclosed methods comprise administering a pharmaceutically effective amount of an agent that stabilizes the non-pathological form of the amyloidogenic polypeptide, wherein the agent alters the NMR spectrum of the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide.

For example, some methods comprise treating, preventing or delaying the onset of Parkinson's disease in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non-pathological form of α-synuclein. In some such methods, the non-pathological monomeric form of α-synuclein is stabilized.

Some methods comprise treating, preventing or delaying the onset of Alzheimer's disease in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non- pathological form of amyloid beta peptide (Aβ). In some such methods, the non-pathological monomeric form of Aβ is stabilized.

Some methods comprise treating, preventing or delaying the onset of familial amyloid polyneuropathy, familial amyloid cardiomyopathy or systemic senile amyloidosis in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non-pathological form of ATTR. In some such methods, the non-pathological tetrameric form of transthyretin is stabilized.

Some methods comprise treating, preventing or delaying the onset of Alzheimer's disease in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non- pathological form of microtubule associated protein tau. In some such methods, the non- pathological monomeric form of tau is stabilized. In some such methods, the hyperphosphorylated form of tau is stabilized.

Some methods comprise treating, preventing or delaying the onset of familial Mediterranean fever, Muckle-Wells syndrome or reactive systemic amyloidosis associated with systemic inflammatory diseases in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non-pathological form of AA. In some such methods, the non-pathological monomeric form of AA is stabilized.

Some methods comprise treating, preventing or delaying the onset of myeloma or macroglobulinemia associated amyloidosis, systemic amyloidosis associated with immunocyte dyscrasia, monoclonal gammopathy, occult dyscrasia, or local nodular amyloidosis associated with chronic inflammatory diseases in a human by administering a pharmaceutically effective amount of an agent that stabilizes a non-pathological form of AL. In some such methods, the non-pathological monomeric form of AL is stabilized.

Other methods comprise treating, preventing or delaying the onset of other diseases listed in Table 1 by administering a pharmaceutically effective amount of an agent that stabilizes a non- pathological form of the amyloidogenic polypeptide associated with the disease in question.

In certain methods, the stabilizing agent may be a compound, for example, a compound having a molecular weight of less than 700 daltons, for example, small molecules having a molecular weight of about 150 to about 300 daltons.

In certain methods, the stabilizing agent may be a peptide.

In certain methods, the stabilizing agent may be an antibody, a monoclonal antibody, or an epitope-binding fragment of an antibody. In some methods, the stabilizing agent may be a nucleic acid molecule.

Certain methods provide for the manufacture of a pharmaceutical comprising providing a pharmaceutically acceptable excipient and a pharmaceutically effective amount of an agent that alters the NMR spectrum of the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide relative to the NMR spectrum and aggregation observed for the amyloidogenic polypeptide in the absence of the agent.

The present disclosure also provides for the use in the manufacture of a medicament of a pharmaceutically effective amount of an agent which stabilizes the non-pathological form of an amyloidogenic polypeptide.

Still other aspects and advantages of the present invention will become readily apparent by those skilled in the art from the following detailed description, wherein it is shown and described preferred embodiments of the invention, simply by way of illustration of the best mode contemplated of carrying out the invention. As will be realized the invention is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, without departing from the invention. Accordingly, the description is to be regarded as illustrative in nature and not as restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed description when read in connection with the accompanying drawing. Included in the drawing are the following figures:

Figure 1 depicts chemical structures of Congo red (A) and Lacmoid (B);

Figure 2 shows that Congo red and lacmoid interact differently with the N-terminus and NAC region of 0C-synuclein. Changes in amide chemical shifts and peak intensities in the α-synuclein ¹H-¹⁵N HSQC spectrum due to addition of Congo red or Lacmoid. A) Relative peak intensity with 2:1 (blue), 5:1 (red) and 10:1 (green) molar excess of Congo red. B) Relative peak intensity with 1 :1 (blue), 4:1 (red), 6:1 (green) and 16:1 (magenta) molar excess of Lacmoid. C) Chemical shift changes with 2:1 (blue), 5:1 (red) and 10:1 (green) molar excess of Congo red. D) Chemical shift changes with 1 :1 (blue), 4:1 (red), 6:1 (green) and 16:1 (magenta) molar excess of Lacmoid. The reported chemical shifts changes are weighted averages of the H and N chemical shift changes ([AO(¹H)² +(Δδ(¹⁵N)/5)² ]^1/2);

Figure 3 shows that Congo red binds to α-synuclein more strongly than does lacmoid. Determination of the affinity for the α-synuclein:small molecule complexes by fluorescence quenching of α-synuclein-conjugated IAEDANS . Titration of 5 μM AS-62C-AEDANS with Congo red (A) and 5 μM AS-24C-AEDANS with Lacmoid (C). Fitting of the fluorescent quenching to a single-site binding model yields relative affinities for the compounds. The calculated K_D is 1.05 ± 0.05 μM for Congo red (B) and 12 ± 2 μM for Lacmoid (B);

Figure 4 is a far-UV CD spectra showing that binding-ligands induce changes in the secondary structure of α-synuclein. Far-UV CD spectrum of 5 μM α-synculein at 10 ⁰C. A) No compound (diamonds), 10 μM Congo red (squares), 0.1 mM Congo red (triangles). B) No compound (diamonds), 5 μM Lacmoid (squares), 50 μM Lacmoid (triangles), 0.5 mM Lacmoid (circles);

Figure 5 depicts an in silco docking of small molecules (binding ligands) on the NMR-derived ensemble of the α-synuclein structure. A, B) Predicted binding mode of Spermidine bound to α- synuclein. C, D) Predicted binding mode of Congo red bound to α-synuclein. E, F) Predicted binding mode of Lacmoid bound to α-synuclein. The small molecules appear as magenta stick and protein as surface representation or blue stick. Residues of α-synuclein in surface representation are coloured accordingly their location in primary sequence. The figure was created using PyMOL (DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlo, CA);

Figure 6 demonstrates that Congo red and Lacmoid form supramolecular aggregates in solution. DLS derived size distributions of Congo red and Lacmoid at 25 ⁰C. A) Size distribution of 1 mM (solid line) and 0.1 mM (broken line) Congo red. B) Representative size distribution of 0.5 mM Lacmoid measured at 532 nm;

Figure 7 is a calorimetric characterization of the interactions between small molecules and α- synuclein. ITC raw data of Congo red and Lacmoid titrated into an α-synuclein solution and pure PBS buffer at 25 ⁰C. A, B) 0.2 mM Congo red titrated into buffer (A) and 2 μM α-synuclein (B). C, D) 1 mM Lacmoid titrated into buffer (C) or 100 μM α-synuclein (D); Figure 8 shows that Congo red and Lacmoid inhibit α-synuclein amyloid aggregation by different mechanisms. Aggregation assay of α-synuclein in the presence of various concentrations of small molecule compounds. A, B) ThioT fluorescence traces as function of time in absence (control) and presence of different concentrations of Congo red (A) and Lacmoid (B). C, D) ThioT fluorescence interference (quenching or competition) determined by incubating pre- formed amyloid fibrils for 30 minutes with different concentrations of Congo red (C) and Lacmoid (D). Results are expressed as relative to the fluorescence of the control (incubation with buffer). E) TEM images of the aggregates corresponding to the end products from the aggregation assays without (control) and with 10 μM Congo red and 5 μM Lacmoid;

Figure 9 Compares NMR spectra of α-synuclein in presence and absence of two binding ligands. A) ¹H-¹⁵N HSQC of 100 μM α-synuclein with (red) and without (blue) 10:1 molar excess of Congo red. B) ¹H-¹⁵N HSQC of 100 μM α-synuclein with (red) and without (blue) 16:1 molar excess of Lacmoid. C) ¹H-¹⁵N SOFAST-HMQC of 1 μM α-synuclein with (red) and without (blue) 4:1 molar excess of Congo red;

Figure 10 is a comparison of ¹H-¹⁵N HSQC (left) and ¹³C direct detected ¹³CO-¹⁵N (right) correlation spectra of 100 μM α-synuclein in the presence and absence of Congo red. Comparison Of ¹H-¹⁵N HSQC (left) and ¹³C direct detected ¹³CO-¹⁵N (right) correlation spectra of 100 μM α-synuclein with (red) and without (green) 9:1 molar excess of Congo red. Peak intensity ratios between bound and free protein are plotted;

Figure 11 presents a PFG-NMR analysis of α-synuclein in the presence of binding ligands. Data was fitted with a single Gaussian function and the rates of decay were converted into hydrodynamic radii. In the case of Congo red and Lacmoid we also fitted the data to a two species model, however this does not reduce residuals (shown in red);

Figure 12 presents a CD characterization of α-synuclein structural transitions. Far-UV CD spectrum of 5 μM α-synculein at 10 ⁰C (A, B), 25 ⁰C (C, D) and 37 ⁰C (E, F). A, C, E) No compound (blue), 10 μM Congo red (red), 0.1 mM Congo red (green). B, D, F) No compound (blue), 5 μM Lacmoid (red), 50 μM Lacmoid (green), 0.5 mM Lacmoid (magenta); Figure 13 correlates the radius of gyration (R_g), solvent accessible surface area (SASA), and the number of non-bonded contacts for 100 analyzed α-synuclein conformations. Correlation between the radius of gyration (R_g), solvent accessible surface area (SASA) and the number of non-bonded contacts. The gray circles represent the α-synuclein structures which were used for the docking calculations;

Figure 14 shows that CR and Lac exhibit different mechanisms of inhibition of αS amyloid formation as evident from aggregation assay of 100 μM αS in the presence of 0.005 to 10 molar equivalents of the compounds. ThioT fluorescence of the samples after 5 days of incubation in absence (control) and presence of varying amounts of CR (A) and Lac (B). ThioT fluorescence interference (quenching or competition) determined by incubating pre-formed amyloid fibrils for 30 minutes with CR (C) and Lac (D). The results are expressed as relative to the fluorescence of the control (incubation with buffer). SDS-PAGE of protein remaining soluble along the aggregation assays in absence (control) and presence of various concentration of CR (E) and Lac (F). G) TEM images of the aggregates corresponding to the end products from the aggregation assays without (control) and with CR or Lac;

Figure 15 is a calorimetric characterization of ligand-binding to α-synuclein. ITC raw data of 100 μM α-synuclein titrated into PBS buffer (A), 5 μM Congo red (B), 20 μM Congo red (C) and 50 μM Congo red (D), and ITC raw data of 0.1 mM Lacmoid titrated into PBS buffer (E) and 5 μM α-synuclein (F);

Figure 16 indicates that small molecules bound to α-synuclein shift the population distribution of monomeric α-synuclein conformations by directly interacting with the residues having observed chemical shifts;

Figure 17 illustrates chemical shift differences as a function of residue number between α- synuclein (AS) alone and α-synuclein (AS) with C14 derived from NMR ¹³CO-¹⁵N correlation spectra;

Figure 18 illustrates that Compound 14 may promote α-synuclein aggregation. The left panel shows an apparent inhibition in AS fibril formation in the presence of Compound 14 as determined by a plate reader assay. As determined by standard fluorimeter measure of ThT fluorescence, Compound 14 promotes AS aggregation in the presence (center) and absence (right) of SDS.

Figure 19 illustrates the dose dependent reduction of α-synuclein aggregation due to the presence of compound C4. The reduction of α-synuclein aggregation was detected by the decrease in Thioflavin T fluorescence;

Figure 20 illustrates chemical shift differences as a function of residue number between α- synuclein (AS) alone and α-synuclein (AS) with C4 derived from NMR CO- ³N correlation spectra;

Figure 21 : Test Compound (Compound 1) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window mσving average (red); (center) chemical shift perturbations (weighted ¹H and ¹⁵N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 22: Test Compound (Compound 2) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window mσving average (red); (center) chemical shift perturbations (weighted ¹H and ¹⁵N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 23 : Test Compound (Compound 3) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 24: Test Compound (Compound 5) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted ¹H and ¹⁵N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 25: Test Compound (Compound 10) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 26: Test Compound (Compound 11) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 27: Test Compound (Compound 14) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of test compound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 28: Test Compound (Compound 16) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of Test

Ccompound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted ¹H and ¹⁵N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 29: Test Compound (Compound 18) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of Test Ccompound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted ¹H and ¹⁵N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 30: Test Compound (Compound 19) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of Test Ccompound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 31 : Test Compound (Compound 21) binds to and inhibits α-synuclein aggregation. An aggregation plot (top) shows kinetic traces of thioflavin-T fluorescence in the presence of α- synuclein and the test compound (yellow and red) and a control in the absence of Test Ccompound (blue, average ± SD, n = 8). The ¹H-¹⁵N HSQC plots (bottom) depict: (left) the perturbation of peak intensities in the presence of Test Compound ± noise (blue) and 5 residue window moving average (red); (center) chemical shift perturbations (weighted H and N ± noise); and, (right) correlation of the chemical shift perturbation and signal to noise ratio for each peak.

Figure 32 presents a detailed analysis of the sequence-dependence of the chemical shift perturbations.

It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

DETAILED DESCRIPTION

Reference is made to the figures to illustrate selected embodiments and preferred modes of carrying out the invention. It is to be understood that the invention is not hereby limited to those aspects depicted in the figures.

A. Amyloidogenic polypeptides and Amyloid Diseases. Amyloidogenic polypeptides are herein defined as peptides and proteins of wide structural diversity which share the property of being transformed into pathological aggregates. This property may be a generic property of the peptide backbone. Structural transformations into pathological multimeric aggregates and fibrillar assemblies have been observed for a range of globular proteins and in particular from intrinsically unstructured poly-peptides. Such structural transformations are characteristic of amyloidogenic polypeptides. A characteristic of many transformed amyloidogenic polypeptides is the adoption of a cross β-sheet structure. Typical, but non-limiting, amyloidogenic polypeptides include serum amyloid A protein (a), immunoglobulin light chain, immunoglobulin heavy chain, apolipoprotein Al (ApoAl), transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, amyloid β protein precursor (β-APP), Beta₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, huntingtin, microtubule associated protein tau, islet amyloid polypeptide, a peptide hormone, and α-synuclein. Such precursors also include mutant proteins, protein fragments and proteolytic peptide products of such proteins, such as those listed below in Table 1. Examples of α-synuclein mutants include A24C and Q62C.

As is known to persons of skill in the art, amyloidogenic polypeptides may exist in one or more non-pathological form(s), typically monomeric form(s). Some amyloidogenic polypeptides exist in non-pathological multimeric forms, such as, for example, thransthyretin, which exists in a non-pathological tetrameric form. As a result of structural transformations, amyloidogenic polypeptides may adopt one or more pathological multimeric forms, including fibrillar form(s). Intermediate between the monomeric form(s) and the fibrillar form(s), there may be one or more prefibrillar, pathological oligomeric forms.

B. Agents. The agents may be small molecules having a molecular weight of less than 1000 daltons, preferably less than 700 daltons, or the test agents may be larger molecules such as polypeptides, antibodies, nucleic acids, lipids, or the agents may be any other substance capable of being subj ected to the conditions of the methods .

C. Methods of Identifying Stabilizers of Non-Pathological Forms of Amyloidogenic polypeptides. The present disclosure provides methods for identifying an agent which stabilizes a non-pathological form of an amyloidogenic polypeptide. The method includes performing a biophysical method for detecting a non-covalent interaction between a test agent and a non- pathological form of an amyloidogenic polypeptide, selecting a test agent that specifically interacts with the non-pathological form of the amyloidogenic polypeptide, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation is indicative of an agent that stabilizes the non-pathological form of the amyloidogenic polypeptide.

Suitable biophysical methods include, for example, nuclear magnetic resonance (NMR) spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy (CD), isothermal titration calorimetry (ITC), computational modeling, dynamic light scattering (DLS), and surface plasmon resonance (SPR).

NMR Spectroscopy. Some methods include determining the effect of one or more agents on the NMR spectrum of an amyloidogenic polypeptide, selecting one or more test agents for which, in their presence, the amyloidogenic polypeptide has an altered NMR spectrum, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide. In some methods, the NMR spectrum of the amyloidogenic polypeptide is observed in the presence and absence of the test agents. In some methods, the NMR spectrum of the amyloidogenic polypeptide in the presence of one or more test agents is compared to a predetermined NMR spectrum of the amyloidogenic polypeptide in the absence of the test agents.

Heteronuclear NMR experiments were performed on a Bruker Avance 700 MHz spectrometer with cryoprobe. The NMR experiment of choice was the H- N HSQC (fast version), which provides a cross peak (signal) for each amide residue in the protein, mapping the chemical environment of every amino acid, except prolines. Temperature of the measurements was set to 15 ⁰C, necessary to reduce the effect of solvent exchange (Hsu, Bertoncini & Dobson, 2009, JACS). Sample conditions were 30 μM protein and 300 μM compound (0.5% DMSO) in a buffer containing 20 mM Tris-HCl pH 7.4 and 100 mM NaCl. The concentration of protein and the ratio of protein to compound was selected based on the expected affinity (kd < 250 μM) and to provide the maximum window of detection without compromising sensitivity. This particular NMR experiment is able to detect bound species as long as they are populated at least 5%. Hence, assuming a 1 :1 binding mode, the experiment is capable of detecting binders up to an affinity of 3 mM.

For each experimental determination, two NMR spectra are recorded, one set as a reference (0.5 % DMSO) and the second with addition of the compound (300 μM, 0.5% DMSO). Three properties were determined for each of the approximately 120 amide cross peaks in each NMR spectrum: I) H and N chemical shift (position of the peak in the spectrum); 2) peak intensity (height of the peak); and 3) signal to noise ratio (contribution of noise to the peak intensity and position).

Three plots are then obtained reflecting the perturbation caused by the compound on the amide resonances of the protein: 1) residue number vs mean weighted ¹H-¹⁵N chemical shift; 2) residue number vs peak intensity ratio; and 3) mean weighted H- N chemical shift vs signal to noise ratio.

The first two plots reflect the perturbation of the chemical environment of the amide bond caused by the binding of the ligand. The third plot permits evaluation of the accuracy of the above determinations based on the noise level of the spectrum.

Preparation of the peptide samples for NMR spectroscopy. In some methods, peptide samples for NMR spectroscopy may be prepared in a buffered salt solution to maintain a substantially constant pH and ionic strength. Some methods utilize a buffer including 25 mM Tris buffer pH 7.4 and 100 mM NaCl. NMR may be performed at a temperature range of about 5⁰C to 37⁰C. In some methods NMR may be performed at about 5⁰C to about 15⁰C, preferably at about 10 ⁰C, for example, to avoid the enhanced line broadening observed at higher temperatures (McNulty et al. 2006 Protein Science 15:602-608). Preferably, diffusion measurements are obtained at about 10⁰C. Persons of ordinary skill in the protein NMR arts may readily determine appropriate conditions for the acquisition of NMR spectra. In some methods, data are recorded at 500 or 700 MHz. The spectrometer may be equipped with a cryoprobe. Persons of ordinary skill in the NMR arts are familiar with many suitable software packages for processing and analyzing NMR spectrographic data. In some methods, data are processed using NMRPIPE (Delaglio et al. 1995 J. Biomol. NMR 6:277-293) or TopSpin (Bruker) and analysed in CCPNMR (Vranken et al. 2005 Proteins 59:687-696), TopSpin (Bruker) or Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco, USA).

In some methods, NMR data comprise 2D ¹H-¹⁵N heteronuclear single quantum correlation (HSQC) spectra (Schleucher et al. 1994 J. Biomol. NMR 4:301-306). The spectra may be recorded, for example, at 500 MHz by obtaining 512 x 128 complex points and spectral widths of 10 x 29.5 ppm. In some methods, a baseline HSQC spectrum is obtained for a amyloidogenic polypeptide in the absence of a test agent. Assignment of the ¹H-¹⁵N correlation spectrum of a free amyloidogenic polypeptide may be performed by methods known to persons of skill in the NMR arts. A suitable method is that of Eliezer et al (2001 JMB 307:1061).

In some methods, peaks in the amyloidogenic polypeptide spectra in the presence of a test-agent are assigned by following the peaks in the correlation map while titrating the amyloidogenic polypeptide with a test-ligand. For example, about 0.1 μM to about ImM of the amyloidogenic polypeptide is titrated with from about 0.1 to about 20 molar equivalents of a test agent. In some methods, about 1 μM to about 100 μM of the amyloidogenic polypeptide, for example, α- synuclein, is used. In some methods, the amyloidogenic polypeptide is titrated with from about 0.5 to 16 molar equivalents of a test agent. Titration with from about 1 to 10 molar equivalents of a test agent may also be performed. For example, NMR spectra of about 1 μM of the amyloidogenic polypeptide may be recorded with and without about 4 μM of a test agent.

Recording of 2D ¹H-¹⁵N HSQC NMR spectra may be done, for example, on a 700 MHz spectrometer using, for example, SOF AST-HMQC (Schanda & Brutscher 2005 JACS 2005 127:8014-8015) with about 512 x 64 complex points, about 12 x 29 ppm spectral widths and about 1152 or 1280 scans per increment. In some methods, the 2D ¹H-¹⁵N HSQC NMR spectra are recorded on a 500 MHz spectrometer with, for example, about 512 x 128 complex points and spectral widths of about 10 x 29.5 ppm.

Recording of CO- N correlation spectra may be done using, for example direct ^JC detection (Bermel et al, 2007 J. Magn. Reson. 188: 301-310) on the 500 MHz spectrometer. Data may be acquired with about 512 x 256 complex points, with spectral widths of about 10 x 40 ppm (to include Pro residues) and with about 16 scans per increment. Two or more spectra may be collected and added to reduce noise. Samples may contain about lμM to about 1 niM, preferably about 100 μM ¹³C¹⁵N-labelled amyloidogenic polypeptide, such as, for example, α-synuclein, with the addition of about .lμM to about 10 mM of the test agent, preferably about 1 μM to about 1 mM of the test agent. Some methods utilize about 0.6 mM to about 0.9 mM of the test agent.

The diffusion coefficient of the amyloidogenic polypeptide with or without the test agents may be measured, for example, using pulse field gradient NMR (Johnson 1999 Prog NMR 34:203-

9? 256) on the 700 MHz spectrometer. Data may be acquired at a temperature of about 5⁰C to about 2O⁰C, preferably about 15⁰C, using an unlabeled protein sample concentration of about lμM to about ImM, preferably about 100 μM. An example of a suitable buffer is about 5OmM phosphate, 100 niM NaCl, in 99.9% ²H₂O and containing 10 rnM dioxane as an internal radius standard and viscosity probe. Multiple ID ¹H spectra may be collected as a function of gradient strengths from about 1.60 Gauss cm^"1 to about 32.0 Gauss cm^"1, in a linear manner. Each ¹H spectrum may comprise about 25 to about 150 scans. In some methods, the H spectrum may comprise about 32 to about 128 scans. In some methods about 8192 complex points are acquired with a spectral width of about 12 ppm. The dioxane peak and selected signals in the aromatic and aliphatic regions of the ¹H protein spectrum may be integrated and the decay of the signal as a function of the gradient strength may be fitted to a Gaussian function using Sigma plot 7.0 to determine the hydrodynamic radii (Wilkins et al. 1999 Biochemistry 38:16424-31) . The radius of hydration for the protein (R-Hprot) may be calculated from the decay rates of the protein and the dioxane peaks, using the formula R_{H pro}t = (d diox /d _prot) ^• RH ώox, whereas R_H ώox is 2.12 A, and d _di_ox and d _prot are the dioxane and protein decay rates, respectively.

Fluorescence Spectroscopy. Some methods include determining the effect of one or more agents on the fluorescence emission of a fluorescently labelled amyloidogenic polypeptide, selecting one or more test agents for which quenching of the fluorescence emission is demonstrated, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide. The fluorescently labeled amyloidogenic polypeptide may be generated, for example, by conjugating the amyloidogenic polypeptide with 1,5-1- AED ANS (5-({2- [(iodoacetyl)amino] ethyl} amino)naphthalene- 1 -sulfonic acid) (I AED ANS) . Other suitable fluorescent dyes include, but are not limited to, MTS-dansyl, dibromobimane, 4 - chloro - 7 - nitrobenzofurazan, 1 - anilinonaphthalene - 8 - sulfonic acid). In some methods, an AS-62C- AEDANS-conjugated amyloidogenic polypeptide is used. An AS-24C-AEDANS-conjugated amyloidogenic polypeptide may also be used. For determination of the affinity of the complex between a test agent and an amyloidogenic polypeptide, the fluorescently labeled amyloidogenic polypeptides may be titrated with increasing amounts of the test agent. Quenching of the fluorescence in a test agent-dependent manner allows the estimation of the amount of complex formed. Titrations may be performed on amyloidogenic polypeptide concentrations of about 1 μM to about 10 μM, preferably about 5 μM, in a suitable buffer such as, for example, 25 rnM Tris buffer pH 7.4, 100 mM NaCl, with the addition of the test agent in concentrations from about 0.1 μM to about 1 mM, preferably about 1 μM to about 0.5 mM. Fluorescence determination may be performed, for example, in Cary-Eclipse spectrofluorimeter (Varian). Fluorescence, for example AEDANS fluorescence, may be recorded with an excitation wavelength of about 330 nm to about 340 nm, preferably about 337 nm, and emission may be collected from about 400 to 600 nm. Control titrations may be performed with test agent alone into buffer and with agent into free fluorophore.

The increase in fluorescence intensity at about 480-520 nm, after subtracting contributions from the free ligand, may be fitted to a one-site ligand binding mode, according to:

Fromequ.Hbrium:

[

F_n - F F_n - F_^, r _^T i !^■„! r,,_T i r_^i F_n - F

From fluorescence : -£-=- = -=²--^ ; [PLL_x = H => [PL] = [P\

IPL] - [PLL_x ' ^{» ">}- - ^» » ^{~* 1^1}- * * F₀ -F,

Re -arranging : + 14 + K + V(H + 14 + Kf ' 44.4

being F the fluorescence intensity observed at a given concentration of ligand, Fo the fluorescence of the free amyloidogenic polypeptide, F the fluorescence at saturation, Po the concentration of amyloidogenic polypeptide, Lo the concentration of the test agent, and k_d the apparent dissociation constant of the complex.

Circular Dichroism Spectroscopy. Some methods include determining the effect of one or more agents on the circular dichroism (CD) spectrum of an amyloidogenic polypeptide, selecting one or more test agents for which, in their presence, the amyloidogenic polypeptide has an altered CD spectrum, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide. Circular dichroism may be measured, for example, on a Chirascan CD spectrometer (Applied photophysics Ltd.) equipped with a Peltier temperature control system. The samples may contain about 1 μM (micromolar) to about 10 μM, preferably about 5 μM of the amyloidogenic polypeptide in a suitable buffer, such as, for example, 25 mM Tris pH 7.4, 100 mM NaCl, with or without about 1 μM to about 1 mM, preferably about 10 μM to about 0.5 mM of the test agents. Samples with the same test agent concentrations but without any amyloidogenic polypeptide may be used as reference. A cell with, for example, 1 mm path length may be used and the measurements may be performed at about 10 ⁰C, about 25 ⁰C and about 37 ⁰C.

Isothermal Titration Calorimetry. Some methods include determining the thermodynamic characteristics of the binding of one or more agents to an amyloidogenic polypeptide, selecting one or more test agents having increased heat effects relative to relative to a control, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide.

Isothermal titration calorimetry (ITC) experiments may be carried out using VP-ITC titration microcalorimeters (MicroCal Inc., MA, USA) at about 25 ⁰C. The amyloidogenic polypeptide and test agents may be dissolved in a suitable buffer, for example, phosphate buffered saline (PBS) pH 7.4, and the samples may be degassed before the measurements. Each experiment may involve a preliminary injection of about 2 μl followed by about 25 to about 27 injections of about 10 μl using, for example, a 300 μL syringe. The cell volumes may be in the range of about 1.2 to about 1.6 ml, preferably about 1.416 ml or about 1.4242 ml. ITC raw data may be analyzed using Origin 7 (OriginLab Corporation, MA, USA). Amyloidogenic polypeptide in a concentration of about 0.5 μM (micromolar) to about 1 mM may be titrated with about 1 μM to about 2 mM of the test agent. For example, about 1 μM to 5 μM, preferably 2 μM of the amyloidogenic polypeptide in the cell may be titrated with about 0.1 mM or 0.2 mM of the test agent. In some methods, about 100 μM of the amyloidogenic polypeptide in the syringe may be titrated with 5 μM, 20 μM or 50 μM of the test agent in the cell. In some methods, about 5 μM of the amyloidogenic polypeptide (in the cell) may be titrated by about 0.1 mM of the test agent. In another example, about 100 μM of the amyloidogenic polypeptide (in the cell) may be titrated by 1 mM of the test agent. Reference experiments may be performed by titrating the solution in the syringe into pure buffer.

Computational modeling of the amyloidogenic polypeptide and a test agent. Some methods include determining the ability of one or more test agents to bind one or more simulated conformations of an amyloidogenic polypeptide, selecting one or more test agents which bind the amyloidogenic polypeptide in silico, and determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide, wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide.

Conformations of the amyloidogenic polypeptide used in the docking calculation may be optimized with the steepest descent minimization using the MMFF94 molecular mechanics force field (Halgren, 1996 J. Comp. Chem., 17: 490-641; Halgren 1999 J. Comp. Chem. 20: 720-748) and a distance dependent dielectric model in MOE (Computational Chemistry Group, 2005). A low energy conformational ensemble of the test agents may be generated, for example, using the Systematic Conformational Search module in MOE. Rotational bonds may be explored by 30 degree intervals. Generated conformations may be minimized using the MMFF94 and a distance dependent dielectric model, preferably keeping only those which have a root mean square deviation of more than about 0.3 A between their heavy atoms. AM1/BCC charges may be used for the small molecules (Jakalian, et al 2000 J. Comput. Chem. 21 : 132 - 146). Docking calculations may be performed, for example, with FRED 2.0 (OpenEye Scientific Software, Inc., Santa Fe, NM, USA, www.eyesopen.com, 2008). FRED may be used with a translational step size of about 0.5 A and a rotational step size of about 0.75 A to exhaustively sample the low energy conformational ensemble of the test agents within the amyloidogenic polypeptide binding site. After the initial round of docking, FRED may optimize about 2500 of the highest scoring docked poses with the ChemGauss3 scoring function. About 300 of the highest scoring docked poses, on the basis of consensus scoring function (Shapegauss, Chemgauss3, Oechemscore, Screenscore), may be optimized further. AM1/BCC charges may be calculated for each test agent conformation used in the subsequent optimization step. Optimization may be performed by minimizing each docked pose inside its rigid protein receptor using, for example, SZYBKI 1.2.0 (OpenEye Scientific Software, Inc., Santa Fe, NM, USA, www.eyesopen.com, 2008). During the SZIBKI minimization the MMFF94 molecular mechanics force field and the Poisson-Boltzman solvation model may be used. The protein may be kept rigid while all atoms of the test agemt may be flexible. Conformations with the lowest E_SZIBKI energy may be considered as the prediction of the binding mode of the new molecule. ES_ZIBKI may be defined by the following equation: ESZIBKI ⁼ E_mt_ermoiecuiai interaction + E_soi_v + Ei_lgand, where E_intermoiecuiai interaction contains all non- bonded interactions between the ligand and the protein, E_soi_v is the interaction energy associated from the electrostatic part of ligand-solvent interactions, and where Ei_lgand is the potential energy of the ligand.

Dynamic Light Scattering. The occurrence of supramolecular structures of the test agents may modulate the interaction with an amyloidogenic polypeptide. Accordingly, some methods include determining the propensity of a test agent to self-associate using dynamic light scattering (DLS).

Dynamic light scattering may be measured using, for example, an ALV/CGS-3 compact gonimeter system equipped with ALV/LSE-5004 multiple tau digital real time correlator, operating at about 632.8 nm wavelength (ALV-GmbH). The samples may be filtered, for example, through 0.2 μm filter before the measurements and the scattered light may be detected at about a 150° angle. The samples may contain about 0.1 mM to about 2 mM, for example, about 0.1 mM, 0.5 mM, about 0.6 mM or about 1 mM of the test agent in a suitable buffer, such as, for example 25 mM Tris pH 7.4, 100 mM NaCl, and the data may be recorded at about 25 ⁰C. The acquired data may be analyzed by the regularization algorithm in the ALV correlator 3.0 software (ALV-GmbH). Other instruments may be used. For example, if the laser wavelength of the instrument overlaps with the absorbance spectrum of the test agent, additional data may be acquired using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd.) operating at about 532 nm and a detection angle of about 173°. The data may be exported and analyzed using the ALV software, for example.

Surface Plasmon Resonance (SPR) Screening. Molecules were screened for α-synuclein binding by high-throughput SPR-based technology. Soluble, monomeric α-synuclein was present in solution and the library compounds to be screened was immobilized at high density on a chip. A suitable SPR-based system may be the RAISE® (Rapid Array Informed Structure Evolution) system (Graffmity Pharmaceuticals GmbH, Heidelberg, DE).

Aggregation Assays. Aggregation of the amyloidogenic polypeptide may be monitored by any of a number of techniques known in the art, including, for example, the Thiofiavin-T (Thio-T, ThT) assay (Conway et al 2000, PNAS 97:571-576.; Hoyer et al, 2002, J. MoI. Biol 322:383).

Aggregation of amyloidogenic polypeptide may be assayed at a concentration of about 1 μM to about 1 mM, preferably about 100 μM in a suitable buffer, such as, for example, 20 mM Tris pH 7.4, 100 mM NaCl with the addition of 0.01% NaN₃. About 100 μl to about 1 ml, preferably 500 μl of the amyloidogenic polypeptide sample may be incubated at about 37 ⁰C, preferably under constant shaking at about 300 rpm. About 10 μl to 100 μl, preferably 50 μl aliquots may be withdrawn regularly, such as, for example, on a daily basis, assayed, for example, for ThioT fluorescence and stored at about 4 ⁰C until the end of the assay for further determinations.

Fibrillation experiments. Aggregation experiments were set up on a 96 well plate. A BMG Fluostar Optima plate reader was employed. Sample conditions were 30 uM protein and 300 uM compound (0.5% DMSO) in buffer Tris-HCl 20 mM pH 7.4, 100 mM NaCl, plus the addition of 350 uM SDS (to promote misfolding and aggregation) and 20 uM ThT (to detect amyloid formation). Temperature of the experiment was set to 37 ⁰C and the experiments were run for 72 hs. Each sample was run in duplicates and 8 DMSO only controls were run in the plate to serve as a reference. The "aggregation curve" is obtained by plotting the ThT signal as a function of time. This curve has, normally, a sigmoidal shape, and can be divided in three intervals: the lag phase (period it takes to reach 10% of the final ThT signal, reflecting formation of the amyloidogenic nuclei), the growing phase (exponential growth phase in which the nuclei are extend into fibrils), and he saturation phase (the equilibrium between the aggregated and soluble protein is reached). An aggregation inhibitor can affect amyloid formation at each of these steps, extending the lag phase, slowing the speed of growing phase (monomer addition) or reducing the final amount of fibrils at saturation.

Fibril formation may be monitored by the Thio-T assay by diluting aliquots of about 10 μl in about 1 ml of about 20 μM Thio-T and measuring the fluorescence, for example, in a FlashScan spectroflourimeter (Jena Analytik), with an excitation wavelength of about 446 nm. Emission wavelengths from about 460 to about 600 nm may be collected and the integrated fluorescence between about 470 and about 490 nm may be employed for the determination of the relative content of fibrils of the amyloidogenic polypeptide in the sample.

Quenching of ThioT fluorescence by test agents may be assayed by incubating pre-formed fibrils for about 30 minutes with various concentrations of a test agent, for example, about 1 nM to about 5 mM, preferably about 0.1 μM to about 2 mM, and measuring the ThioT fluorescence of the sample. In some methods, concentrations of the test agent of about 1 μM to about 1 mM are used.

The relative amount of aggregated (insolubilized) amyloidogenic polypeptide may be assayed by centrifuging the samples at 16,00Og and resolving the supernatant fraction in about 4-12 % SDS- PAGE (No vex, Invitrogen). The aggregated amyloidogenic polypeptide material may be determined, for example, by resistance to solubilization with 1 % Sarkosyl, by resolving the soluble fraction (non-amyloid) in a SDS-PAGE. Image quantization may be performed, for example, on Coomassie-stained gels with the software Image J (NIH).

Transmission electron microscopy (TEM) of aggregated samples may be performed by depositing about 10 μl of an about 1:10 dilution sample on Formvar-coated nickel grids (Agar scientific), and staining with about 2 % (w/v) uranyl acetate. Negative-staining images may be obtained at 25,000 x magnification using a Phillips CEMlOO transmission electron microscope (Imaging facility, Dept. of Pathology, University of Cambridge).

Other aggregation assays may be utilized for the detection of the formation of oligomers and fibrils of amyloidogenic polypeptides, for example assays utilizing light scattering (Palecek et. al., Analyst. 2008 Jan;133(l):76-84), fluorescence spectroscopy (Dusa et. al, Biochemistry. 2006 Feb 28;45(8):2752-60.) FRET spectroscopy (Kaylor et. al, J MoI Biol. 2005 Oct 21;353(2):357-72) and electrochemical methods (Palecek et. al., Analyst. 2008 Jan;133(l):76- 84).

Protein Preparation. The amyloidogenic polypeptides may be produced by any of a number of techniques known in the art, including, for example, recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to the coding sequences for the amyloidogenic polypeptides, including naturally-associated or heterologous promoter regions. The expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly expression vectors contain selection markers, e.g., ampicillin-resistance or hygromycin- resistance, to permit detection of those cells transformed with the desired DNA sequences. Suitable vectors may have expression control sequences, an origin of replication, termination sequences, enhancers, necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and the like as desired.

For example, the peptides may be expressed in Escherichia coli or other bacterial cells, such as, for example, BL21 cells, using an appropriate vector. Examples of suitable vectors include pT7-

7.

Alternatively the peptides may be expressed in a eukaryotic host, such as yeast, for example, Saccharomyces, or mammalian cells. Typical promoters for yeast expression include 3- phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Some suitable mammalian cell lines include CHO cell lines, COS cell lines, HeLa cells, L cells and myeloma cell lines. See, for example, Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). Some expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148:1149 (1992). The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, lipsomes, electroporation, and microinjection (see generally, Sambrook et al.).

Once expressed, the amyloidogenic polypeptides can be purified according to standard procedures of the art, including HPLC purification, column chromatography, gel electrophoresis and the like (see generally, Scopes, Protein Purification (Springer- Verlag, NY, 1982)).

The amyloidogenic polypeptides can be labeled, for example, following purification. In such labeling methods, a fluorescent dye may be added following standard protocols, for example as described in Example 1. IAEDANS (Sigma) is an example of a suitable dye. Other suitable fluorescent dyes include, but are not limited to, MTS-dansyl, dibromobimane, 4 - chloro - 7 - nitrobenzofurazan, 1 - Anilinonaphthalene - 8 - sulfonic acid).

In certain medical and pharmacological methods, contacting the amyloidogenic polypeptide with the monomer-stabilizing compound is achieved by administration of the monomer- stabilizing compound to a mammal. The contacting may be by any route of administration known to, and approved by, persons of skill in the medical arts.

C. Pharmaceutical Compositions. The disclosure also provides pharmaceutical compositions comprising an agent the non-pathological form of an amyloidogenic polypeptide, such as, for example, an amyloidogenic polypeptide listed in Table 1. In some pharmaceutical compositions the stabilizing agent has been identified by any of the methods described above. In some pharmaceutical compositions, the stabilizing agent binds specifically to an aggregation promoting region of an amyloidogenic polypeptide, and stabilizes the amyloidogenic polypeptide in a non-pathological form. For example, the stabilizing agent decreases the aggregation of the amyloidogenic polypeptide relative to the aggregation observed for the amyloidogenic polypeptide in the absence of the stabilizing agent. The stabilizing agent may also have one or more of the following properties: (i) ability to alter the NMR spectrum of the amyloidogenic polypeptide relative to the NMR spectrum in the absence of the stabilizing agent; (ii) ability to alter the CD spectrum of the amyloidogenic polypeptide relative to the CD spectrum in the absence of the stabilizing agent; (iii) thermodynamic characteristics consistent with specific binding of the stabilizing agent to the non-pathological form of the amyloidogenic polypeptide; and (iv) in silico binding to a desired conformation of the non-pathological amyloidogenic polypeptide generated by computational modeling. For example, an α-synuclein stabilizing agent may cause chemical shift perturbations in the NMR spectrum of α-synuclein within the region of amino acid residues 1-100. Some α- synuclein stabilizing agents stabilize the region of amino acid residues 1-40, some stabilizing agents stabilize the region of amino acids 50-100, some stabilizing agents stabilize the NAC region, and some stabilizing agents stabilize the region of amino acids 50-77.

D. Methods of Treatment. The disclosure also provides methods of treating, preventing or delaying the onset of a disease characterized by the conversion of an amyloidogenic polypeptide from a non-pathological form to a pathological form, such as, for example, by oligomerization, aggregation or deposition of the amyloidogenic polypeptide. The methods comprise administering to a mammal in need thereof a pharmacologically effective amount of an agent that stabilizes a non-pathological form of an amyloidogenic polypeptide, for example as part of any of the pharmaceutical compositions described above. In some methods, the stabilizing agent has been identified by any of the identification methods described above. According to the disclosure, the mammal may be a human or non-human mammal.

EXAMPLES

Example 1. asynuclein Protein preparation, α-synuclein was expressed in Escherichia coli BL21 cells using a pT7-7 vector as described in (Hoyer et al. 2002 JMB 322:383). ¹⁵N- and ¹³C- ¹⁵N-labelled protein was produced using M9 minimal medium supplemented with ¹⁵NH₄Cl and ¹³C-glucose (Spectra gases isotopes). The protein was purified using heat treatment, ammonium sulphate precipitation, anion exchange and size exclusion chromatography as described in (Hoyer et al. 2002 JMB 322:383). Purified protein samples were dialyzed against water, flash frozen in liquid nitrogen and stored at -80 ⁰C. Production of A24C and Q62C α-synuclein mutants has been reported elsewhere (Dedmon et al., 2005, JACS).

Labelling with the fluorescent dye IAEDANS (Sigma) was performed following standard protocols. Briefly, 1 mg of purified Cys-containing protein was reduced by incubation in 10 mM DTT for 30 min. The reducing agent was by removed by fast desalting in PD-IO columns (GE healthcare) and 10:1 molar excess of the dye was immediately added. Proteins were incubated with the dye for 1 h at 4 ⁰C and the excess of fiuorophore was removed with another fast desalting step in PD-IO columns. Due to the unfolded nature of α-synuclein, the yield of labeling reached 99 %.

Example 2. Preparation of Chemicals. Congo red, Lacmoid (Figure 1) and the polyphenol EGCG (Ehrnhoefer et al 2008 Nat Struct MoI Biol. 15:558-566) have recently been reported to affect fibril formation of several amyloidogenic proteins, including α-synuclein and Aβ (Masuda et al. 2006 Biochemistry 45:6085, Necula et al. 2007 J. Biol. Chem. 282:10311) and to interact with monomeric α-synuclein (Rao et al 2008 Biochemistry 47:4651). Lacmoid resembles the phenothiazine class of compounds which serve as scaffolds for several neuroleptic antipsychotic drugs such as thorazine, prolixin, and phenothiazine. Lacmoid contains polyphenol groups which renders it similar to ECGC. Lacmoid and Congo red (Aldrich) were dissolved in buffer to prepare stock solutions of 1 - 50 mM. The stock solutions were carefully sonicated before further dilution.

Example 3. Measurement of the change in NMR spectra for a-synuclein upon addition of Lacmoid and Congo red. The interaction of α-synuclein with Lacmoid and Congo red was first investigated by heteronuclear NMR spectroscopy. NMR samples were prepared in 25 mM Tris buffer pH 7.4 with 100 mM NaCl. Data were recorded on Bruker Avance 500 MHz, and 700 MHz spectrometers equipped with cryoprobes and processed using NMRPIPE (Delaglio et al. 1995 J. Biomol. NMR 6:277-293) or TopSpin (Bruker) and analysed in CCPNMR (Vranken et al. 2005 Proteins 59:687-696), TopSpin (Bruker) or Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco, USA). All NMR experiments, except for the diffusion measurements, were performed at 10 ⁰C. 2D H- N heteronuclear single quantum correlation (HSQC) (Schleucher et al. 1994 J. Biomol. NMR 4:301-306) spectra were recorded on the 500 MHz spectrometer with 512 x 128 complex points and spectral widths of 10 x 29.5 ppm. Figure 2 shows HSQC spectra for 100 μM α- synuclein monitored during addition of from 0.5 to 16 and 1 to 10 molar equivalents of Lacmoid and Congo red respectively. Assignment of the ¹H-¹⁵N correlation spectrum of free α-synuclein has previously been reported (Eliezer et al 2001 JMB 307:1061). The assignments of the α- synuclein spectra in the presence of Lacmoid or Congo red were done by following the peaks in the correlation map during the titration.

2D H- N correlation spectra of 1 μM α-synuclein with and without 4 μM Congo red was recorded on the 700 MHz spectrometer using SOFAST-HMQC (Schanda & Brutscher 2005 JACS 2005 127:8014-8015) with 512 x 64 complex points, 12 x 29 ppm spectral widths and 1152 or 1280 scans per increment.

The CO- N correlation spectra were recorded using direct C detection (Bermel et al, 2007 J. Magn. Reson. 188: 301-310) on the 500 MHz spectrometer. Data were acquired with 512 x 256 complex points, with spectral widths of 10 x 40 ppm (to include Pro residues) and with 16 scans per increment. Two spectra were collected and added to reduce noise. Samples contained 100 μM ¹³C^bN-labelled α-synuclein with the addition of 0.9 mM Congo red or 0.6 mM Lacmoid.

The diffusion coefficient of α-synuclein with and without a test ligand was measured using pulse field gradient NMR (Johnson 1999 Prog NMR 34:203-256) on a 700 MHz spectrometer. Data was acquired at 15 ⁰C on a 100 μM unlabeled protein sample in 50 mM phosphate buffer (pH 7.6) (uncorrected), 100 mM NaCl, in 99.9 % ²H₂O and containing 10 mM dioxane as an internal radius standard and viscosity probe. 24 ID H spectra were collected as a function of gradient strengths from 1.60 Gauss cm^"1 to 32.0 Gauss cm^"1, in a linear manner. Each ¹H spectrum comprised 32 scans (or 128 scans in the case of Congo red). 8192 complex points were acquired with a spectral width of 12 ppm. The dioxane peak and selected signals in the aromatic and aliphatic regions of the ¹H protein spectrum were integrated and the decay of the signal as a function of the gradient strength was fitted to a Gaussian function using Sigma plot 7.0 to determine the hydrodynamic radii (Wilkins et al. 1999 Biochemistry 38:16424-31) (Supporting material figure S3). The radius of hydration for the protein (R_{H prot}) was calculated from the decay rates of the protein and the dioxane peaks, using the formula RHprot = (d dioχ/d _prot) ' R_H ώox, whereas R_H diox is 2.12 A, and d _di_0X and d _prot are the dioxane and protein decay rates, respectively.

Results of NMR Spectroscopy. In general, the vast majority of compounds caused slight perturbations on the backbone of the protein. In most cases such perturbations were clustered at the N-terminus and NAC region of the protein. The high resolution of the spectrometer (700MHz) allowed precise determination of small chemical shifts. Perturbations caused by the compounds were similar to those observed for previously for Lacmoid (Lendel, Bertoncini et al, 2009, Biochemistry), and much smaller than those seen by polyamines (Fernandez, 2003, EMBO J).

Titrations of the protein with 0.5 to 16 molar equivalents of Lacmoid and 1 to 10 molar equivalents of Congo red allowed characterization of the binding events on a residue-specific basis. The gradual changes in chemical shifts and intensities observed for several peaks in the ¹H-¹⁵N correlation NMR spectrum of α-synuclein indicate that Lacmoid and Congo red bind to the protein in a concentration dependent manner (Figures 2 and 11). The changes are more pronounced for Congo red than for Lacmoid, which suggests different affinities for the agents. The chemical shift perturbations directly report on changes in the chemical environment and/or in the relative populations of different conformations in the α-synuclein structural ensemble. The perturbations in the NMR observables are not uniform along the backbone of α-synuclein. Residues in the C-terminal part of the protein seem to be significantly less affected by both compounds (Figure 2). Within the sequence 1-100, two distinct binding domains can be identified, with the first domain being approximately residues 1-40 and the second residues 50- 100. The latter of these domains includes the NAC region, which is believed to be the most aggregation promoting segment of the protein (Han et al Lansbury 1995 Chem. Biol. 2:163-169; Du et al 2003 Biochemistry 42:8870-8878; El-Agnaf & Irvine 2002 Biochem. Soc. Trans.

30:559-565; Giasson et al 2001 J. Biol. Chem. 276:2380-2386). The most N-terminal binding domain experiences a higher degree of line broadening for both compounds while the region showing the largest chemical shift changes differs between the two binders. For Congo red, the largest shift changes are observed in the second binding domain (the region 50-77) while Lacmoid mainly affects residues in the most N-terminal part. The chemical shift changes with Lacmoid are rather small, and changes of the same magnitude are also observed in the N- terminal region with Congo red.

The binding of the agents causes slow to intermediate time-scale conformational changes in α- synuclein. Without being bound by a particular theory, the changes in peak intensity (line width) observed in the H- N- correlation spectrum of α-synuclein may be due to several processes: i) increase in the rotational and translational correlation times (e.g. by binding to large ligand aggregates or induced compaction or oligomer formation of the protein); H) conformational exchange on the μs-ms time scale due to changes in the conformational behaviour of the α- synuclein polypeptide chain and/or interaction with the ligands; Hi) changes in amide hydrogen exchange rates.

Figure 10 displays data for protonless NMR spectra of α-synuclein bound to Congo red. Comparison Of ¹H-¹⁵N HSQC (left) and ¹³C direct detected ¹³CO-¹⁵N (right) correlation spectra of 100 μM α-synuclein with (red) and without (green) 9:1 molar excess of Congo red. Peak intensity ratios between bound and free protein are plotted.

Changes in peak intensity resulting from hydrogen exchange can be expected from interactions that alter the exposure of the peptide backbone. In order to discard this effect as a cause for the changes in peak intensity, ¹³C direct detected NMR experiments were performed on ¹³C-¹⁵N- labelled α-synuclein with and without Congo red and Lacmoid. ¹³CO-¹⁵N correlation spectra showed the same profiles for intensity changes as the Η-^N-correlation (Figure 10) suggesting that hydrogen exchange is not a cause of line broadening.

Figure 11 is an analysis of PFG-NMR data measured for α-synuclein in the presence of small molecule compounds. Data was fitted with a single Gaussian function and the rates of decay were converted into hydrodynamic radii. In the case of Congo red and Lacmoid the data was also fitted to a two species model, however this does not reduce residuals (shown in red).

Pulse field gradient NMR (PFG-NMR) experiments were used to monitor the diffusion properties, and thereby the molecular dimensions, of α-synuclein in absence and presence of the two agents. R_H of free α-synuclein was found to be 29.2 ± 0.2 A (Figure 11). Addition of 0.9 mM (9 molar equivalents) Congo red induces a more compact conformational ensemble (R_H = 25.0 ± 0.3 A) while the R_H for α-synuclein in presence of 0.6 mM (6 molar equivalents) Lacmoid is slightly higher than without the compound (30.6 ± 0.3 A). The data thus suggest that the line broadening observed upon binding of the agent is not due to significant increases in the molecular size of the complex, and points towards slow conformational exchange as the major cause for decreased peak intensity. For Congo red, without being bound to a particular theory, the compaction of the structural ensemble may also contribute to the line broadening.

Figure 9 presents data comparing the 2D-NMR spectra of α-synuclein in presence and absence of Congo red and Lacmoid. A) ¹H-¹⁵N HSQC of 100 μM α-synuclein with (red) and without (blue) 10:1 molar excess of Congo red. B) ¹H-¹³N HSQC of 100 μM α-synuclein with (red) and without (blue) 16:1 molar excess of Lacmoid. C) ¹H-¹⁵N SOF AST-HMQC of 1 μM α-synuclein with (red) and without (blue) 4:1 molar excess of Congo red.

Binding affinities of the α-synuclein:test agent complexes was assessed. To further explore the apparent high affinity binding of Congo red to α-synuclein (see above) additional NMR experiments were carried out at 100 times lower protein concentration. The ¹H-¹⁵N correlation spectrum of 1 μM α-synuclein with 4 μM Congo red shows substantial line broadening of the 100 most N-terminal residues, most of them beyond the detection limit (Figure 9). This result is similar to the results obtained at high protein concentration data (see above), and confirm that Congo red interacts with monomeric α-synuclein even at a low μM concentration which is most likely below its critical aggregating concentration (Edwards and Woody, 1979 Biochemistry 18:5197).

Example 4: Measurement of the change in AEDANS fluorescence ofa-synuclein in the Presence of Congo red or Lacmoid. To estimate the relative affinities of the α-synuclein: small molecule complexes, the quenching of the fluorescence emission of AEDANS-conjugated α- synuclein upon titration with the compounds was monitored. For determinations of the affinity of the α-synuclein-small molecule complexes, AS-62C-AEDANS (for Congo red) or AS-24C- AEDANS (for Lacmoid) were titrated with increasing amounts of a ligand. AEDANS fluorescence was quenched in a ligand dependent manner, allowing the estimation of the amount of complex formed. Titrations were performed on 5 μM AS-AEDANS proteins in 25 mM Tris buffer pH 7.4, 100 mM NaCl, with addition of Congo red or Lacmoid in concentrations spanning from 1 μM to 0.5 mM. Fluorescence determinations were performed in a Cary-Eclipse spectrofluorimeter (Varian). AEDANS fluorescence was recorded with an excitation wavelength of 337 nm and emission was collected from 400 to 600 nm. Control titrations were performed with ligand alone into buffer and with ligand into free IAEDANS. The increase in fluorescence intensity at 480-520 nm, after subtracting contributions from the free ligand, was fitted to a one- site ligand binding mode, according to:

Fromequ.Hbrium:

[^pi] = ^([P]₀ + [L]₀ + k_d + V([4 +.4 + ^)² - 4MM

From fluorescence : [ I/P\ Jo ^F° ^F

F 0 - ^±F sat

Re -arranging : A_£- = -^-(H + [L]₀ +k_d + <J([^p]₀ + [L]₀ + kj - 4[P]₀[L]₀ Fo -F_sat 2[PJ₀ V

being F the fluorescence intensity observed at a given concentration of ligand, Fo the fluorescence of the free protein, F the fluorescence at saturation, Po the concentration of protein, Lo the ligand concentration, and k_d the apparent dissociation constant of the complex.

Fluorescence Spectroscopy Results. Figure 3 shows that Congo red binds to α-synuclein more strongly than does Lacmoid. The fluorophore was specifically attached to cysteine residues introduced in the vicinity of the NMR-derived binding regions (position 24 for Lacmoid and 62 for Congo red). 5 μM of the fluorescently-labelled proteins were titrated with 0.25 to 100 molar equivalents of the compounds (Figure 3). Fitting of the data to a single site binding model resulted in dissociation constants (K_D) of 1.05 ± 0.05 μM for Congo red and 12 ± 2 μM for Lacmoid. These data thus confirm the relative affinities for the protein: ligand interactions observed by NMR.

Control experiments of free IAEDANS titrated with Lacmoid or Congo red also showed strong ligand concentration dependent quenching of the fluorophore. The apparent K_D values for these interactions are an order of magnitude higher than for the protein binding (19 ± 2 μM and 154 ± 6 μM for Congo red and Lacmoid respectively). Fitting the protein titration data to two independent binding events using the K_D derived from the control experiments were not successful indicating that specific binding of the compounds to the attached AEDANS is not likely to occur. However, the affinities of the protein for the two compounds are most likely affected by the presence of the fiuorophore and the measured K_D thus might not agree with those derived from other techniques.

Example 5. Circular dichroism spectroscopy of a-synuclein. Circular dichroism (CD) was measured on a Chirascan CD spectrometer (Applied photophysics Ltd.) equipped with a Peltier temperature control system. The samples contained 5 μM α-synuclein in 25 mM Tris buffer pH 7.4, 100 mM NaCl with or without 10 μM to 0.5 mM of the compounds. Samples with the same compound concentrations but without any protein were used as reference. A cell with 1 mm path length was used and the measurements performed at 10 ⁰C, 25 ⁰C and 37 ⁰C.

Circular Dichroism Results. Circular dichroic spectra (CD) reveal changes in α-synuclein backbone conformations upon ligand binding. Figure 4 shows that binding of small molecules induces changes in secondary structure content of α-synuclein. CD spectroscopy was employed to probe whether complex formation perturbs the content of secondary structure of the ensemble of α-synuclein conformations. The far-UV CD spectrum of 5 μM α-synuclein at 10 ⁰C indicates an unstructured protein (Figure 4).

Figure 11 presents a detailed characterization of structural transitions in α-synuclein by CD.

Increased temperature results in positive changes in the amplitude of the negative peak at 196 nm and also in smaller, negative, changes around 220 nm (Figure 11). The CD spectrum of α- synuclein in presence of 10 μM Congo red at 10 ⁰C shows decreased amplitude of the negative peak at 196 nm with minor changes in the rest of the spectrum (Figure 4A). At 0.1 mM Congo red concentration, the amplitude changes below 200 nm are enhanced. In addition, the position of this minimum is altered towards longer wavelengths and there are significant negative changes in the spectrum around 220 nm. Similar but less significant changes in the α-synuclein CD spectrum are observed at higher temperatures (Figure 12). The changes in the 196-200 nm region of the CD spectrum observed in the presence of Lacmoid are similar to what is seen with Congo red but with lower magnitude (Figure 4B). No significant changes are observed at 5 μM and 50 μM Lacmoid concentrations at all three assayed temperatures. However, at 0.5 mM Lacmoid concentration, changes of similar amplitude to the ones observed for 10 μM Congo red are detected, which is in agreement with the lower binding affinity of Lacmoid observed by NMR and fluorescence quenching. In summary, the CD data suggest that the ensemble of conformations populated by α-synuclein is strongly perturbed upon binding to Congo red but less affected by Lacmoid.

Example 6. Isothermal titration calorimetry performed on a-synuclein with Lacmoid and Congo red. Isothermal titration calorimetry (ITC) experiments were carried out using VP-ITC titration microcalorimeters (MicroCal Inc., MA, USA) at 25 ⁰C. α-synuclein, Lacmoid and Congo red were dissolved in PBS buffer pH 7.4 and the samples were degassed before the measurements. Each experiment involved a preliminary 2 μl injection followed by 25-27 injections of 10 μl using a 300 μL syringe. The cell volumes were 1.416 ml or 1.4242 ml. ITC raw data was analyzed using Origin 7 (OriginLab Corporation, MA, USA).

In the first set of Congo red experiments 2 μM α-synculein (in the cell) was titrated with 0.1 mM or 0.2 mM Congo red. Additional experiments with 100 μM α-synuclein in the syringe were performed with 5 μM, 20 μM and 50 μM Congo red in the cell. In the Lacmoid experiments, 5 μM, and 100 μM α-synuclein (in the cell) was titrated by 0.1 mM and 1 mM Lacmoid respectively. For all the experiments, relevant reference experiments were performed by titrating the solution in the syringe into pure buffer.

Results of calorimetric investigation of the α-synuclein:ligand interactions. Figure 7 presents a calorimetric characterization of the interactions between small molecules and α-synuclein. Two types of experiments were performed for the Congo red:α-synuclein system. In the first set of experiments, Congo red (0.1 mM and 0.2 mM) was titrated into 2 μM α-synuclein. Control titration of Congo red into buffer showed substantially larger endothermic heats for the fist few injections compared to later ones (Figure 7A), suggesting that Congo red is in an aggregated state in the syringe at such concentrations. This type of behaviour is typically observed for disaggregation of micellar surfactants [Bijma et al 1997 J. Chem. Soc, Faraday Trans. 93:1579; Chanamai & McClements 2001 Colloids and Surfaces A: Physicochemical and Engineering Aspects 181:261-269) and similar results have previously been reported for Congo red (Kim et al 2003 JBC 278:10842).

Titrations of Congo red into solutions of α-synuclein indicate a complex interaction mechanism with several ligand molecules binding to the protein. Under these conditions, complete saturation of the binding site on α-synuclein was achieved in the 0.2 mM Congo red titration (Figure 7B).

Figure 14 is a calorimetric characterization of ligand-binding to α-synuclein. ITC raw data of 100 μM α-synuclein titrated into PBS buffer (A), 5 μM Congo red (B), 20 μM Congo red (C) and 50 μM Congo red (D), and ITC raw data of 0.1 mM Lacmoid titrated into PBS buffer (E) and 5 μM α-synuclein (F). Congo red at various concentrations (5 μM, 20 μM and 50 μM) was titrated with 100 μM α-synuclein (Figure 14). Control runs, with the protein titrating into buffer, showed that the heat effects are first exothermic and then become endothermic as the titration proceeds. This may reflect the presence of some oligomeric species in the α-synuclein sample. The 5 μM Congo red titration with 100 μM α-synuclein shows that the heats for the first three injections are slightly larger than those of the control run only, suggesting that Congo red, possibly in the monomer state, binds to α-synuclein weakly. At higher Congo red concentrations (20 μM and 50 μM), when Congo red is more likely to be in an aggregated state, the heat effects for the first three injections increased substantially suggesting a much stronger interaction under these conditions. Calculations of reliable stoichiometry and thermodynamic parameters of binding are prohibited by the complex binding mechanism.

ITC experiments of the Lacmoid: α-synuclein system confirm the lower affinity of this compound compared to Congo red. Titration of 0.1 mM Lacmoid into 5 μM α-synuclein resulted in no significant heat changes (supporting material figure S6); however, exothermic heat effects were generated when 1.0 mM Lacmoid was titrated into 100 μM α-synuclein (Figure 7D). The lack of binding site saturation on α-synuclein in these experiments prohibits the determination of any dissociation constant, and could be explained by the protein binding to large Lacmoid aggregates. However, it could also be a result of a reduced effective concentration of binding competent small species due to the presence of large assemblies. Control titration of 0.1 mM (Figure 14) and 1.0 mM (Figure 7C) Lacmoid into buffer generated exothermic and endothermic heat effects, respectively. Exothermic heat effects indicate a process dominated by the heat of dilution, while endothermic heat effects suggests additional contributions from dissociation of aggregated Lacmoid, as previously described for Congo red.

Example 7. Formation of supramolecular assemblies of Lacmoid and Congo red shown by dynamic light scattering. The ability of Congo red to form aggregates or micelle-like species is well known (Iyer & Singh 1970 Kolloid-Z. u, Z. Polymere 242:1196-1200; Edwards and Woody, 1979 Biochemistry 18:5197, Skorownek et al 1998 Biopolymers 46:267-281, McGovern et al. 2002 J Med. Chem. 45:1712-1722 ; Stopa et al Acta. Biochim. Pol. 5:1213) and self-assembly has as well been reported for some phenothiazines (Barbosa et al. 2008 J. Phys. Chem. B. 112:4261). The occurrence of supramolecular structures of Lacmoid and Congo red could certainly modulate the interaction with α-synuclein and their propensities to self-associate were therefore investigated using DLS.

Dynamic light scattering (DLS) was measured using an ALV/CGS-3 compact gonimeter system equipped with ALV/LSE-5004 multiple tau digital real time correlator, operating at 632.8 nm wavelength (ALV-GmbH). The samples were filtered through 0.2 μm filter before the measurements and the scattered light was detected at 150° angle. The samples contained 1 mM or 0.1 mM Congo red or 0.5 - 0.6 mM Lacmoid in 25 mM Tris pH 7.4, 100 mM NaCl and the data was recorded at 25 ⁰C. The acquired data was analyzed by the regularization algorithm in the ALV correlator 3.0 software (ALV-GmbH). Since the laser wavelength of this instrument overlaps with the absorbance spectrum of Lacmoid, additional data was acquired using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd.) operating at 532 nm and a detection angle of 173°. Data were exported and analyzed using the ALV software. The presented data represents averages of several measurements.

Results of DLS Analysis. In 1 mM solution, Congo red forms two types of species. The smaller has a R_H distribution centred around 1.9 nm (Figure 6A), which is in good agreement with the value of 1.4 nm reported by Stopa and co-workers (Stopa et al. Acta Biochim. Pol. 2003). The larger particles have a R_H of approximately 40 nm (Figure 6A). The results obtained for 0.1 mM Congo red are similar to the ones at 1 mM but with the average size of the smaller species slightly shifted towards smaller particles (~1.4 nm) and the relative populations are altered in favour of the smaller particles (Figure 6A). The use of DLS to investigate Lacmoid self-assembly was problematic due to the overlap of its absorbance spectrum with the laser wavelengths of the DLS instruments. However, measurements from two instruments, operating at different wavelengths, indicate that Lacmoid, at 0.5 mM concentration, indeed forms supramolecular aggregates. The average R_H of these species appears to be approximately 15 nm and 90 nm for the two populations respectively (Figure 6B). The DLS data recorded for 0.1 mM Lacmoid were inconclusive, most likely because of sample absorbance, and neither confirms nor excludes the presence of aggregated species.

Example 8. Computational modeling ofa-synuclein interactions with certain compounds. Recent progress in NMR and computational methodologies used to characterize the conformational space of intrinsically unstructured proteins has produced ensemble structure representations of α-synuclein (Dedmond et. al. 2005 JACS 127:476; Bertoncini et al. 2005 PNAS 102: 1430-1435). Applicants have developed methodology for utilizing such structure representations as a basis for computational docking, α-synuclein conformations used in the docking calculation were optimized with the steepest descent minimization using the MMFF94 molecular mechanics force field (Halgren, 1996 J. Comp. Chem., 17: 490-641; Halgren 1999 J. Comp. Chem. 20: 720-748) and a distance dependent dielectric model in MOE (Computational Chemistry Group, 2005). The low energy conformational ensemble of Putrescine, Spermidine, Congo red and Lacmoid was generated using the Systematic Conformational Search module in MOE. Rotational bonds were systematically explored by 30 degree intervals. Each generated conformation was minimized using the MMFF94 and a distance dependent dielectric model and only those were kept which had a root mean square deviation of more than 0.3 A between their heavy atoms. AM1/BCC charges were used for the small molecules (Jakalian, et al 2000 J. Comput. Chem. 21 : 132 - 146). Docking calculations were performed with FRED 2.0 (OpenEye Scientific Software, Inc., Santa Fe, NM). FRED was used with a translational step size of 0.5 A and a rotational step size of 0.75 A to exhaustively sample the low energy conformational ensemble of the ligands within the protein binding site. After the initial round of docking FRED optimized the 2500 highest scoring docked poses with the ChemGauss3 scoring function. The 300 highest scoring docked poses, on the basis of consensus scoring function (Shapegauss, Chemgauss3, Oechemscore, Screenscore), were optimized further. First AM1/BCC charges were calculated for each ligand conformation used in the subsequent optimization step. Optimization was performed by minimizing each docked pose inside its rigid protein receptor using SZYBKI 1.2.0 (OpenEye Scientific Software, Inc.). During the SZIBKI minimization the MMFF94 molecular mechanics force field and the Poisson-Boltzman solvation model was used. The protein was kept rigid while all atoms of the ligand were flexible. Conformations with the lowest E_SZIBKI energy were considered as the prediction of the binding mode of the new molecule. ESZIBKI was defined by the following equation: E_SZIBκi = E_intermoi_ecuiar interaction + E_soi_v + E_llgand, where E_intermoiecuiai interaction contains all non-bonded interactions between the ligand and the protein, E_soi_Y is the interaction energy associated from the electrostatic part of ligand-solvent interactions, and where Ei;_gand is the potential energy of the ligand.

Results of computational modeling of the α-synuclein: ligand interactions. Applicants modeled the α-synuclein:ligand interactions by docking two polyamines, Congo red and Lacmoid onto 22 selected α-synuclein structures. Selected α-synuclein structures were from among 100 conformations derived randomly from about 40,000 conformations generated with restrained molecular dynamics simulations using NMR derived paramagnetic relaxation enhancement distance thresholds (Dedmond et. al. JACS 2005, 127, 476). Figure 13 in the supporting material illustrates the correlation between the radius of gyration (R_g), solvent accessible surface area (SASA) and the number of non-bonded contacts of the 100 analyzed conformations. Out of the 22 conformations selected for docking simulations, 18 are compact and 4 are less compact.

First the polyamines, putrescine and spermidine, were docked to α-synuclein as a positive control for the employed methodology. These compounds have previously been reported to bind to the C-terminal part of the protein with weak binding affinity (Fernandez et al 2004 EMBO J. 23:2039). Both polyamines were predicted by the docking results to bind exclusively to the C- terminal region of several of the α-synuclein conformations. Figure 5 A and B illustrate the predicted highest affinity binding mode of Spermidine, in which the ligand forms hydrogen bonds with side- chain hydroxyl group of Serl29, backbone carbonyl group of Alal24, and salt bridges with the side chain carboxyl groups of Aspl21, Aspl30, Aspl35, and Glul23. The docking predictions reproduce the reported NMR results (Fernandez et al 2004 EMBO J. 23:2039) remarkably well. Congo red was found to bind to only one of the tested conformations, which indicates some degree of specificity. This protein conformation is well folded and compact in the N-terminal and NAC regions, while quite unfolded in the C-terminus. The Congo red molecule binds in a pocket created mostly by parts of the N-terminal, NAC and the beginning of the C-terminal regions (Figure 5C). The bi-phenyl part of Congo red is deeply buried in a hydrophobic pocket formed by residues Gly7, Leu8, Ser9, Ala53, Val74 and GlulO9 while one of the charged sulfonate groups of the molecule is solvent exposed (Figure 5D). The results clearly show how a single Congo red molecule could interact with residues that are sequentially well separated and thus provide an explanation for the changes in NMR observables across sequence of α-synuclein. Good agreement is observed between the regions of α-synuclein predicted to be responsible for Congo red interaction in the in silico data and the binding profile deduced from the NMR experiments.

Lacmoid is observed to bind to three different α-synuclein conformations approximately equally well. It binds mostly to the N-terminal regions of two of the α-synuclein conformations and also to the C-terminal of the third conformation. Interestingly, Lacmoid was found to bind to the same α-synuclein conformation as Congo red did but to an alternate site. In this conformation Lacmoid is predicted to bind to the N-terminal region and to the beginning of the NAC and C- terminal regions (Figure 5 E). It forms hydrogen bonds with the backbone carbonyl group of Ala 18 and the side chain hydroxyl group of Ser9 and a T-shaped aromatic-aromatic interaction with Tyr39 (Figure 5F). There is considerable agreement between the computational and the NMR results as both studies find the N-terminal part of α-synuclein to be most affected by Lacmoid binding.

Example 9. Congo red and Lacmoid modulate the aggregation of a-synuclein. Aggregation of α-synuclein was assayed in 100 μM protein samples in 20 mM Tris buffer pH 7.4, 100 mM NaCl with the addition of 0.01% NaN₃. 500 μl of protein sample were incubated in at 37 ⁰C under constant shaking at 300 rpm. 50 μl aliquots were withdrawn on a daily basis, assayed for ThioT fluorescence and stored at 4 ⁰C until the end of the assay for further determinations (SDS- PAGE and TEM). Fibril formation was monitored by the Thioflavin-T (Thio-T) assay (Conway et al 2000, PNAS 97:571-575.; Hoyer et al, 2002, J. MoL Biol 322:383). Briefly, 10 μl aliquots were diluted in 1 ml of 20 μM Thio-T and the fluorescence was measured in a FlashScan spectroflourimeter (Jena Analytik), with an excitation wavelength of 446 nm. Emission wavelengths from 460 to 600 nm were collected and the integrated fluorescence between 470 and 490 nm was employed for determination of the relative content of α-synuclein fibrils in the sample.

Quenching of ThioT fluorescence by Lacmoid and Congo red was assayed by incubating preformed fibrils for 30 minutes with various concentrations of compound (1 μM to 1 mM), and measuring the ThioT fluorescence of the sample.

The relative amount of aggregated (insolubilized) protein was assayed by centrifuging the samples at 16,00Og and resolving the supernatant fraction in 4-12 % SDS-PAGE (Novex, Invitrogen). The amyloid-aggregated material was determined by resistance to solubilization with 1 % Sarkosyl, by resolving the soluble fraction (non-amyloid) in a SDS-PAGE. Image quantization was performed on Coomassie-stained gels with the software ImageJ (NIH).

Transmission electron microscopy (TEM) of aggregated samples was performed by depositing 10 μl of a 1 :10 dilution sample on Formvar-coated nickel grids (Agar scientific), and staining with 2 % (w/v) uranyl acetate. Negative-staining images were obtained at 25,000 x magnification using a Phillips CEMlOO transmission electron microscope (Imaging facility, Dept. of Pathology, University of Cambridge).

Figure 2 shows NMR data for the binding of Lacmoid and Congo red to monomelic α- synuclein. The data indicate that Congo red interacts differently with the N-terminus and NAC region of α-synuclein than does Lacmoid. The interaction of these small molecule binding ligands with α-synuclein are indicated by changes in amide chemical shifts and peak intensities in the α-synuclein ¹H-¹⁵N HSQC spectrum. Figure 2A plots the relative peak intensity with a 2:1 (blue), 5:1 (red) and 10: 1 (green) molar excess of Congo red over α-synuclein. Figure 2B plots the relative peak intensity with 1 :1 (blue), 4:1 (red), 6:1 (green) and 16:1 (magenta) molar excess of Lacmoid. Figure 2C shows chemical shift changes with 2:1 (blue), 5:1 (red) and 10:1 (green) molar excess of Congo red. Figure 2D shows chemical shift changes with 1:1 (blue), 4:1 (red), 6:1 (green) and 16:1 (magenta) molar excess of Lacmoid. The reported chemical shifts changes are weighted averages of the ¹H and ¹⁵N chemical shift changes ([AO(¹H)² +(Δδ(¹⁵N)/5)² f'²).

Results of the Aggregation Assays. Lacmoid and Congo red have previously been reported to inhibit amyloid fibril formation of α-synuclein (Masuda et al. 2006 Biochemistry 45:6085). The effect of the two compounds on α-synculein aggregation and fibrillation was investigated by incubating the protein in the presence of 0.5 μM to 1 mM concentration of the compounds (corresponding to 0.005 to 10:1 ligand:protein molar ratios). ThioT fluorescence was monitored ex-situ and inhibition of amyloid formation was determined from the reduced fluorescence of this amyloid-specific dye. According to this, both compounds seem to have substantial effect on α-synuclein fibril formation, even at very low stoichiometric ratios (Figure 8 A, B). For Congo red the ThioT fluorescence is significantly reduced already at 10 μM concentration and at 0.1 mM there is almost no observable time dependence in the fluorescence intensity (Figure 8A). Lacmoid shows a somewhat less potent inhibition profile (Figure 8B) but as the concentration is increased there is an accompanying decrease in the fluorescence intensity.

In contradistinction to the ThioT fluorescence results, protein aggregates were visible at almost all the concentrations assayed. This may be due to a population of ThioT -negative, non-amyloid aggregates or simply to the specific reduction of ThioT fluorescence by the compounds as a result of quenching or competition for binding competent sites in the fibrillar species. The possibility of interference with ThioT fluorescence by the compounds was checked by adding dye to mature compound- free α-synuclein fibrils, pre-incubated with Lacmoid or Congo red, and compare the observed fluorescence with a negative (compound free) control. The results clearly show that high concentrations of the compounds alter the final ThioT fluorescence of the fibrils, either by collision quenching with the dye, or by inhibiting its ability to bind the fibrils (Figure 8C, D). However, the data at low concentrations (0.5 - 10 μM) is still valid since interference with ThioT is not as strong, and indicates a slight reduction in fibril formation by both compounds.

To further verify the presence of aggregated material, the amount of soluble protein in the samples was evaluated at different time points of the aggregation assays by spinning an aliquot at 16,00Og and resolving the protein remaining in the supernatant using SDS-PAGE. The results show that even though the ThioT fluorescence indicates reduced formation of fibrils, the protein still aggregates and insolubilizes (Figure 15). Only in samples containing Lacmoid at 0.5 mM concentration (stoichiometry 5:1) does α-synuclein remain in a monomeric soluble form until the end of the assay.

Indeed, treatment of aggregated protein with 1 % Sarkosyl, a detergent capable of solubilising amorphous aggregates but not mature amyloid fibrils, does show an increased amount of soluble protein both in control experiments and with the compounds. While Lacmoid addition does not show differences with the control at concentrations of 50 μM or lower Congo red seems to increment the amount of Sarkosyl soluble protein even at low concentrations (Figure 15).

Such observations are corroborated by TEM of the aggregated samples at the end of the assays. In the control experiment, without any compounds added, α-synuclein formed fibrils similar to what is reported in other studies (Figure 8E) (Hoyer et al. 2002 JMB 322:383; Uversky et al. 2001 JBC 276:10737-10744). Incubation with low concentration (5 μM) of Lacmoid does not change the nature of the fibrillar material that is formed (Figure 8E). However, incubation with 10 μM Congo red produces amorphous aggregates as well as amyloid fibrillar species, supporting the notion of distinct modes of action by the different compounds (Figure 8E). At higher concentrations of Congo red, some fibrils are visible but amorphous aggregates are still the dominating morphology (Figure 14). Interestingly, in the samples incubated with 0.5 mM Lacmoid, no aggregates are visible (Figure 14), confirming its ability to reduce the overall protein insolubilization at high compound concentrations.

In summary, the results show that Congo red is able to alter the aggregation pathway of α- synuclein by increasing the population of amorphous aggregates and reducing the formation of fibrils. Lacmoid, on the other hand, functions mainly through reducing the overall self- association rate of the protein, and is capable of inhibiting formation of both intermediates and mature fibrils.

Example 10: NMR Screening of Compounds. 50 μM ¹⁵N-labeled α-synuclein was used. Compounds were added to the protein sample individually or in pooled samples of 5 compounds at the same time. The final concentration of each compound was 500 μM (i.e. 10:1 compared to α-synuclein). HSQC spectra were recorded on the 500 MHz NMR spectrometer equipped with cryo probe. The compounds were originally from a 20 mM DMSO stock solution. The effect of DMSO on the NMR HSQC spectra was insignificant. The pH of the NMR samples were set for each measurement. One hit, Compound C 14, having a molecular weight of about 251 was obtained from the ten compounds screened one by one and sixty compounds screened in multiplexed samples. As shown in Figure 17, Compound C 14 binds monomeric α-synuclein (indicated by the spectral shifts detected above the reference line in the 13CO-15N correlation spectra). Computational modeling suggests that Compound C 14 binds α-synuclein in the regions of about amino acid residues 43-51, 34-50 and 88-98.

Example 11: Evaluation of anti-aggregation capabilities of compounds corresponding to in silico hits. Aggregation of α-synuclein was assayed in 20 μM protein samples in 20 mM Tris- HCl, pH 7.4, 100 mM NaCl, 350 μM SDS, 20 μM ThioT with 0.01% Na-azide. Compounds were screened at two concentrations, 20 μM and 200 μM, in duplicates. The protein-small molecule samples were incubated at 37⁰C under constant shaking at 300 rpm. Under these conditions α-synuclein aggregates within 60 hours and forms long amyloid fibrils. Fibril formation was assayed by ThioT fluorescence. As a secondary screen, aggregation of α- synuclein was assayed in 100 μM protein samples in 20 mM Tris-HCl, pH 7.4, 100 mM NaCl buffer, no SDS. Compounds were screened at 200 μM, in duplicates. Thio T fluorescence was performed ex-situ (5 μl diluted in 200 μl of 20 μM ThioT) To date Compounds 1-30 have completed both the primary screen and the secondary screen, yielding 3 validated hits.

Compounds 30-57 have completed the primary screen, yielding 2 hits. Compound C4 strongly inhibits α-synuclein aggregation as shown in Figure 19. In addition, Compound C4 binds to monomeric α-synuclein as shown in the NMR spectra depicted in Figure 20. Some compounds bound monomeric α-synuclein as determined by the NMR spectra but did not inhibit α-synuclein aggregation.

Example 12. Small molecule inhibitors of a-synuclein aggregation increase lag phase of ThT fluorescence. Of 110,000 compounds screened by surface plasmon resonance, 500 hits were obtained. For proof of concept studies, 30 were chosen for follow-up analyses as broadly representative of the classes of positively-reacting compounds present in the screen. Selection was based on a class analysis which included factors such as SPR signal strength, structural diversity, and the presence of desirable physico-chemical properties. Soluble forms of reactive compounds were synthesized for further analysis.

Overall analysis of the amyloid aggregation results. Several compounds displayed a very interesting anti-amyloid effect, extending the lag phase of Thioflavin-T fluorescence by 20 to 60 hours, before aggregation of the protein occurs, without altering the final amyloid content at the end of the assay, as determined by the final fluorescence value. This suggests that the compounds retard effectively the initial steps in the self-oligomerization of the protein, prior to amyloid formation.

Compounds 1, 2, 3, 5, 10, 11, 14, 16, 18, 19, and 21 showed a significant extension of the lag phase for the aggregation of α-synuclein. Compounds 5 and 10 showed, in addition, reduction of the final ThT levels. Compound 26 showed only reduction of the ThT levels.

Figures 21-32 present NMR analysis of the binding of Compounds 1, 2, 3, 5, 10, 11, 14, 16, 18, 19, and 21 to α-synuclein.

INCORPORATION BY REFERENCE

Throughout this application, various references including publications, patents, and pre-grant patent application publications are referred to. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. It is specifically not admitted that any such reference constitutes prior art against the present application or against any claims thereof. All publications, patents, and pre-grant patent application publications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the case of inconsistencies the present disclosure will prevail. The publication: Lendel C, Bertoncini CW, Cremades N, Waudby CA, Vendruscolo M, Dobson CM, Schenk D, Christodoulou J, and Gergely Toth, On the Mechanism of Nonspecific Inhibitors of Protein Aggregation: Dissecting the Interactions of α-Synuclein with Congo Red and Lacmoid, 48 Biochemistry 8322-8334 (2009), is specifically incorporated by reference for all purposes. INDUSTRIAL UTILITY

The present invention has industrial applicability in providing methods for determining drugs that stabilize non-pathological forms of amyloidogenic polypeptides. The present invention has industrial utility in providing drugs that stabilize non-pathological forms of amyloidogenic polypeptides.

Claims

CLAIMS We claim:

1. A method to identify an agent which stabilizes a non-pathological form of an amyloidogenic polypeptide, comprising:

(a) performing at least one biophysical method for detecting an interaction between a test agent and a non-pathological form of an amyloidogenic polypeptide;

(b) selecting a test agent that specifically interacts with the non-pathological form of the amyloidogenic polypeptide; and

(c) determining the effect of the selected test agent on aggregation of the amyloidogenic polypeptide,

wherein a decrease in aggregation is indicative of an agent that stabilizes the non- pathological form of the amyloidogenic polypeptide.

2. The method of Claim 1 , wherein the biophysical method is nuclear magnetic resonance (NMR) spectroscopy.

3. The method of Claim 1 , wherein the biophysical method is fluorescence spectroscopy.

4. The method of Claim 1, wherein the biophysical method is circular dichroism (CD) spectroscopy.

5. The method of Claim 1 , wherein the biophysical method is isothermal titration calorimetry (ITC).

6. The method of Claim 1 , wherein the biophysical method is computational docking.

7. The method of Claim 1 , wherein specific interaction of the selected test agent with the non-pathological amyloidogenic polypeptide is demonstrated by at least two biophysical methods.

8. The method of Claim 7, wherein the at least two biophysical methods include computational docking and NMR spectroscopy.

9. The method of Claim 2, wherein the selected test agent alters the NMR spectrum of the amyloidogenic polypeptide.

10. The method of Claim 1 , wherein the non-pathological form is a monomelic form.

11. The method of Claim 1 , wherein the non-pathological form is a tetrameric form.

12. The method of Claim 1, wherein the amyloidogenic polypeptide is a proteolytic peptide product of transthyretin.

13. The method of Claim 10, wherein the amyloidogenic polypeptide is α-synuclein.

14. The method of Claim 10, wherein the amyloidogenic polypeptide is amyloid beta peptide (Aβ).

15. The method of Claim 10, wherein the amyloidogenic polypeptide is microtubule associated protein Tau.

16. The method of Claim 13 , wherein the selected test agent interacts with the amyloidogenic polypeptide at an N-terminal region.

17. The method of Claim 13 , wherein the selected test agent interacts with the amyloidogenic polypeptide at a region within amino acid residues 1-40.

18. The method of Claim 13 , wherein the selected test agent interacts with the amyloidogenic polypeptide at a region within amino acid residues 50-100.

19. The method of Claim 13 , wherein the selected test agent interacts with the amyloidogenic polypeptide at a region within the NAC region.

20. The method of Claim 13, wherein the selected test agent interacts with the amyloidogenic polypeptide at a region within amino acid residues 50-77.

21. The method of Claim 1 , wherein the effect on aggregation is determined by monitoring ThioT fluorescence, and wherein a reduced fluorescence is indicative of an agent that stabilizes the non-pathological form of the amyloidogenic polypeptide.

22. The method of Claim 21 , wherein the agent is a stabilizer of the non-pathological form of the amyloidogenic polypeptide if ThioT fluorescence is reduced in the presence of less than 1 mM of the agent.

23. The method of Claim 21 , wherein the agent is a stabilizer if ThioT fluorescence is reduced in the presence of less than O.lmM of the agent.

24. The method of Claim 2, wherein said amyloidogenic polypeptide comprises ¹⁵N.

25. A method to identify an agent which stabilizes a non-pathological form of an amyloidogenic polypeptide, comprising:

(a) determining the effect of a test agents on the NMR spectrum of an amyloidogenic polypeptide;

(b) a test agent for which, in its presence, the amyloidogenic polypeptide has an altered NMR spectrum; and

26. The method of Claim 25, wherein the NMR spectrum is an HSQC spectrum.

27. The method of Claim 1, wherein said amyloidogenic polypeptide is selected from the group consisting of serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, apolipoprotein Al (ApoAl), transthyretin, lysozyme, fibrogen a chain, gelsolin, cystatin C, amyloid β protein precursor (β-APP), β₂-microglobulm, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, microtubule associated protein Tau, huntingtin, α-synuclein, mutants thereof, and proteolytic peptide products thereof.

28. The method of Claim 27, wherein said amyloidogenic polypeptide is α-synuclein.

29. The method of Claim 24, wherein said amyloidogenic polypeptide is uniformly ¹⁵N- labeled.

30. A pharmaceutical composition comprising an agent that stabilizes the non-pathological form of an amyloidogenic polypeptide, wherein the stabilizing agent:

(a) decreases the aggregation of the amyloidogenic polypeptide relative to the aggregation observed for the amyloidogenic polypeptide in the absence of the stabilizing agent; and

(b) has one or more of the following properties:

(i) ability to alter the NMR spectrum of the amyloidogenic polypeptide relative to the NMR spectrum in the absence of the stabilizing agent;

(ii) ability to alter the CD spectrum of the amyloidogenic polypeptide relative to the CD spectrum in the absence of the stabilizing agent;

(iii) thermodynamic characteristics consistent with specific binding of the stabilizing agent to the non-pathological form of the amyloidogenic polypeptide; and

(iv) in silico binding to a desired conformation of the non-pathological amyloidogenic polypeptide generated by computational modeling.

31. The pharmaceutical composition of Claim 30, wherein the amyloidogenic polypeptide is α-synuclein, Aβ, AA, AL or tau.

32. The pharmaceutical composition of Claim 30, wherein the amyloidogenic polypeptide is α-synuclein, the stabilized non-pathological form of the α-synuclein is monomeric α-synuclein, and the stabilizing agent produces chemical shift changes in the NMR spectrum of α-synuclein in the region comprising amino acid residues 1-100.

33. A method of treating or preventing a disease characterized by oligomerization, aggregation or deposition of an amyloidogenic polypeptide, comprising:

administering a stabilizing agent that alters the NMR spectrum of the amyloidogenic peptide and decreases aggregation of the amyloidogenic peptide relative to the NMR spectrum and aggregation observed for the amyloidogenic peptide in the absence of the stabilizing agent.

34. The method of claim 33, wherein the stabilizing agent is a small molecule having a molecular weight of less than about 1000 daltons.

35. The method of claim 33, wherein the stabilizing agent is a polypeptide.

36. The method of claim 34, wherein said stabilizing agent is an antibody.

37. The method of Claim 33, wherein said amyloidogenic polypeptide is selected from the group consisting of serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, apolipoprotein Al (ApoAl), transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, Amyloid β protein precursor (β-APP), β₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, alpha-synuclein, microtubule associated protein Tau, huntingtin, mutants thereof, and proteolytic peptides thereof.

38. The method of claim 37, wherein said amyloidogenic polypeptide is alpha-synuclein.

39. A method of stabilizing the monomeric form of an amyloidogenic polypeptide, comprising:

contacting said amyloidogenic polypeptide with an agent identified by the method of

Claim 1.

40. The method of claim 39, wherein the contacting is achieved by administration of the agent to a mammal.

41. The method of Claim 39, wherein said amyloidogenic polypeptide is selected from the group consisting of serum amyloid A protein (ApoSSA), immunoglobulin light chain, immunoglobulin heavy chain, apolipoprotein Al (ApoAl), transthyretin, lysozyme, fibrogen α chain, gelsolin, cystatin C, Amyloid β protein precursor (β-APP), β₂ microglobulin, prion precursor protein (PrP), atrial natriuretic factor, keratin, islet amyloid polypeptide, alpha-synuclein, microtubule associated protein Tau, huntingtin, mutants thereof, and proteolytic peptides thereof.

42. The method of claim 41 , wherein the amyloidogenic polypeptide is alpha-synuclein.

43. The method of Claim 410, wherein the amyloidogenic polypeptide is amyloid beta peptide (Aβ).

44. A method of manufacturing a pharmaceutical comprising providing a pharmaceutically acceptable excipient and an agent that alters the NMR spectrum of the amyloidogenic polypeptide and decreases aggregation of the amyloidogenic polypeptide relative to the NMR spectrum and aggregation observed for the amyloidogenic polypeptide in the absence of the agent.

45. A method to identify an agent which stabilizes a non-pathological form of an amyloidogenic polypeptide, comprising:

(a) comparing the NMR spectrum of said amyloidogenic polypeptide in the presence and absence of at least one test agent;

(b) selecting one or more test agents for which, in their presence, the amyloidogenic polypeptide has an altered NMR spectrum; and

(c) comparing aggregation of the amyloidogenic polypeptide in the presence and absence of the selected test agent,

wherein a decrease in aggregation in the presence of the selected test agent relative to that observed for the amyloidogenic polypeptide in the absence of the selected test agent is indicative of an agent which stabilizes a non-pathological form of the amyloidogenic polypeptide.

46. The method of Claim 7, where the at least two biophysical methods include NMR spectroscopy and ITC.

47. The method of Claim 1 , wherein the biophysical method is surface plasmon resonance.

48. The method of Claim 7, where the at least two biophysical methods include NMR spectroscopy and surface plasmon resonance.

49. The method of Claim 1 , wherein the biophysical method is dynamic light scattering.