Amytoidosis-mhihiting Polypeptides and Their Use

Cross-Reference

This application claims priority to U.S. Provisional Patent Application Serial No. 61/785,356 filed March 14, 2013, incorporated by reference herein in its entirety.

Statement of Government Support

This invention was made with government support under grant number CBET- 0966977 awarded by the National Science Foundation. The government has certain rights the invention.

Background

There are now over 40 different human amyloid diseases, each linked to the buildup of a specific precursor protein or peptide. These diseases involve the conversion of a protein from its soluble native state into insoluble amyloid fibrils, or, in the case of peptides, the conversion from a soluble, loosely structured form to fibrils. Given that many different sequences can form amyloid fibrils of similar architecture, there may be some common structural features of the prefibriilar amyloidogenic intermediates. X-ray fiber diffraction indicates that the insoluble, mature amyloid fibrils are composed of cross β-sheet structure. Therefore, it is widely held that the formation of amy loid fibrils involves a transition to β- sheet structure in the amy loidogenic intermediate. However, the mechanism of self-assembly at the atomic level remains elusive. Another feature of these diseases is that soluble oligomeric intermediates, not the insoluble well-ordered fibrils, are preferentially responsible for cellular toxicity. Similarly, the soluble oligomeric forms of the prion protein are the most infectious per unit protein. As such, fibrils may be protective, at least up to a point, as their breakdown to smaller aggregates yields greater toxicity and infectivity.

Summary of the Invention

In a first aspect, ihe invention provides isolated polypeptides, comprising or consisting of 12-23 contiguous amino acids according to the general formula X1-X2-X3-X4- X5 (SEQ ID NO: 1 ), wherein XI is 0-7 contiguous amino acid residues that do not alternate between L and D residues:

X2. is 5-9 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 0-5 contiguous amino acid residues that do not alternate bet een L and D residues;

X4 is 4-12 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 0-4 contiguous amino acid residues that do not alternate between L and D residues;

wherein the isolated polypeptide is not:

RGE(m)N(l)S(w)MNEYSGW(t)M(n)L(k)MGR (SEQ ID NO: 2).

In one embodiment, I is 0-2 contiguous amino acid residues that do not alternate between L and D residue. In another embodiment, all XI amino acid residues are L amino acids. In a further embodiment, X2 is 6-8 contiguous amino acid residues alternating between D amino acids and L amino acids. In another embodiment, X3 is 1-5 contiguous amino acid residues that do not alternate between L and D residues. In a further embodiment, all X3 amino acids are L amino acids. In various other embodiments X4 is 6- 12 contiguous amino acid residues alternating between D amino acids and L amino acids; X5 is 0-3 contiguous amino acid residues that do not alternate between L and D residues and all X5 amino acids are L amino acids.

In one embodiment, XI is absent; X2 is 5-7 contiguous amino acid residues alternating between D amino acids and L amino acids X3 is 0-3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 6-1 1 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is 0 or 1 amino acid residue.

In another embodiment, X 1 is absent; X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 9 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In a further embodiment, XI is absent; X2 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 10 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent. In a still further embodiment, Xi is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids; X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 9 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids.

In another embodiment, XI is absent; X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 4 contiguous amino acid residues that do not alternate between L and D residues; X4 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In one embodiment, XI is absent; X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is I contiguous amino acid residues that do not alternate between L and D residues: X4 is 4 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In a further embodiment, XI is absent; X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 5 contiguous amino acid residues that do not alternate between L and D residues; X4 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In another embodiment, XI is absent; X2 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 1 1 contiguous amino acid residues alternating between D amino acids and L amino acids; and XS is 1 amino acid residue.

In a still further embodiment, XI is absent; X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 1 amino acid residue that do not alternate between L and D residues; X4 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In one embodiment, XI is absent; X2. is 6 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 10 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is 1 amino acid residue.

In another embodiment, XI is absent; X2 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues; X4 is 12 contiguous amino acid residues alternating between D amino acids and L amino acids; and X5 is absent.

In various embodiments, the polypeptide comprises or consists of 14-2.3, 17-23, or 21- 23 contiguous amino acids according to the recited formula.

In another aspect, the invention provides pharmaceutical compositions, comprising:

(a) the isolated polypeptide of any embodiment or combination of embodiments of the invention; and

(b) a pharmaceutically acceptable carrier.

In a further aspect, the invention provides methods for treating an amyloid disease, comprising administering to a subject with an amyloid disease an amount effective of the isolated polypeptide of any embodiment or combination of embodiments of the invention, or the pharmaceutical composition of the invention to treat the amyloid disease. In various embodiments, ihe amyloid disease may be selected from the group consisting of Creutzieldt - Jakob disease, spongiform encephalopathy, Huntington's disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado- Joseph disease, Alzheimer's disease, bovine spongiform encephalopathy, scrapie, t e 2. diabetes, amyloidosis caused by transthyretin (ATTR), Parkinson's disease, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, and multiple myeloma.

In another aspect, the invention provides methods for diagnosing or prognosing an amyloid disease, comprising

(a) contacting a tissue sample from a subject at risk of having an amyloid disease with the isolated poKpeptide of any one of claims 1-48, or the pharmaceutical composition of claim 49, under conditions suitable for binding of the isolated polypeptide with an amyloid intermediate, if present in the tissue sample, to produce a binding complex

(b) def ecting binding complexes in the tissue sample; and

(c) diagnosing or prognosing an amyloid disease based on the detecting.

In various embodiments, the tissue sample may be a blood sample, serum sample, nasal secretions, urine or a cerebral spinal fluid sample. Description of the Figures

Figure 1. a-sheet conversion, conformational properties and peptide designs. (A) β- to a-sheet conversion of transthyretin (TTR), which is implicated in systemic amyloidosis and familial peripheral polyneuropathy, as reported by Armen and co-workers (11, 13). The protein backbone is shown in cartoon representation with the region of interest (residues 105- 121 ) shown as sticks. At 0 ns (top left) the residues of interest form a β-hairpin. The dihedral angles for 1 ns of dynamics of these residues are found mainly in the β-region of the Ramachandran plot (top left quadrant, lower left panel; increasing frequency of occupancy is shown from blue through red) with several turn residues in the Q conformation (bottom left quadrant). After 30 ns (top right) the β-sheet has converted to an a-sheet. The dihedral angles for 1 ns of dynamics of the same residues reveal that the majority of residues have moved from the β-region to the O,L (top right quadrant) or C R region of the Ramachandran plot (lower right panel). (B) Intrinsic residue propensities for L- and D-alanine were calculated from 100 ns of MD simulations of a GGXGG (SEQ ID NO: 3) peptide system (18) (D- alanine was simulated using the same protocol). The backbone structure (upper panels) as well as the Ramachandran plot of the conformation of the alanine residue during ihe entire simulation, demonstrate the conformational preference for L-alanine to adopt the GIR conformation and for D-alanine to favor the ο¾ _. . conformation (lower panels). (€) Peptide designs reported in Example 1. Designs are linear single turn hairpins or closed cyclic hairpins, i.e., a cyclic peptide backbone resulting in two turns, as illustrated by o:3. Hairpin peptides are N- and C-terminally acetylated and amidated, respectively, except for β, which had a free N -terminus. D-amino acids are denoted by lower case and underlined, and turn residues are identified in red and blue.

Figure 2. a~Sheet designs inhibit amyloid formation and selectively bind toxic species. (A) Peptide designs (800 μΜ) were co-incubated at pH 4.5 with 40 μΜ TTR (monomer) at 37 °C and aggregation was monitored by Congo red binding. Error bars indicate the standard deviation. (B) Toxicity of the TTR solution after 2.4 hr pre-incubation at pH 4.5 against the human neuroblastoma cell line SH-SY5Y in a MTT metabolic viability assay. (C) Thioflavin T (ThT) monitoring of 10 μΜ of the A-beta (1 -42) peptide implicated in Alzheimer's Disease (hereafter referred to as Αβ) aggregation and inhibitory effects of 100 μΜ designed peptides present from the beginning of the aggregation at 37 °C. (D) Αβ toxicity after 6 hrs of aggregation, as probed using the MTT assay and the SH-SY5Y cell line. Ail data represent average ± s.d. and (* indicates P < 0.05, determined using Student's t-test). Figure 3. Immobilized designs bind species from solutions. Peptide designs were immobilized via lysine residues on aldehyde- functionalized agarose beads and their ability to bind TTR or Αβ from solution at various stages of aggregation was probed using dot blot analysis. The presence of TTR or Αβ in the initial flow through (FT), sequential buffer washes (W), and sequential guanidine hydrochloride elution steps (E1 -E2 and E3-E4) (x- axes) was detected by the integrated peak density of the dot blot analysis (y-axes). E1-W9 are within the linear range of the immunoehemistry. (A) All three a-sheet designs, al, a2 and aJ more strongly bound species from the 24 h pre-aggregated, toxic TTR solutions than did either control. (B) Similar results were observed with the α-sheet designs binding to toxic Αβ solutions pre- ggregated for 6 hrs. Despite the inhibitory effects seen with the β design in the A assay, little Αβ from a pre- aggregated solution bound to the immobilized β design. (C) Comparison of binding from a fresh (0 h), or pre-aggregated (6 h), toxic Αβ solution, ? is the only design that preferentially bound fresh Αβ over the aggregated toxic form, indicating that the inhibition observed was due to interactions with monomelic Αβ, (D) In contrast to the β control, al preferentially bound the pre-aggregated, toxic form of Αβ compared to fresh, monomeric Αβ.

Figure 4. Designed peptides display unique spectroscopic signatures expected for a-sheet. (A) CD spectra for peptide designs reveal a random coil structure for rc, and characteristic β-stracture spectrum for β _ν with some random coil from the ends of the structure. In contrast, al, a,2 and a3 have largely featureless CD spectra with some random coil content expected to arise from the turns and tail residues. Note the different scales for the y-axes. Ail spectra are presented as molar ellipticity, highlighting the difference in intensity of the random coif component for each design. (B) FTTR spectra of the peptide designs, displayed as both absorbance (black line) and the second derivative (colored line), correlate well with the CD spectra. The β design shows a strong signal at 1632 cm ^"1 , as expected for β- structure. The a-sheet designs have signals near 1640 and 1675-1680 cm ^"1 and the absorption is more intense for a! and o2. (C) Fingerprint (top) and NH region (bottom) of the ^J H NOESY spectra for al. Sequential assignments are shown in red and multiple sequential NOEs are observed and labeled. (D) Fingerprint (top) and NH region (bottom) of the Ή NOESY spectra for a.3. The NH region reflects the predominance of NH-NH interactions and lack of other main-chain interactions characteristic of the common secondary structures. Mapping backbone NOEs on computational models as green bars (E, al and F, aS) reveal in-plane alignment of the peptide groups along the majority of the sheet. OEs in the turn regions determined whether the carbonyl or amide hydrogens pointed up in the structures as oriented in the figure (N-terminus top left). Ca, C, N, H and O atoms are shown in grey, grey, blue, white and red, respectively.

Figure 5. Table showing properties of designed peptides.

Figure 6A-C. Graphs showing inhibitory properties of polypeptides of the invention against Amyloid β 1 -42 peptide (Αβ42 or Αβ) (panel A), islet amylin polypeptide i I A PI') (panel B), and transthyretin (TTR) (panel C) amyloidosis. Aggregation is tracked over time using ThT or Congo Red without inhibitor in black and addition of inhibitor (colored traces) leads to a drop in aggregation. Inhibitory activity was observed for all peptides featuring alternating L- and D- amino acids.

Figure 7. Graph summarizing the inhibitory activity and "random coif'-charaeter for exemplary polypeptides of the invention plotted against their sequence identity relative to the al, ldas90 polypeptide. The top y-axis reflects the random coil character as reflected in the CD minimum at -195-198 nm and that axis should read Θ]™ _Γ , {TO ⁴ deg cm ² dmol ^{" 1} ].

Detailed Description of the Invention

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboraiory Manual (Sambrook, et al,

1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 1 85, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), "Guide to Protein Purification" Methods in Enzymology (M.P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al.

1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2 ^nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109- 128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, TX).

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise.

As used herein, the amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutanrine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleueine (He; I), leucine (Leu: L), lysine (Lys; K), methionine (Met: M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Val; V).

All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides isolated polypeptides, comprising or consisting of 12-23 contiguous amino acids according to the general formula 1 X1 -X2-X3- X4-X5 (SEQ ID NO: 1), wherein

XI is 0-7 contiguous amino acid residues that do not alternate between L amino acids and D amino acids;

X2 is 5-9 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 0-5 contiguous amino acid residues that do not alternate between L amino acids and D amino acids;

X4 is 4 - 12 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 0-4 contiguous amino acid residues that do not alternate between L amino acids and D amino acids;

wherein the isolated polypeptide is not:

GE(m)]sl(l)S(w ^' )MNEYSGW(t)M(n)L(k)MG (SEQ ID NO: 2).

In the various general formulae and sequences disclosed herein, amino acid residues recited in the uppercase and not within parentheses are in the "L" configuration ("L amino acids"), while those in lower case and within parentheses are in the \D" configuration ("D amino acids"). In the individual polypeptide sequences provided herein, amino acid residues in uppercase are L amino acids, while amino acid residues in lowercase are D amino acids.

As is disclosed in detail below, the inventors have discovered that the polypeptides of the invention adopt an a-sheet structure, regardless of the primary amino acid sequence and thus can be used, for example, in the therapeutic and diagnostic methods of the invention described herein. As demonstrated herein, the polypeptides of the invention are useful for inhibiting aggregation and/or binding of the toxic oligomeric form of amyloidogenic intermediates. As further demonstrated herein, the primary amino acid sequence of the polypeptides does not per se dictate the inhibitory activity (here defined as inhibition of aggregation and'or binding of ioxic oligomer). Instead it is ihe ability of the polypeptides to adopt a stable a-sheet stmcture that is operative here, and that is achieved through the recited polypeptide generic structure and the recited arrangement of alternating D/L amino acids. As demonstrated herein, rando primary sequences of amino acids are active in the polypeptides of the invention. These same primar '- sequences are not active if the polypeptide is composed entirely of L-amino acids or if the L/D amino acids are randomly distributed within the polypeptide such that they are not alternating. The primary amino acid sequence of ihe polvpeptides does however modulate the stability, solubility and degree of activity of the polypeptide.

As used throughout the present application, the term "polypeptide" is used in its broadest sense to refer to a sequence of sublimit amino acids. The polypeptides of the invention comprise regions of alternating L-amino acids and D-amino acids. "L amino acids" are L-steroisomers (left-handed isomers), while 'D amino acids" are D steroisomers (right- handed isomers), as is known to those of skill in the art. The amino acids may be any of ihe naturally occurring L- or D-amino acids, analogues thereof, or may be any non-natural or uncoded amino acids. Exemplary non-natural/uncoded amino acids include, but are not limited to aminoisobuteric acid, cyclocexylalanine, napt ylalanine, norvaline, norieucine, aminoheptanoic acid, aminoctanoic acid, and selenomethionine. Those of skill in the art are well aware that many such non-natural/uncoded amino acids exist, and it is well within the level of skill in the art to use such non-natural/uncoded amino acids in the polypeptides of the invention, based on the teachings herein.

As used herein, "alternating" means a stretch of at least 3 amino acids that alternative between L isomers and D isomers (i.e., L-D-L; D-L-D; etc.)

As used herein, "do not alternate between L and D residues" means that the region does not include a stretch of at least 3 amino acids that alternate between L isomers and D isomers. Such regions (XI , X3, and X5) may thus include both D and L residues. For example, the polypeptide eLkSwTNAAAaWtMqLlDpNR (SEQ ID NO: 4) (amino acid residues in uppercase are L amino acids, while amino acid residues in lowercase are D amino acids) has the following subunit structure according to the present invention:

I is 0 contiguous amino acid residues that do not alternate between L amino acids and D amino acids;

X2 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids (eLkSwT) (SEQ ID NO: 5);

X3 is 3 contiguous amino acid residues that do not alternate between L amino acids and D amino acids ( AA): X4 is 1 1 contiguous amino acid residues alternating between D amino acids and L amino acids (AaWtMqLlDpN) (SEQ ID NO: 6); and

X5 is 1 amino acid residue that does not alternate between L amino acids and D amino acids (R).

In another example, the polypeptide RGEmNlSwMNEYSNWtMnLkMGR (SEQ ID

NO: 7) (amino acid residues in uppercase are L amino acids, while amino acid residues in lowercase are D amino acids) has the following subunit structure according to the present invention:

XI is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids (R.G);

X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids (EmNlSwM) (SEQ ID NO: 8);

X3 is 5 contiguous amino acid residues that do not alternate between L amino acids and D amino acids (NEYSN) (SEQ ID NO: 9);

X4 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids (WtMnLkM) (SEQ ID NO: 10); and

X5 is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids (GR).

Those of skill in the art will recognize how other polypeptides sequences fit within the various general formulae described herein. The polypeptides described herein may be chemically synthesized using standard techniques.

In various embodiments, XI is 0-6, 0-5, 0-4, 0-3, or 0-2 contiguous amino acid residues that do not alternate between L and D residues. In other embodiments, I is 1 amino acid or is absent. In a further embodiment of any of these embodiments, all XI amino acid residues are L amino acids.

In various embodiments, X2 is 5-8, 5-7, 6-9, 6-8, 6-7, 5, 6, 7, 8, or 9 contiguous amino acid residues alternating between D amino acids and L amino acids.

In various further embodiment X3 is 0-4, 0-3, 0-2, 1-5, 1-4, 1-3, 1-2, 2-5, 2-4, 2-3, 3- 5, 3-4, 4-5, 5, 4, 3, or 2 contiguous amino acid residues that do not alternate between L and D residues. In various further embodiments, X3 is a single amino acid residue or is absent. In a further embodiment of any of these embodiments, ail X3 amino acids are L amino acids.

In various embodiments, X4 is 4- 11 , 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-12, 5-1 1, 5-10, 5- 9, 5-8, 5-7, 5-6, 6-12, 6-1 1, 6- 10, 6-9, 6-8, 6-7, 7- 12, 7-1 1, 7-10, 7-9, 7-8, 8-12, 8-1 1, 8-10, 8- 9, 9-12, 9-1 1, 9- 10, 10-12, 10- 1 1, 1 1 - 12, 12, 1 1 , 10, 9, 8, 7, 6, 5, or 4 contiguous amino acid residues alternating between D amino acids and L amino acids.

In various embodiments, X5 is 0-3, 0-2, 1-4, 1 -3, 1-2, 2.-4, 2-3, 3-4, 4, 3, or 2.

contiguous amino acid residues that do not alternate between L and D residues, in a further embodiment of any of these embodiments, ail X5 amino acids are L amino acids.

In various further embodiments, at least one of the following (i.e.: 1, 2, 3, 4, or all 5) is/are true about polypeptides according to general formula 1 :

(a) XI is 0 or 1 amino acid;

(b) X2 is 5, 6, 8, or 9 contiguous amino acid residues alternating between D amino acids and L amino acids;

(c) X3 is 0-4 contiguous amino acid residues that do not alternate between L and D residues;

(d) X4 is 6, 8, 9, 10, 1 L or 12 contiguous amino acid residues alternating between D amino acids and L amino acids; or

(e) X5 is 0, 1, 3, or 4 contiguous amino acid residues that do not alternate between L and D residues.

In one embodiment, the polypeptides are linear and possess an c -strand - turn - a-strand structure, in one such embodiment that can be combined with any other embodiment herein unless the context clearly dictates otherwise, X2 and X4 are of different length (i.e.: they are not symmetrical). For example, where X2 is 5 contiguous amino acid residues alternating between D amino acids and L amino acids, X4 might be 7 contiguous amino acid residues alternating between D amino acids and L amino acids.

In one such embodiment:

XI is absent;

X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 3 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 9 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

In another embodiment, the isolated polypeptide is not

rgeMrisWmneysgwTmNlKmgr (SEQ ID NO: 2).

I I Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

EqQISwTNAAAaWtQqLkQ (SEQ ID NO: 1 1 );

EqQISwKNAAAaWtQqLkQ (SEQ ID NO: 12);

EqQISwTNAAAaWtKqLkQ (SEQ ID NO: 13);

EqQISwTNAAAaWtQqLk (SEQ ID NO: 14);

EqQISwKNAAAaWtQqLkK (SEQ ID NO: 15);

EqQ 1 S wTNAAAaWt qLkK (SEQ ID NO: 16);

EqQISwKNAAAaWtKqLkQ (SEQ ID NO: 17);

EqQISwTNP AAaWtQqLkQ (SEQ ID NO : i 8) ;

EqQISwTNAPAaWtQqLkQ (SEQ ID NO: 19);

EqQlEwTNAAAaWkQqLkQ (SEQ ID NO: 20);

EqQlEwTNAAAaWkQqLkQ (SEQ ID NO: 21);

EqQlSlTNAAAaLtQqLkQ (SEQ ID NO: 22);

EqQlSiTNAAAaltQqLkQ (SEQ ID NO: 23);

EqQiSiTNAAAaltQqLkQ (SEQ ID NO: 24);

NmNlSiMNEYSaLtMnLqM (SEQ ID NO: 25);

NmNlSIMNAAAaLtMnLqM (SEQ ID NO: 26);

NmNlSiMNAASaLtMnLqM (SEQ ID NO: 27); and

QqQqQqQNAAAaQqQqQqQ (SEQ ID NO: 28).

As will be understood by those of skill in the art, the "reverse chiral counterpart" of a listed polypeptide is a polypeptide with an identical amino acid sequence but wherein each residue has the opposite chirality (e.g., each "L" residue is changed to a 'D" residue and each Ό" residue is changed to an "L" residue. By way of non-limiting example, the reverse chiral counterpart of EqQISwTNAAAaWtQqLkQ (SEQ ID NO: 1 1 ) is eQqLsWtnaaaAwTqQIKq (SEQ ID NO: 1 1). Based on the teachings herein, the identity of the reverse chiral counterpart of each specific polypeptide disclosed herein will be clear to those of skill in the art.

In another embodiment:

XI is absent;

X2 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 3 contiguous amino acid residues that do not alternate between L and D residues:

X4 is 10 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

kEqQlSwTNAAAaWtQqLkQq (SEQ ID NO: 29);

kEq Q 1 S wTNP AAa W tQqLkQq (SEQ ID NO: 30);

kEqQISwTNAPAaW tQqLkQq (SEQ ID NO: 31 );

kEqQlEwTNAAAaWkQqLkQq (SEQ ID NO: 32);

kEqQlSwTNAPAaWtQqLiQq (SEQ ID NO: 33); and

kEqQlEwTNAPAaWkQqLkQq (SEQ ID NO: 34).

In another embodiment:

XI is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids:

X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 3 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 9 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

RGEqQlSwTNAAAaWtQqLkQGR (SEQ ID NO: 35);

RGEmNlSwMNAAAaWtMnLkMGPv (SEQ ID NO: 36);

RGEqQlSwTNAAAaWtMqLkQGR (SEQ ID NO: 37);

RGEmNlSwMNEYSGWtMnLkMGR (SEQ ID NO: 38); and

RGEmNlSwMNEYSNWtMnLkMGR (SEQ ID NO: 39).

In another embodiment:

XI is absent; X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 4 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

m lSlMAEYSGItMnLqM (SEQ ID NO: 40);

Nm SIMNETSGltMnL-qM (SEQ ID NO: 41).

In another embodiment:

XI is absent;

X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 1 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 4 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

QlSwEpNKWtQk (SEQ ID NO: 42);

QqSwEpNKWtLk (SEQ ID NO: 43); and

SmTlEpNKLtLk (SEQ ID NO: 44).

In another embodiment that can be combined with any other embodiment herein unless the context clearly dictates otherwise, X2 and X4 are the same length (i.e.: they are symmetrical). For example, X2 and X4 may each be 5, 6, 7, 8, or 9 contiguous amino acid residues alternating between D amino acids and L amino acids.

In one non-limiting embodiment of such symmetrical polypeptides:

XI is absent;

X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; X3 is 5 contiguous amino acid residues that do not alternate between L and D residues:

X4 is 7 contiguous amino acid restdues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chirai counterparts:

NmNlSlMNEYSNLtMnLqM (SEQ ID NO: 45);

NmNlSiMNEYSDLtMnLqM (SEQ ID NO: 46);

NmNlSlMNEYSGLtMnLqM (SEQ ID NO: 47);

NmNlSiMLEYSOLtMnLqM (SEQ ID NO: 48);

EmNlSlMNEYSGLtMnLkM (SEQ ID NO: 49);

NniNlSf NEYSGFtMnLqM (SEQ ID NO: 50);

NmNISwMNEYSGWtMnLqM (SEQ ID NO: 51);

MnLnLsFNEYSGMfTlNmQ (SEQ ID NO: 52);

NmNlSiMGEYSGLtMnLqM (SEQ ID NO: 53);

NmNlSlMGEYSNLtMnLqM (SEQ ID NO: 54);

NmNlSlMNEASGLtMnLqM (SEQ ID NO: 55);

NwNIS wMNEY S G WtMnWqM (SEQ ID NO: 56);

EmNlSwMNEYSGWtMnLkM (SEQ ID NO: 57);

NtNlSIMNETSGLtMnTqK (SEQ ID NO: 58);

NmNlSlMNKTSGLtMnLqM (SEQ ID NO: 59);

NfNiSwMNETSGWtMiiTqK (SEQ ID NO: 60);

NtNlSlMNEYSGLt nTqK (SEQ ID NO: 61);

N tN 1 S wMNE Y S G WtMnTqK (SEQ ID NO: 62);

NmNiSlMNAATGLtMiiLqM (SEQ ID NO: 63);

NmNlSlMNAATGLiM-nLqM (SEQ ID NO: 64);

NmN 1 S1MGD Y SN LtMnLqM (SEQ ID NO: 65);

NmNiSlMGAASNLtMnLqM (SEQ ID NO: 66);

QmQlSlMNEYSGLtMqLqM (SEQ ID NO: 67); and

QmQlSiQNEYSGLtMqLqQ (SEQ ID NO: 68).

In another non-limiting embodiment of such symmetrical polypeptides:

XI is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids; X7 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 5 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 2 contiguous amino acid residues that do not alternate between L amino acids and D amino acids.

Exemplary polypeptides according to this embodiment include, but are not limited io the following polypeptides or their reverse chiral counterparts:

RGNm SwMNEYSGWtMnLqMGR (SEQ ID NO: 69);

RGEmN 1 S wMNE Y S G Wt MnLkMGR (SEQ ID NO: 70);

RGErnNlSwMNEYYGWtMnLkMGR (SEQ ID NO: 71);

RGEmAlSwMNEYSGWtMnLkMGR (SEQ ID NO: 72);

RGErnNlSwMNEYYGWtCnLkMGR (SEQ ID NO : 73);

RGEmNlSwMNEYYGWtMnCkMGR (SEQ ID NO: 74);

RGEmNlSwMNEYYGWtMnLkCGR (SEQ ID NO: 75);

RGErnNlSwMNEYYGWtMnLkMGR (SEQ ID NO: 76);

RGEmNlSwMNEYYGWcMnLkMGR (SEQ ID NO: 77);

RGEmN IS wMNEYYGWtMcLkMGR (SEQ ID NO: 78);

RGEmN ISwMNEYY G WtMnLcMGR (SEQ ID NO: 79);

RGEmNlFwMNEYYGWtMnLkMGR (SEQ ID NO: 80);

RGEmNlSwMNEYYGWtKnLkMGR (SEQ ID NO: 81);

RGEmNlSwMNEYYGWtMnKkMGR- (SEQ ID NO: 82);

RGEmNlFwMNEYYGWtMnCkMGR (SEQ ID NO: 83);

RGEmNlFwMNEYYGWtRnCkMGR (SEQ ID NO: 84);

RGEmN IF wMNEYYGWtKnCkMGR (SEQ ID NO: 85);

RGEmN IFwMLEYYGWtMnCkMGR (SEQ ID NO: 86);

RGEmNlFwMHEYYGWtMnCkMGR (SEQ ID NO: 87);

RGEmNlFwMMEYYGWtMnCkMGR (SEQ ID NO: 88);

AQQiEcItNVWDKEItMyFnVSE (SEQ ID NO: 89); and

WRPwGiDqMNTVKQRkAsVyLQP (SEQ ID NO: 90).

RGNwNeSkMNEYSGWmLmLtMGR (SEQ ID NO: 91). In another embodiment, the polypeptides comprise or consist of cyclic a-sheet hairpins that have a second turn engineered to close the sequence. In one such embodiment that can be combined with any other embodiment herein unless the context clearly dictates otherwise, X2 and X4 are of different length (i.e.: they are not symmetrical). In one such embodiment, XI is 0 or 1 amino acid, and X5 is 0 or 1 amino acid, in another such embodiment,

XI is absent;

X2 is 5-7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 0-3 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 6-1 1 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 0 or 1 amino acid residue,

In another such embodiment,

XI is absent;

X2 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 3 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 1 1 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is I amino acid residue.

Exemplary polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

qQkSwTNAAAaWtMqLkEpNK (SEQ ID NO: 92);

qWkSwTNAAAaWtMqLwDpNR (SEQ ID NO: 93);

qQkSwTNAAAaWtMqLkDpNR (SEQ ID NO: 94);

lQkSwTNAAAaWt qLkDpNR (SEQ ID NO: 95);

qLkSwTNAAAaWtMqLIDpNR (SEQ ID NO: 96);

wQkSwTNAAAaWtMqWkDpNR (SEQ ID NO: 97);

eWkSwTNAAAaWtMqLwDpNR (SEQ ID NO: 98);

eQkSwTNAAAaWtMqLkDpNR (SEQ ID NO: 99); qQlSwTNAAAaWt qLkEpNK (SEQ ID NO: 100);

eLkSwTNAAAaWtMqLlDpNR (SEQ ID NO: 4):

eWkSwTNAAAaWtEqLwDpNR (SEQ ID NO: 102);

qQkSwTNEYSaWtMqLkEpNK (SEQ ID NO: 103);

qQkSwTNEYYaWtMqLkEpNK (SEQ ID NO: 104);

qQkFwT E Y Ya WtMqLkEpN K (SEQ ID NO: 105);

qQkSwTNEYYaWtMqCkEpNK (SEQ ID NO: 106);

qQkS TNEYY aWtCqLkEpNK (SEQ ID NO: 107);

qQkFwTNAAAaWtMqLkEpNK (SEQ ID NO: 108); and

qQkFwTNAAAaWtEqLkEpNK. (SEQ ID NO: 109).

In another such embodiment,

XI is absent;

X2 is 7 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 1 amino acid residue that do not alternate between L and D residues;

X4 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplaiy polypeptides according to this embodiment include, but are not limited to the following polypeptides or their reverse chiral counterparts:

SdTlDpNRLtRkpP (SEQ ID NO: 1 10);

SnTlDpNRLtRkpP (SEQ ID NO: 1 11 );

SITlDpNRLtLkpP (SEQ ID NO: 112); and

S 1 TnDp RRtLkpP . (SEQ ID NO: 1 13).

In another such embodiment,

XI is absent;

X2 is 6 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 3 contiguous amino acid residues that do not alternate between L and D residues:

X4 is 10 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is 1 amino acid residue, Exemplary polypeptides according to this embodtmem include, but are not limited to the following polypeptides or their reverse ehiral counterparts:

qQkFwTNAAAaWtMqkEpNK (SEQ ID NO: 1 14); and

qQkFwTNEYYaWtMqkEpNK (SEQ ID NO: 1 15).

In another such embodiment,

XI is absent;

X2 is 8 contiguous amino acid residues alternating between D amino acids and L amino acids;

X3 is 3 contiguous amino acid residues that do not alternate between L and D residues;

X4 is 12 contiguous amino acid residues alternating between D amino acids and L amino acids; and

X5 is absent.

Exemplary polypeptides according to this embodtmem include, but are not limited to the following polypeptides or their reverse ehiral counterparts:

kEqQISwTNAPAaWtQqLkQqPp (SEQ ID NO: 1 16); and

kEqQISwTNAAAaWtQqLkQqPp (SEQ ID NO: 1 17).

In various embodiments, the polypeptide comprises or consists of 13-23, 14-23, 15- 23, 16-23, 17-23, 18-23, 19-23, 20-23, 21-23, 22-23, 12-22, 13-22, 14-22, 15-22, 16-22, 17- 22, 18-22, 19-22, 20-22, 21-22, 12-21 , 13-21, 14-21, 15-21, 16-21, 17-21 , 18-21, 19-21, 20- 21, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, 19-20, 12- 19, 13- 19, 14-19, 15-19, 16- 19, 17-19, 18-19, 12- 18, 13-18, 14- 18, 15-18, 16- 18, 17-1 8, 12-17, 13- 17, 14-17, 15-17, 16- 17, 12- 16, 13- 16, 14-16, 15-16, 12-15, 13- 15, 14-15, 12-14, 13-14, 12- 13, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids of general formula 1.

In another embodiment the isolated polypeptide features a first Fourier-transform infrared (FTIR) signal between about 1675-1680 cm ^" ' and a second FTIR signal at about 1640 cm ^'1 , wherein the first signal FTIR signal is stronger than the second FTIR signal. Alternatively, the isolated polypeptide may teature a first Fourier-transform infi'ared (FTIR) signal between about 1675-1680 cm ^{" 1} and a second FTIR signal at about 1640 cm ^{" 1} , wherein the first signal FTIR signal is weaker than the second FTIR signal. As shown in the examples, polypeptides of the invention often possess such FTIR signal patterns. In another embodiment, the polypeptides of the invention comprise or consist of a primary amino acid sequence according to the general formula 2:

R1-R2-R3-R4-R5-R6-R7-R8-R9-R10-R 11 -R12-R13-R14-R15-R16-R17-R 18-R19- R20-R21-R22-R23 (SEQ ID NO: 1 18), wherein at least residues R2-R12 are present in the isolated peptide, and wherein

Rl is selected from the group consisiing of R, M, E, (k), N, K, Q, S, V, L, W (w), (q), and A, or their reverse chiral counterparts, or is absent;

R2 is selected from the group consisting of G, (q), E, (m), (n), (w), (t), (1), A, S, Q, (k), and N, or their reverse chiral counterparts;

R3 is selected from the group consisting of E, Q, (q), N, T, S, (1), (k), W, and P, or their reverse chiral counterparts;

R4 is selected from the group consisiing of (q), (m), M, (1), Q, (w), K, (t), E, (i), and (a) , or their reverse chiral counterparts;

R5 is selected from the group consisting of Q, N, E, S, (1), L, (t), (k), (w), (q), A, W, and G, or their reverse chiral counterparts;

R6 is selected from the group consisting of (1), G, (w), S, (i), E, (f), and (s) , or their reverse chiral counterparts;

R7 is selected from the group consisting of S, N, T, K, (w), M, F, Q, L, (k), (t), W, (I), E, G, I, Y, and D, or their reverse chiral counterparts;

R8 is selected from the group consisting of (w), R, N, T, G, A, (n), (a), K, (q), E, (k),

L, (t), and (1) , or their reverse chiral counterparts;

R9 is selected from the group consisting of T, M, (k), A, N, P, E, K, D, L, W, (w), and Q, or their reverse chiral counterparts:

RIO is selected from the group consisting of N, E, S, A, P, Y, T, (t), (1), V, (q), L, H, and M, or their reverse chiral counterparts;

Rl i is selected from the group consisting of A, E, (p), (n), S, T, L, Q, Y, P, R, W, and N, or their reverse chiral counterparts;

R12 is selected from the group consisting of A, Y, N, M, W, (a), D, G, (k), (q), L, and V, or their reverse chiral counterparts;

R13 is selected from the group consisting of A, S, K, L, W, (a), I, F, W, M, (1), Y, V,

R, and Q, , or their reverse chiral counterparts, or is absent;

R14 is selected from the group consisting of (a), A, W, N, (t), (k), (f), G, Q, S, (I), (w), (q), T, Y, and E, or their reverse chiral counterparts or is absent; R 15 is selected from the group consisting of (q), (k), Q, (w), (t), T, W, G, I, M, L, R, and K, or their reverse chiral counterparts, or is absent;

R16 is selected from the group consisting of (t), M, G, (q), Q, (n), (1), K, (k), L, T, (c), and (m), or their reverse chiral counterparts, or is absent;

R17 is selected from the group consisting of Q, M, (n), (1), L, (q), , W, T, S, (t), (k),

(m), C, K, R, and A, or their reverse chiral counterparts, or is absent;

R l 8 is selected from the group consisting of (q), (ri), L, (w), (k), (m), H, D, W, (1), Q, E, L, T, S, (y), (c), and (s), or their reverse chiral counterparts, or is absent;

R19 is selected from the group consisting of L, (k), S, Q, K, M, F, (q), T, W, (r), (t), C, N, and V, or their reverse chiral counterparts, or is absent;

R20 is selected from the group consisting of (k), M, T, S, (n), (c), (y), (t), N, Q, and (q), or their reverse chiral counterparts or is absent;

R21 is selected from the group consisting of Q, M, G, W, N, (q), T, E, (t), (k), (1), V, C, and L, or their reverse chiral counterparts, or is absent;

R22 is selected from the group consisting of G, R, Y, H, (n), S, C, and Q, or their reverse chiral counterparts, or is absent; and

R23 is selected from the group consisting of R, M, E, and P, or their reverse chiral counterparts, or is absent. In the various general formulae disclosed herein, amino acid residues recited in the uppercase and not within parentheses are in the "L" configuration ("L amino acids"), while those in lower case and within parentheses are in the Ό" configuration ("D amino acids"). In the specific sequences recited, amino acid residues recited in the uppercase are "L amino acids", while those in lower case are "D amino acids".

In one embodiment RI is an L amino acid residue. In another embodiment the C~ terminal residue is an L amino acid residue.

In another embodiment, the primary sequence of cyclic polypeptides according to the invention comprise or consist of a primary amino acid sequence according to the general fonnuia 3 :

R1 -R2-R3-R4-R5-R6-R7-R8-R9-R10-R1 1 -R12-R13-R14-R15-R16-R17-R18-R 19-

R20-R21 -R22-R23 (SEQ ID NO: 1 19), wherein at least residues R1 -R14 are present in the isolated peptide, and wherein

Rl is selected from the group consisting of S, (q), (1), (w), (e), Q, and (k) , or their reverse chiral counterparts; R2 is selected from the group consisting of (1), Q, W, L, E, (d), and (n) , or their reverse chiral counterparts:

3 is selected from the group consisting of T, (k), (1), (q), and S, or their reverse chirai counterparts;

R4 is selected from the group consisting of (1), S, Q, (w), (n), and F, or their reverse chirai counterparts;

R5 is selected from the group consisting of D, (w), (1), and T, or their reverse chiral counterparts;

R6 is selected from the group consisting of (p), T, S, N, and W, or their reverse chiral counterparts;

R7 is selected from the group consisting of N, (w), and A, or their reverse chiral counterparts;

R8 is selected from the group consisting of R, A, T, and E, or their reverse chirai counterparts:

R9 is selected from the group consisting of (1), L, A, N, and Y, or their reverse chiral counterparts;

RIO is selected from the group consisting of T, (t), A, (a), S, and Y, or their reverse chirai counterparts;

Rl i is selected from the group consisting of (1), L, (a), P, A, R, and W, or their reverse chiral counterparts;

Rl 2. is selected from the group consisting of K, (k), W, A, and (t) , or their reverse chiral counterparts;

R13 is selected from the group consisting of (p), (t), (a), and M, or their re verse chiral counterparts;

R14 is selected from the group consisting of P, M, W, (q), E, and C, or their reverse chirai counterparts;

R15 is selected from the group consisting of (q), (t), and L, or their reverse chiral counterparts, or is absent;

R 16 is selected from the group consisting of L, W, Q, P, (k), and C, or their reverse chiral counterparts, or is absent;

R17 is selected from the group consisting of (k), (w), (1), (q), (p), P, and E, or their reverse chirai counterparts, or is absent;

R1S is selected from the group consisting of E, D, L, (p), and P, or their reverse chiral counterparts, or is absent; R19 is selected from the group consisting of (p), (k), and N, or their reverse chiral counterparts, or is absent;

R20 is selected from the group consisting of N, Q, and K, or their reverse chiral counterparts, or is absent;

R21 is selected from the group consisting of K, R, and (q), or their reverse chiral counterparts, or is absent;

R22 is P, or its reverse chiral counterpart, or is absent; and

R23 is (p), or its reverse chiral counterpart, or is absent.

In various embodiments, Rl is a D amino acid residue and/or the C- terminal residue is an L-amino acid residue. In a further embodiment of the cyclic polypeptides of the invention,

Rl is selected from the group consisting of S, (q), (e), and Q, or their reverse chiral counterparts:

R2 is selected from the group consisting of (!) and Q, or their reverse chiral counterparts;

R3 is selected from the group consisting of T and (k), or their reverse chiral counterparts;

R4 is selected from the group consisting of (1), S, and F, or their reverse chiral counterparts;

R5 is selected from the group consisting of D and (w), or their re verse chiral counterparts:

R6 is selected from the group consisting of (p) and T, or their reverse chiral counterparts;

R7 is N, or its reverse chiral counterpart;

R8 is selected from the group consisting of A and E, or their reverse chiral counterparts;

R9 is selected from the group consisting of L, A, and Y, or their reverse chiral counterparts:

R 10 is selected from the group consisting of (t), A, and Y, or their reverse chiral counterparts;

Rl 1 is (a), or its reverse chiral counterpart;

R12 is selected from the group consisting of (k) and W, or their reverse chiral counterparts; s selected from the group consisting of (p) and (t) , or their reverse chiral counterparts

R14 s selected from the group consisting of P and M, or their reverse chiral counterparts

R15 s (q), or its reverse chiral counterparts;

R16 s L, or its reverse chiral counterparts;

R 17 s selected from the group consisting of (k), (1) and E, or their reverse chiral counterparts

R18 s selected from the group consisting of E and D, or their re verse chiral counterparts

R19 s (p), or its reverse chiral counterpart;

R20 s selected from the group consisting of N and K, or their reverse chiral counterparts

R21 5 selected from the group consisting of K and R, or their reverse chiral counterparts

R22 s absent; and

R23 s absent.

In various further embodiments, the cyclic polypeptide comprises or consists of a primary amino acid sequence selected from the group consisting of the following polypeptides or their reverse chiral counterparts:

Table A

S ITlDp RITlKpP (SEQ ID NO: 120)

S ITlDp RLtLk P (SEQ ID NO: 121 )

qQkSwTNAAAaWtMqLkEpNK

(SEQ ID NO: 92)

qWkSwTNAAAaWtMqLwDpNPv

(SEQ ID NO: 93)

qQkSwTNAAAaWtMqLkDpNR

(SEQ ID NO: 94)

IQkS wTNAAAa W t MqLkDpNR

(SEQ ID NO: 95)

qLkSwTNAAAaWl qLlDpNR (SEQ ID NO: 96)

wQkSwTNAAAaWtMqWkDpNR

(SEQ ID NO: 97)

eWkSwTNAAAaWtMqLwDpNR (SEQ ID NO: 98)

cQkSwTNAAAaWtMqLkDpNR (SEQ ID NO: 99)

qQlSwTNAAAaWiMqLkEpNK (SEQ ID NO: 100)

kEqQISwTNAPAaWtQqLkQqPp (SEQ ID NO: 1 16)

kEqQISwTNAAAaWtQqLkQqPp (SEQ ID NO: 1 17)

SdTlDpNRLtRkpP (SEQ ID NO: 1 10)

QlSwTNAAAaWtMqLPp(SEQ ID NO: 101)

QlSnTWAAAaWtMqLkPp (SEQ ID NO: 122)

SnTlDpNRLtRkpP (SEQ ID NO: i l l )

SlTlDpNRLtLkpP (SEQ ID NO: 1 12) eLkS wTNAAAaWtMqLlDpNR (SEQ ID NO: 4)

eWkSwTNAAAaWtEqLwDpNR (SEQ ID NO: 102)

qQkSwTNEYSaWtMqLkEpNK (SEQ ID NO: 103)

qQkSwTNEYYaWtMqLkEpNK (SEQ ID NO: 104)

qQkFwTNEYYaWtMqLkEpNK (SEQ ID NO: 105)

qQkSwTNEYYaWtMqCkEpNK (SEQ ID NO: 106)

qQkSwTNEYYaWtMqLEpNK(8EQ

ID NO: 123)

qQkSwTNEYYaWtCqLkEpNK(SEQ

ID NO: 107)

qQkF ^' wTNAAAaWtMqLkEpNK(SEQ

ID NO: 108)

qQkFwTNAAAaWtEqLkEpNK (SEQ

ID NO: 109)

qQkFwTNAAAaWtMqkEpNK (SEQ

ID NO: 1 14)

qQkFwTNEYYaWtMqkEpNK (SEQ

ID NO: 1 15)

qQlSwTNAAAaWtMqLkPp (SEQ ID

NO: 124)

SITnDpN RtLkpP (SEQ ID NO:

125)

In another embodiment, the polypeptide comprises or consists of a primary amino acid sequence selected from the group consisting of the following polypeptides or their reverse chiral counterparts:

Table B

RGEqQlSwTNAAAaWtQqLkQGR (SEQ ID NO:

35)

RGEmNl SwMNEYS AWtMiiLkMGR (SEQ ID

NO: 126)

RGEmNlSwMNAAAaWiM-nLkMGR (SEQ ID

NO: 36)

RGEmNlSwMEpNKWtMiiLkMGR(SEQ ID NO:

127)

MGEMEGNRkSnMLNRGlwStWYm(SEQ ID NO:

128)

EqQlSwTNAAAaWiQqLkQiSEQ ID NO: 1 1 ) EqQlSwKNAAAaWtQqLkQ(SEQ ID NO: 12) EqQlSwTNAAAaWtKqLkQ(SEQ ID NO: 13) EqQlSwTNAAAaWtQqLkK(SEQ ID NO: 14) kEqQlSwTNAAAaWtQqLkQq(SEQ ID NO: 29) EqQlSwKNAAAaWtQqLkK(SEQ ID NO: 15) EqQlSwTNAAAaWtKqLkK(SEQ ID NO: 16) EqQ3S KNAAAaWtKqLkQ(SEQ ID NO: 17) qQlSwTNAAAaWtQqLk(SEQ ID NO: 129) EqQlSwTNPAAaWtQqLkQ(SEQ ID NO: 18) EqQlSwTNAPAaWtQqLkQiSEQ ID NO: 19) EqQlEwTNAAAaWkQqLkQ(SEQ ID NO: 20) EqQlEwTNAAAaWkQqLkQ(SEQ ID NO: 21) EqQlSlTNAAAaLtQqLkQ(SEQ ID NO: 22) EqQI SiTN AAAaItQqLkQ(SEQ ID NO: 23) EqQiSiTNAAAaItQqLkQ(SEQ ID NO: 24) kEqQlSwTNPAAaWtQqLkQq(SEQ ID NO: 30) kEqQlSwTNAPAaWtQqLkQg(SEQ ID NO: 31) kEqQlEwTNAAAaWkQqLkQq(SEQ ID NO: 32) NinNl S1MNEY SNLtMnLqMf SEQ ID NO: 45) NmNlSlMNEYSDLtMiiLqMiSEQ ID NO: 46) NmNlS1MNEYSGLlMnLqM(SEQ ID NO: 47) NmNlSlMLEYSGLtMnLqM(SEQ ID NO: 48) EniN IS IMNEYS GLtMnLldVI ( SEQ ID NO: 49) NmNlSiMNEYSOFtMnLqM(SEQ ID NO: 50) NmNlSwMNEYSGWtMnLqM(SEQ ID NO: 51) MnLnLsFNEYSGMfTlNmQ(SEQ ID NO: 52) NmNlSlMGEYSGLtMiiLqMiSEQ ID NO: 53) NmNlS1MGEYSNLlMnLqM(SEQ ID NO: 54) NmNlS3MNEASGLtMnLqM(SEQ ID NO: 55) NmNlS3MAEYSGltMnLqM(SEQ ID NO: 40) NmNlSlMNETSGltMnLqM(SEQ ID NO: 41) NwNlSwMNEYSGWtMnWqM(SEQ ID NO: 56) EmNlSwMNEYSGWtMnLkM(SEQ ID NO: 57) NtNlSlMNETSGLtMnTqK(SEQ ID NO: 58) NmNlSlMNKTSGLtMnLqM(SEQ ID NO: 59) N†.N 1 S wMN ET S G WtMnTqK (S EQ ID NO: 60) NtNlSlMNEYSGLtMnTqK(SEQ ID NO: 61)

NlSwMNEYSGWiMnTqKiSEQ ID NO: 62) RGN-mNiSwMNEYSGWiMnLqMGR(SEQ ID NO: 69)

NmNlSlMnEYSGLtMnLqM(SEQ ID NO: 47) NmNlSlMNEYSaLtMnLqM(SEQ ID NO: 25)

NmNlSlMNAATGLtMnLqM(SEQ ID NO: 63) NmNlSlMNAATGLtMnLqM(SEQ ID NO: 64) NmNi81MNAAAaLtMnLqM(SEQ ID NO: 26) NmN ISlMNAASaLtMnLqM( SEQ ID NO: 27) NmNiS1MaAAAaLtMnLqM(SEQ ID NO: 130) NmNlSlMGDYSNLtMnLqM(SEQ ID NO: 65) NmNiSlMGAASNLtMnLqM(SEQ ID NO: 66) RGEmNlSwMNEYSGWtMnLkMGR(SEQ ID NO: 38)

QmQl SlMNEYSGLtMqLqM( SEQ ID NO: 67) QmQlSlQNEYSGLtMqLqQ(SEQ ID NO: 68) SmTlEpNKLtLk(SEQ ID NO: 44)

RGEqQlSwTNAAAaWtMqLkQGR(SEQ ID NO: 37)

QISwEpNKWtQk(SEQ ID NO: 42)

QqSwEpNKWtLk(SEQ ID NO: 43)

RGEmNlSwMNEYSGWtMnLkMGR(SEQ ID NO: 70)

RGEmNlSw NEYSNWt nLkMGR(SEQ ID NO:

7)

RGEmNyFwMNEYYGWt nkMGR(SEQ ID NO: 131)

VALKLHKNEYYGVALKLHK(SEQ ID NO: 132) LALKSDFNEYYGLALKSDF(SEQ ID NO: 133) Q wEqSLQkNAPAakQl tWqqTf SEQ ID NO: 134) WSqQtqLwNAPAaQqkklQTE(SEQ ID NO: 135) QqEtkwkTNAPAaSqL1QqQW(SEQ ID NO: 136) wqEtLlQqNAPAakqQkWQST(SEQ ID NO: 137) kQqWwqtTNAPAalkqLEQSQ(SEQ ID NO: 138) QkEWqwSqNAPAa.lqQQLTkt(SEQ ID NO: 139) SQltQqkTNAPAawqLWEqQkfSEQ ID NO: 140) QtkqSwWENAPAaqQTLqQkl(SEQ ID NO: 141 ) klQtEqwqNAP AaS QLkQ WTq( SEQ ID NO: 142) qEWqLQIkNAPAaqwQkTStQCSEQ ID NO: 143) kEqQlSwTNAPAaWtQqLrQq(SEQ ID NO: 33) LlqqQkEwNAPAaQTkQStqW(SEQ ID NO: 144) qqEwSQLkNAPAaqWklQtQT(SEQ ID NO: 145) kQQqwEqLNAPAaTklqQtSW(SEQ ID NO: 146) KEQQLSWTNAPAaWTQQLKQQ(SEQ ID NO: 31)

kEqQlEwTOAPAaWkQqLkQqCSEQ ID NO: 34) MNWESkGEwlRMRYGtmLSNGnM(SEQ ID NO :

147)

AQQiEcItNVWDKEItMyFnVSE(SEQ ID NO: 89) QqQqQqQN AAAaQqQqQqQiSEQ ID NO: 28) QqQqEpNKQqQq(SEQ ID NO: 148)

RGEniNlSwMNEYYGWtMnLkMGR(SEQ ID NO: 71)

RGEmA3S MNEYSGWtMnLkMGR(SEQ ID NO: 72)

RGEmN18 MNEYYGWiCnLkMGR(SEQ ID NO: 73)

RGEmNlSwMNEYYGWtMnCkMGR(SEQ ID NO:

74)

RGEniNlSwMNEYYGWtMnLkCGR(SEQ ID NO: 75)

RGEmNlSwMNEYYGWtMnLkMCR(SEQ ID NO: 76)

RGEmNlSwMNEYYGWcMnLkMGR(8£Q ID NO: 77)

RGEmN3S MNEYYGWtMcLkMGR(SEQ ID NO:

78)

RGEmN18 MNEYYGWiM-nLcMGR(SEQ ID NO: 79)

RGEmNlFwMNEYYGWtMnLkMGR(SEQ ID NO:

80)

RGEmN1SwMNEYYGWtKnLkMGR(SEQ ID NO:

81)

RGEmNlSwMNEYY GWiMnKkMGR(SEQ ID NO: 82)

RGEmNlFwMNEYYGWtMnCkMGR(S ^' EQ ID NO:

83)

RGEmNlFwMNEYYGWtRiiCkMGR(SEQ ID NO: 84)

RGEmN3F MNEYYGWtKnC.kMGR(SEQ TD NO: 85)

RGEmNlFwMLEYYGWtMnCkMGR(SEQ ID NO: 86)

RGEmNlFwMHEYYGWtMnCkMGR(SEQ ID NO:

87)

RGEmN3FwMMEYYGWtMnCkMGR.(SEQ ID NO: 88)

RGEaWmY3KNNLSETmMsNMWGR(SEQ ID NO: 149)

WRP GiDqMNTVKQRkAsVyLQP(SEQ ID NO: 90)

RGNwNeSklVlNEYSGWmLniLtMGR(SEQ ID NO: 91)

RGEaWmY3KNNLSETmMsNMWGR(SEQ ID NO: 149) The inventors have discovered that the polypeptides of the invention adopt an a-sheet structure, regardless of the primary amino acid sequence and thus can be used, for example, in the therapeutic and diagnostic methods of the invention described herein. The

polypeptides of the invention are useful for inhibiting aggregation and/or binding of the toxic o igomeric form of amyloidogenic intermediates, as well as for use as diagnostic/imaging agents

Referring to Figure 5, and as detailed in the examples that follow, evidence that the primary amino acid sequence of the polypeptides does not per se dictate the inhibitory activity (here defined as inhibition of aggregation and/or binding of toxic oligomer) of the polypeptides is presented. Instead it is the ability of the polypeptides to adopt a stable a-sheet structure that is operative, which is achieved through the recited polypeptide generic structure and the recited arrangement of alternating D/L amino acids. Random primary sequences of amino acids are active in the polypeptides of the invention; these same primary sequences are not active if it the polypeptide is provided in an entirely L-amino acid peptide or if the L/D amino acids are randomly distributed within the polypeptide such that they are not alternating (i.e.: if they are not polypeptides according to the invention).

Based on all of the above, the inventors have clearly demonstrated that the primary amino acid sequences are not determining the α-sheet structure and associated activity, or in other words, there is degeneracy and many primary amino acid sequences are active within the templated structure provided by alternating L/D amino acids. The specific amino acids do modulate the stability, solubility and degree of activity, however.

In a further aspect, the present invention provides pharmaceutical compositions, comprising one or more (!, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) polypeptides of any embodiment or combination of embodiments of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the invention can be used, for example, in the methods of the invention described below. The pharmaceutical composition may comprise in addition to the polypeptidefs) of the invention (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent: (e) a stabilizer; (i) a preservative and/or (g) a buffer.

In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosai, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate- 60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer- 188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monosiearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The

pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood.

Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

The polypeptides of the invention may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.

The pharmaceutical compositions described herein are substantially free of non-pharmaceutically acceptable components, i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by US regulatory requirements at the time of filing this application. In some embodiments of this aspect, if the compound is dissolved or suspended in water, the composition further optionally comprises an additional

pharmaceutically acceptable carrier, diluent, or excipient. In other embodiments, the pharmaceutical compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).

These compositions can be prepared in a manner well known in the pharmaceutical art, and can be formulated for administration by any suitable route. The pharmaceutical compositions can be in any suitable form, including but not limited to tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to .10% by weight of the active compound, soft and hard gelatin capsules, sterile injectable solutions, and sterile packaged powders. The polypeptides of the invention can be capped or uncapped, as most appropriate for any given use. In various embodiments, one or both of the N- terminus or the C -terminus of the polypeptide is acetylated or amidated. In other embodiments, neither the N-terniinus nor the C-terrmnus is capped. The polypeptides may be finked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASyiation, or glycosylation. Such linkage can be eovalent or non-covaleni as is understood by those of skill in the art.

In one embodiment, the polypeptide comprises the recited amino acid structure; in this embodiment, the polypeptides may, for example, be linked (covalently or non- covalentiy) onto other polypeptides to increase stability, reduce half-life, provide bifunctionality, etc.; or onto other compounds or moieties as desired, including but not limited to detectable labels. The label(s) can be linked to the polypeptide through ovalent bonding, including, but not limited to, disulfide bonding, hydrogen bonding, electrostatic bonding, recombinant fusion and conformational bonding. Alternatively, the label(s) can be linked to the polypeptide by means of one or more linking compounds. Techniques for conjugating labels to polypeptides are well known to the skilled artisan. Polypeptides comprising a detectable label can be used diagnosficaily to, for example, assess if a subject has toxic amyloidogenic intermediates in a tissue or bodily fluid sample, or monitor the development or progression of an a treatment regimen to eradicate toxic amyloidogenic intermediates (i.e., determine the efficacy of a given treatment regimen. However, they may also be used for other detection and/or analytical and/or diagnostic purposes. Any suitable detection label can be used, including but not limited to enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions. The label used will depend on the specific detection/analysis/diagnosis techniques and/or methods used such as immuiiohistochemical staining of (tissue) samples, flow cytometric detection, scanning laser cytometric detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassay s (RIAs), bioassays (e.g., neutralization assays). Western blotting applications, etc.

The polypeptides of the invention can also be attached to solid supports, which are particularly useful for in vitro assays or therapeutic procedures to remove toxic

amyloidogenic intermediates from a subject. Such solid supports might be porous or nonporous, planar or nonplanar and include, but are not limited to, glass, nanoparticles, quantum dots, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene supports. The polypeptides can also, for example, usefully be conjugated to filtration media, such as NHS-activated Sepharose or CNBr-activated Sepharose, agarose beads for purposes of affinity chromatography. They can also usefully be attached to paramagnetic microspheres, typically by biotin-streptavidin interaction. As another example, the polypeptides of the invention can usefully be attached to the surface of a microtiter plate for ELISA.

In a third aspect, the present invention provides methods for treating an amyloid disease, comprising administering to a subject with an amyloid disease an amount effective of the isolated polypeptide of any embodiment or combination of embodiments of the invention, or a pharmaceutical composition thereof, to treat the amyloid disease,

As described in the examples that follow, while the alpha sheet polypeptides of the present invention were not designed against a specific protein target, inhibition of aggregation was observed in three very different amyloid systems. These results support the hypothesis that the a-sheet structure may constitute a broad-based inhibitor of amy loidosis, and thus the polypeptides of the invention a novel class of amyloid inhibitors that specifically target the toxic soluble oligomeric state of different amyloidogenic peptides and proteins. In various non-limiting embodiments, the amyloid disease may be selected from the group consisting of Creutzfeldt- Jakob disease, spongiform encephalopathy, Huntington's disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy (transthyretin), Kennedy disease, Machado- Joseph disease, Alzheimer's disease, bovine spongiform encephalopathy, scrapie, type 2 diabetes, amyloidosis caused by transthyretin (ATTR), Parkinson's disease, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dystrophy, and multiple myeloma.

As used herein, "treating an amyloid disease" means accomplishing one or more of the following: (a) reducing the severity of the amyloid disease; (b) limiting or preventing development of symptoms characteristic of the amyloid disease(s) being treated; (c) inhibiting worsening of symptoms characteristic of the amyloid disease(s) being treated; (d) limiting or preventing recurrence of the amyloid disease(s) in patients that have previously had the disorders); (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the amyloid disease(s); and (f) limiting development of the amy loid disease in a subject at risk of developing the amyloid disease, or not y et showing the clinical effects of the dental disease.

As used herein, an "amount effective" refers to an amount of the polypeptide that is effective for treating and/or limiting amyloid disease. The polypeptides are typically formulated as a pharmaceutical composition, such as those disclosed above, and can be administered via any suitable route, including orally, parentally, by inhalation spray, rectaliy, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes, subcutaneous, intravenous, intra- arterial, intramuscular, intraster ai, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques or inrraperitoneally.

Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). A suitable dosage range may, for instance, be 0.1 ug/kg- 100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight, The polypeptides can be delivered in a single bolus, or may be administered more than once (e.g., 2, 3, 4, 5, or more times) as determined by an attending physician.

In a fourth aspect, the present invention provides methods for diagnosing or prognosing an amyloid disease, comprising

(a) contacting a tissue sample from a subject at risk of having an amyloid disease with any embodiment or combination of embodiments of the invention, or a pharmaceutical composition thereof, under conditions suitable for binding of the isolated polypeptide with an amyloid intermediate, if present in the tissue sample, to produce a binding complex;

(b) detecting binding complexes in the tissue sample; and

(c) diagnosing or prognosing an amyloid disease based on the detecting.

The methods of this aspect of the invention can be used to more accurately diagn ose or prognose patients that may be suffering from an amyloid disease and to thus provide more informed determination of treatment options by an attending caregiver. Individuals at risk of an amyloid disease are those exhibiting one or more signs, symptoms, or risk factors for an amyloid disease, including but not limited to Creutzfeidt- Jakob disease, spongiform encephalopathy, Huntington's disease, amyotrophic lateral sclerosis (ALS), senile systemic amyloidosis, familial amyloid polyneuropathy, Kennedy disease, Machado- Joseph disease, Alzheimer's disease, bovine spongiform encephalopathy, scrapie, type 2. diabetes, amyloidosis caused by transthyretin (ATTR), Parkinson's disease, atherosclerosis, rheumatoid arthritis, aortic medial amyloid, prolactinomas, dialysis-related amyloidosis, cerebral amyloid angiopathy, Finnish amyloidosis, lattice corneal dy strophy, and multiple myeloma. The tissue sample may be any suitable tissue sample including, but not limited to blood, serum, cerebral spinal fluid, nasal secretions, urine or other biological material from a subject at risk of an amyloid disease.

Conditions suitable for binding the polypeptides of the invention with an amyloid intermediate, if present in the tissue sample, to produce a binding complex will depend on specifies of the polypeptide(s) being used, the tissue sample, and the technique employed. Determining such suitable conditions are within the level of skill in the art based on the teachings herein.

The formation of such complexes, if any, indicating the presence of amyloid intermediates in the sample, is then detected and measured by suitable means. Such methods include, but are not limited to homogeneous and heterogeneous binding immunoassays, such as radioimmunoassays (RJA), ELISA, immunofluorescence, immunohistochemistry, FACS, BIACORE and Western blot analyses.

The polypeptides of the invention for use in this aspect may comprise a conjugate as disclosed above, to provide a tag useful for any detection technique suitable for a given assay. The tag used will depend on the specific detection/analysis/diagnosis techniques and/or methods used. The methods may be carried in solution, or the polypeptide(s) of the invention may be bound or attached to a carrier or substrate, e.g., microtiter plates (ex: for ELISA), membranes and beads, etc. Carriers or substrates may be made of glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose, or teflon, etc. The surface of such supports may be solid or porous and of any convenient shape.

Example 1. Designed a-Sheet Peptides Inhibit Amyloid Formation by Targeting the Toxic Oligomeric Form

Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Here the experimental characterization of peptides designed to be complementary to the a-sheet conformation observed in the simulations is reported. Inhibition of aggregation is demonstrated in two different amyloid systems, β-amyloid peptide 1-42 (Αβ) and transthyretin (TTR), by these designed a-sheet peptides. When immobilized the a-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non- aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondar structures, supporting cc-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics.

There are now over 40 different human amyloid diseases, each linked to the buildup of a specific precursor protein or peptide (/). These diseases involve the conversion of a protem from its soluble native state into insoluble amy loid fibrils, or, in the case of peptides, the conversion from a soluble, loosely structured form to fibrils. Given that many different sequences can form amyloid fibrils of similar architecture, there may be some common structural features of the prefibriilar amyloidogenic intermediates. X-ray fiber diffraction indicates that the insoluble, mature amyloid fibrils are composed of cross β-sheet structure (2). Therefore, it is widely held that the formation of amyloid fibrils involves a transition to β-sheet structure in the amyloidogenic intermediate. However, the mechanism of self- assembly at the atomic level remains elusive. Another feature of these diseases is that soluble ofigomeric intermediates, not the insoluble well-ordered fibrils, are preferentially responsible for cellular toxicity (3-6). Similarly, the soluble oligomeric forms of the prion protein are the most infectious per unit protein (7). As such, fibrils may be protective, at least up to a point, as their breakdown to smaller aggregates yields greater toxicity and infectivity. The discovery of a compound that promotes inclusion formation while reducing toxicity and cellular pathology supports this hypothesis (8),

Research on the soluble oligomers has become critically important since there is a consensus that the soluble oligomer species is more toxic than mature fibrils (5, 6). In fact, structural similarities between soluble oligomers from a range of unrelated protems/peptides has been suggested based on an antibody that recognizes a common backbone structure (5, 6, 9). Glahe and co-workers developed an antibody (denoted Al 1 ) that binds soluble oligomeric intermediates derived from a variety of peptides and proteins, including Αβ(Ί -42), α- synuclein, islet amyloid polypeptide, polyglutamine, lysozyme, human insulin, and a prion peptide (10), The antibody does not, however, bind the corresponding insoluble fibrils (cross-β structure) or the natively folded precursors (various structures). Based on the specificity of the antibody for soluble oligomers with various sequences, it was proposed that the antibody recognizes a unique conformation of the backbone. This antibody inhibits toxicity associated with the intermediates, implying a common mechanism of toxicity and offering hope for a broad-based therapeutic agent. Some years ago the inventors "discovered" a novel secondary structure, which is called "a-sheet, that is populated during molecular dynamics (MD) simulations of a range of amyloid proteins (and peptides) with different structures and sequences under amyloidogenic conditions (Fig. la) (/ 1-14). The position where the a-sheet forms along the sequence coincides with the most amyloidogenic regions of the proteins, as determined experimentally (13). An α-sheet is similar to a β-sheet, but instead of alternating main chain NH and C=0 groups along the strands, an α-sheet has the NH groups aligned on one side and the carbonyls on the other. As such, the a-sheet has a molecular dipole and a very different hydrogen- bonding pattern across the sheet compared to a β-sheet. Interestingly, the main chain (Φ,Ψ) dihedral angles of the a-sheet alternate between the OIL and <XR conformations (15) (Fig. la). Although locally helical, the alternating dihedral angles form an extended chain resulting in the carbonyi groups and amide groups aligning in a plane. Such an arrangement creates uniform electrostatic faces to aid in the addition of further strands (11). Once a sheet is formed, a simple peptide plane flip could convert the α-sheet into a β-sheet and ultimately a mature fibril (15). Extensive a-sheet formation has not been observed in native proteins.

To investigate the role of a-sheet in amyloid formation, numerous small, stable a- hairpin peptides were computationally designed. It was reasoned that if a-sheet is the novel backbone structure in toxic oligomers, then a-sheet peptides designed to be complementary to the structure in the oligomer should bind to the toxic oligomer and inhibit amyloidosis. The designs began with a backbone template in an ideal a-sheet conformation. Investigation was then carried out on combinations of residues with high propensities to populate desired regions of conformational space using the Structural Library of Intrinsic Residue Propensities (SLIRP), which is part of the Dynameoraics project (18, 19). Owing to the expected transient nature of the OIL conformation, the structure was stabilized using D-amino acids, which essentially have inverted conformational propensities compared with their normal L- counterparts (Fig. lb). Peptides containing alternating D- and L-amino acids have previously been shown to form extended structures (20, 21), and they populate similar conformational space as the MD-identified a-sheet sequences in amyloid systems consisting solely of L- amino acids. MD simulations (22) were performed to assess the stability of the de novo designed amino acid sequences. Sequences designed to adopt β-sheet and random coil conformations were included in the experiments as controls.

Several of the most promising peptide designs were selected for experimental characterization in two different amyloid systems: transthyretin (TTR) and beta amyloid 1-42 [Αβ(1-42) or Αβ for short]. Both are associated with amyloid diseases but have completely different sequences and structures. Αβ is a largely unstructured peptide fragment in aqueous solution, but it aggregates to become the dominant component of plaques from patients with Alzheimer's Disease. In contrast, TT is a tetramer composed of immunoglobulin-like β- sandwich domains; TTR aggregation is associated with peripheral polyneuropathy and systemic senile amyloidosis. Both systems have well characterized aggregation profiles and aggregate under mild, non-denaturing conditions (23-25), Here the focus is on five peptide designs (β, rc, al, o2, a3) (Fig, lc). β is the Trpzip 3 peptide (26) but with Trp to Leu substitutions to improve spectroscopic properties. The β-design was intended to be a negative control for TTR and a positive control for Αβ, as β -hairpins are known inhibitors of Αβ aggregation (27). rc was designed to be an unstructured random coil to provide a negative control; it is a scrambled, random sequence of the β-sheet design, β. The remaining three peptides were designed to adopt a-sheet structure, al and oc2 are linear hairpins containing a sheet of alternating D- and L-amino acids, a! was designed to have high a-sheet propensity. a2 is a derivative of al with modifications aimed to improve stability and the introduction of a Cys for coupling experiments. a3 consists of a sheet of alternating D- and L-amino acids and two turns, creating a cyclic amide backbone. It was not possible to design a soluble, random coif control based on the a-sheet peptides, i.e. the same composition and length. Shuffling of the amino acid sequences resulted in insoluble peptides that were unusable in the solution-phase assays, so it was determined to use smaller but well-defined controls.

First the peptides were tested for anti-amyloidogenic activity in a fibrillization assay using transthyretin (TTR). Four of the five designed peptides (a2 was sparingly soluble and therefore not tested in any solution-phase experiments) were co-incubated with TTR (in excess 20: 1 , expressed relative to TTR monomer) at pH 4.5 to trigger dissociation of the native tetramer followed by aggregation (23, 24), The aggregation was monitored via binding of Congo red (Fig. 2a). It was not possible to use thiofiavin T (ThT) as an extrinsic dye to monitor the aggregation of transthyretin, as addition of ThT to freshly dissolved and filtered TTR gives high background fluorescence. For this reason Congo red was used, which did not bind monomelic TTR. The percentage inhibition of aggregation was determined after 48 hours at 37 °C, after the aggregation stabilized. At these concentrations, al and a3 resulted in 72 and 56% inhibition, respectively, relative to TTR alone at low pH. In contrast, the rc and β controls resulted in little to no inhibition. The neutral pH tetrameric TTR control changed little over time. Moreover, the designs in the absence of TT did not bind Congo red and were indistinguishable from the buffer-only controls (data not shown).

Since the aim is to target the toxic oligomer, it was determined when the toxic oligomeric species was present during the course of aggregation. The toxicity of TTR was assessed by monitoring ceil viability using the MTT assay after treating SH-SY5Y neuroblastoma cells with TTR that had been allowed to aggregate for different periods of time at pH 4.5. Toxicity began to build up around 24 hrs, and the results from this time point are shown in Figure 2b. Under these conditions, the v iability of the treated cells was reduced by approximately 20%, indicating that TTR was aggregating via the toxic pathway. Addition of the controls, rc or β, to a 24-hr pre-incubated TTR sample had no little to no effect on further aggregation, as detected with the Congo red assay. However, when al and a.3 were mixed with 24-hour pre-incubated, toxic TTR, they inhibited 81% and 77%, respectively, of the remaining TTR aggregation observed in the absence of inhibitor.

Similarly, the designs were co-incubated (in 0: 1 excess) with Αβ at pH 7.4 and followed fibril formation with ThT fluorescence changes upon binding at 37 °C (Fig, 2c). As observed for TTR, both al and a3 inhibited aggregation. Again al was more efficient, inhibiting approximately 87% of fibril formation compared with 66% for o3, when measured after the reaction stabilized at 22 hours. The β-sheet design, β, also resulted in 62% inhibition of Αβ fibrillizaiion, and it exerted its effect primarily between 0 and 6 hrs. In contrast, al and o3 showed a distinct lag, becoming inhibitory after aggregation proceeded for approximately 6 hrs. The peptide designs alone did not alter the fluorescence of ThT, and, as with Congo red, they were indistinguishable from buffer-only controls over the time course of the assay (data not shown). Also note that the a-sheet designs are effective at lower concentrations and the efficacy increases at lower temperature; for example, a 4-fold excess of al essentially completely abolishes aggregation at 25 °C. The 37 °C studies are reported here, though, for comparison with TTR, which aggregates very slowly at 25 °C.

Αβ toxicity was also assessed using the MTT assay and found to reduce the viability of treated SH-SY5Y cells to less than 50% after 6 hrs incubation (Fig. 2d). Addition of the c - sheet designs to a pre-aggregated (6 hrs), toxic sample of Αβ showed essentially complete inhibition, amounting to 97 and 96%, respectively for a! and a3, compared with the extent of aggregation observed in the absence of inhibitor.

To further probe which species the peptide designs are binding to, the designs were immobilized on agarose beads and applied solutions of either fresh or pre-incubated, toxic samples of Αβ and TTR, Immobilization in this manner also allowed us to test the sparingly soluble a2 design by limiting self- aggregation. All three immobilized a-sheet designs (al, al and a3) bound pre-incubated, toxic oligomer-containing TTR solutions (pre --incubated at low pH for 24 hrs) to a greater extent than the rc and β controls (Fig, 3a). al, o2 and a3 also bound pre-aggregated, toxic Αβ preferentially, whereas rc did not (Fig. 3b). In the case of the β design, it preferentially bound the fresh, monomeric Αβ (Fig. 3c), indicating that inhibition was due to interactions with the "native" form, not the toxic oligomer, β-hairpins are known inhibitors of Αβ aggregation (2.7). In contrast, α-sheet, as demonstrated with al, did not bind native, tetrameric TTR nor fresh, monomeric Αβ but instead preferentially bound species from the toxic, aggregated samples (shown for Αβ in Fig. 3d). Moreover, the a-sheet designs did not bind the fibrillar forms of Αβ or TTR acquired by allowing the aggregation reactions to continue for over 3 weeks (data not shown).

The instability of α-sheet structure in proteins and peptides containing solely L- amino acids leaves no established spectroscopic signatures with which to assess the structures. However, predictions can be made and tested based on the unique conformational and electronic environments resulting from this structure. Circular dichroism (CD) signals arise from the differential absorption of left- and right-hand polarized light by chiral molecules. For proteins, the orientation of individual amide bonds is responsible for the resulting CD spectra in the far-UV region. Mirror image structures, formed by replacing whole L-amino acid sequences with D-amino acids, such as gramicidin A, produce mirror-image CD-spectra (29). The a-sheet structure is expected to be effectively invisible due to near equal absorbance of both left and right polarized light, with any residual signal emanating from the turns and terminal residues.

The electrostatic interactions between aligned amide groups in an a-sheet (13, 15) are expected to give rise to strong Fourier-transform infrared (FTIR) signals. Torii recently performed density functional theory calculations of three slightly different orientations of a- sheet structures (30). All models featured a strong high-frequency absorbance in the 1675- 1680 cm ^"1 region, with a weaker band around 1640 cm ^": , which is distinct from a-helix (-1650- 1658 cm ^" ') and β-sheet (-1620- 1640 cm ^"1 ) and instead overlaps somewhat with turn structures (-1670 cm ^"1 ) (31).

Nuclear magnetic resonance (NMR) spectroscopy can provide site-specific confor mational information. Owing to the unique alignment of the amide groups in an a- sheet, to see strong sequential d.w Nuclear Overhauser Effect (NOE) crosspeaks are expected along the backbone since the NH groups are aligned on one side of the chain instead of alternating between opposite faces as in β-sheet structure. Furthermore, the long -range or the strong sequential d«N NOEs characteristic of β-sheets would not be expected. Thus, CD, FTIR and NMR studies were performed to assess the structures of the peptide designs.

The CD spectrum of rc shows a random coil signal with a strong negative absorption around 195 ran (Fig. 4a). In its FTIR spectrum multiple small absorbance peaks not corresponding to a predominance of any specific structure are observ ed (Fig. 4b). The β- sheet control is a modified version of the Trpzip peptide, which forms a relatively stable β- hairpin in solution. In agreement with previous structural work (32), β exhibits a CD spectrum reflective of β-strueture with some random coil, with a minimum near 220 nm and another near 195 nm (Fig, 4a). The substitution of Leu for Tip removed the strong exciton- coupling between the Trp residues observed in the parent peptide thereby 'exposing' the β- stracture CD signal. The β-sheet structure was confirmed by FTIR through its strong absorbance at 1632. cm ^" ' (Fig. 4b). The CD spectra for al, oc2 and a3 are essentially featureless, as expected for the cancellation of OIL and C/.R signals, except for a slight dip around 195 nm consistent with turn formation (Fig, 4a). a2 has a particularly featureless spectrum with a slight positive inflection around 195 nm, possibly due to a better ordered a- hairpin turn, al and a2 exhibited the predicted FTIR -sheet bands at 1640 and 1675-1680 cm ^" ' (Fig. 4b). These bands were less pronounced in a3. Cyclization of the amide backbone of <x3 through a non-optimal turn may have caused distortion of the structure, as has been reported in other peptide systems (33) and suggests an area for improvement in future designs. Altogether, these results prove that the designed a-sheet peptides do not form a- hefix or β-sheet structure, and that the random coil content is not large (compare against the scale of the rc control CD spectrum). Thus, these results are consistent with and supportive of the designed α-sheet structure.

Further structural studies were performed utilizing homonuclear NMR spectroscopy. Multiple sequential NOEs were observed along the backbones of both al and a3) (Fig. 4c,d). The al and a.3 designs were well-behaved monomers under the conditions of the NMR experiments, which include very high sample concentrations in solvent conditions similar to those of the aggregation assays. In contrast, a2 was not soluble enough for NMR. Consequently the focus was on al and ai here, and the observed NH....NH NOEs are mapped onto structural models of al and o3 in Figures 4e and f, highlighting the stretches of α-strand structure in both designs. No long-range dm or d« NOEs indicative of a-helical or β- sheet structure (34) were observed. The breaks in the a-strand structure and the deformed turns highlight vulnerabilities in the design and provide suggestions for improved designs. No long-range side chain-side chain NOEs were observed despite increasing the mixing time of the NOES Y experiments up to 400 ms, perhaps due to residual dynamics in the peptide. The lack of these distance restraints prevented the generation of a well-converged solution structure; however, the NMR data are consistent with -sheet secondary structure and inconsistent with a-helix, random coil, and β-sheet structures.

Ten years ago a common conformation was demonstrated among soluble oligomeric species from amyloid proteins/peptides of diverse sequence and structures that cross react with the A! 1 antibody (9). Also at that time a novel target structure, α-sheet, was identified through MD simulations and proposed that it is the defining feature of the toxic oligomeric species (13, 15). Unfortunately, the precise structure of this toxic intermediate remains elusive, and it has become clear that the oligomers are conformationally heterogeneous (35, 36 and references therein). Here another approach was taken to probe these soluble oligomeric species and experimentally test the a-sheet hypothesis through peptides designed to be complementary to the proposed a-sheet structure. Three of these computationally derived designs have been synthesized and characterized experimentally, and they do indeed appear to adopt a-sheet structure (as shown by FT1R, CD and NMR). The two soluble a- sheet designs (oil and a3) inhibited both TTR and Αβ aggregation in solution, in addition, conformation- specific binding was confirmed by passing solutions of nontoxic and toxic TTR and Αβ over all three α-sheet designs immobilized to agarose beads, which preferentially bound species from the toxic preparations, in contrast, the ^control formed a β-hairpin, as supported by CD and FTIR, and it preferentially bound the monomelic form of Αβ and it did not react with TTR in any form. When mature fibrils were applied to the

immobilized a-sheet designs, the fibrils did not bind and appeared to have no affinity for the a-sheet structure.

While these a-sheet peptides were not designed against a specific protein target, inhibition of aggregation was observed in two very different amyloid systems. These findings extend to many other sequences and a third independent amyloid system, as summarized in Figure 5 and discussed further in Example 2. These results support the hypothesis that a- sheet structure is involved in the toxic oligomer stage of aggregation, and they provide a reference to determine spectroscopic signatures that can now be used to investigate the structural changes amyloid proteins undergo during amyloidosis. Having demonstrated that the α-sheet stmcture may constitute a broad-based inhibitor of amyloidosis, the a-sheet designs introduce a novel class of amyloid inhibitors that specifically target the toxic soluble ofigomeric state of different amyloidogenic peptides and proteins.

References and Notes:

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25. W. Colon, J. W. Kelly, Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro, Biochemistry 31, 8654-8660 (1992).

26. A. G. Cochran et al, A Minimal Peptide Scaffold for β-Turn Display: Optimizing a Strand Position in Disulfide-Cyclized β-Hairpins, J. Am. Chem. Soc. 123, 625-632 (2001).

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Structure, Function, and Bioinformatics 12, 49-62 (1992). 30. H. Torii, Amide I Infrared Spectral Features Characteristic of Some Untypical

Conformations Appearing in the Structures Suggested for Amyloids, J. Phvs. Chem. B 112, 8737-8743 (2008).

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32. A. G. Cochran, N. J. Skelton, M. A. Starovasnik, Tryptophan zippers: stable, monomelic beta -hatpins, Proc. Nail. Acad. Sci. U.S.A. 98, 5578-5583 (2001 ).

33. R. Clark el al, Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the alpha-conotoxin Mil, Proc. Natl. Acad. Sci. U.S.A. 162, 13767-13772 (2005).

34. K, Wuthrieh, in NMR of Proteins and Nucleic Acids, (Wiley, New York, 1986), pp. 162— 176.

35. N. Carulla, M. Zhou, M. Arimon, M. Gairi, E. Giralt, C.V. Robinson, and CM. Dobson, Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation. Proc. Natl. Acad. Sci. USA 106, 7828-7833 (2009).

36. G, Bitan, E.A. Fradinger, S.M. Spring, D.B. Teplow, Neurotoxic protein oligomers - what you see is not always what you get. Amyloid - J. ofProl. Fold. Disorders, 12, 88-95 (2005).

37. M. Levitt, M. Hirshberg, R. Sharon, Potential energy function and parameters for simulations of the molecular dynamics of proteins and nucleic acids in solution, Computer Physics Communications (1995).

38. M. Levitt, M. Hirshberg, R. Sharon, K. Laidig, V. Daggett, Calibration and testing of a water model for simulation of the molecular dynamics of proteins and nucleic acids in solution, ,/. Phys. Chem. B 101, 5051-5061 (1997).

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40. W. E, Klunk, J. W. Pettegrew, D. J. Abraham, Two simple methods for quantifying low- affinity dye-substrate binding, J Histochem. Cylochem. 37, 1293-1297 (1989).

41. N. Reixach, S. Deechongkit, X. Jiang, J. W. Kelly, J. Buxbaum, Tissue damage in the amyloidoses: Transthyretin monomers and normative oligomers are the major cytotoxic species in tissue cultu e, Proc. Natl. Acad. Sci. USA 181 , 2817-2822 (2004).

Materials and Methods for Example 1

Computational Design The a-sheet peptides were designed in silico using a database of amyloid protein MD simulations to determine preferred backbone geometries. The SLIRP database (18, 19) was used to select residues with high propensity to adopt the desired structure. MD simulations were performed to assess both turn and a-sheet stability. Ideal a- sheet and β-sheet templates were created and sequences were chosen based on their intrinsic conformational preferences and to consist of a mix of polar and nonpolar amino acids to maintain good inter-strand inieractions and solubility. Intrinsic conformational propensities of all 20 L-amino acids in a GGXGG (SEQ ID NO: 3) peptide were determined by extensive molecular dynamics (MD) simulations (18). D-amino acid propensities were determined in a similar manner (unpublished results). MD simulations were then performed to assess the stability of the designs. Multiple short simulations were performed, at least 3 x 20 ns, for each peptide at 25 °C using an in-house MD package in lucem molecular mechanics (//mm) (22), with the Levitt et a!. all atom force field (37) and the F3C water model (38). Standard simulation protocols were followed (39). -Sheet stability was assessed by monitoring both the secondary structure and ihe turn structure, based on hydrogen bonding over the duration of the simulations. Results were expressed as the fraction of simulation time the atoms were within hydrogen bonding distance. Co. RMSD was used qualitatively to monitor backbone deviation from the ideal hairpin structure in conjunction with the hydrogen bond scoring function to determine promising designs. An iterative approach was used, with the results of the analyses used to modify and refine sequences for further simulation and evaluation. Several sequences were chosen from a pool of well-behaved simulations for experimental evaluation. High scoring designs were synthesized and their inhibitory effects were determined in TTR and Αβ fibrii!ization assays at 20- or 10-fold molar excess of inhibitor, respectively.

TTR Fibril!ization Assay Aliqouts of transthyretin (TTR) (496-1 1; Lee Biosoluiions, St, Louis MO) were made from a 1 mg/mL solution 20 mM ammonium carbonate, pH 8.

Aliquots were lyophilized and stored at - 18 °C. Prior to use, TTR was dissolved to 80 μΜ (monomer) in acetate buffer (50 mM potassium acetate, 100 mM potassium chloride pH 4.5) and sonicated for 10 minutes. The stock solution was centrifuged before use. Peptide designs were added to stock TTR to a final TTR concentration of 40 μ.Μ (monomer) in acetate buffer (pH 4.5) in 500 μΐ, microcentrifuge tubes, which were incubated at 37 ^,J C, Periodically, samples were collected from the TTR:peptide mixture by briefly centrifuging, and then carefully pipetting the solution up and down prior to withdrawing a 10 L sample and diluting it in 190 xL of 10 μΜ Congo red in an individual well of a 96- well assay plate. Each measurement was performed in triplicate. Absorbance measurements were taken at 477 and 540 nm. Relative Congo red binding was determined using the method of Klunk et aL ^' using the relationship: rCb = (Abs ₅ 4o/25,295)-(Abs477/46,3()6) (40). All datapoints were normalized to the value recorded for TTR alone pH 4.5 at 48 hrs.

Αβ Fibrillizaiioi! Assay. Αβ(1-42) (AMYD-002; CPC Scientific, Sunnyvale CA) was stored as 2 mg/mL stock in hexaiiuoroisopropanol (HFTP) at - 18°C, Prior to use, the stock solution was thawed, an aliquot taken and the HFTP was removed under a gentle stream of air. A 1 mg/mL stock solution of Αβ was made in 5 mM NaOH and sonicated for 5- 10 minutes. The stock was filtered through a 0,22 μηι cellulose filter (Costar Spin-X; Coming Inc, NY). The concentration of stock Αβ was determined by first diluting the stock 1 :50 in 5 mM NaOH then taking the absorbance at 2.20 nm (ε ₂₂ ο ⁼ 50,000 cm ^"1 M ^{" 1} ). Aliquots of the NaOH stock were placed in separate wells of a 96-weil black-walled fluorescence plate (Nunc) and immediately diluted in PBS (T 1 mM phosphate) containing 20 μ,Μ Thioflavin T (ThT) to give 150 uL of 10 μΜ Αβ at pH 7.5. Peptide inhibitors were added directly to 10 μΜ Αβ samples from concentrated aqueous stocks. Covered plates were incubated at 37 °C and were periodically removed for fluorescence measurements. ThT fluorescence was measured at λ _6Χ -450 nm and λ _βϊΙΪ =480 nm using a Tecan 8afire2 plate reader,

immobilization and Solution Binding Peptide designs were immobilized to the Pierce Amino Link resin following the manufacturer's instructions. Peptides were immobilized in a volume of 2.00 μΐ, of coupling buffer (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) and 2 uL cyanoborohydride solution (5 M sodium cyanoborohydride in 1 M NaOH) at a concentration of 358 uM overnight at 4°C. Any residual active sites were blocked with 400 μΐ, quenching buffer (1 M tris hydrochloride, pH 7.4) and 4 μί _^ cyanoborohydride solution for 4 hours at 25°C. Binding experiments were performed from 2.00 uL amyloid solution (5 μΜ Αβ or 20 μΜ TTR (monomer) diluted to the desired concentration in coupling buffer), which was allowed to bind to the peptide -bound agarose beads for 2 h at 25°C. The solution was then collected by centrifugation (flow through, FT). The beads were resuspended in 300 xL coupling buffer, vortexed to obtain an uniform slurry, and the solution was again collected by centrifugation (wash 1 , Wl ). The wash step was performed an additional five times (W2-W6). One final wash step was performed but after resuspending the resin, the solution was allowed to sit for 5 minutes before centrifugation ( W7). The resin was next resuspended in 100 μL 2M guanidine hydrochloride, incubated for 5 minutes at room temperature, then collected as before. This was performed twice (E1-E2). The resin was washed again with 300 Τ coupling buffer (W8) followed by two eiution steps with 6M guanidine hydrochloride (E3-E4). One final wash step was performed with 300 μL coupling buffer (W9). All collected eluents were analyzed by applying triplicate 1 uL spots to nitrocellulose, and then performing standard dot blot analysis as described by Kayed et al. (9) with an anti-TTR (sc- 13098, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-Αβ (ab39377; Abeam Inc, Cambridge, MA) primary antibody diluted 1 : 1,000 in 5% or 10% nonfat milk, respectively.

SH-SY5Y Cell Viability The toxicit '- of aggregates was tested against the human neuroblastoma cell line SH-SY5Y in an MTT cell viability assay. The hitman neuroblastoma cell line SH-SY5Y (CRL-226; American Type Culture Collection) was grown in 75 cm ¹ flasks in 1 :1 DMEM F12 (CellGro) supplemented with 10% FBS and 50 units/mL penicillin/50 itg/mL streptomycin (complete media), and incubated at 37 °C in humidified 5% CO _? environment. Cells were routinely passaged when they reached 90% confluence by addition of trypsin (Gibco) and replated at a ratio of 1 : 10 in complete media. Cells were plated to a density of 25,000 cells/well in a 96-well plate (100 uL/well) and allowed to attach overnight. The cell assay was performed as described by Reixach et al. (41),

CD Spectroscopy Far UV CD spectra were recorded on an A viv model 420 spectrometer (Aviv Biomedical) over 190-260 rim in a 1 mm quartz cuvette. Ail samples were prepared at 100 μΜ in 50 mM phosphate, 100 mM KG buffer, pH 5.8, and were recorded at 25 °C with a resolution of 0,5 nm, a scan speed of 20 nm min, and a 2 nm bandwidth. Average values from three scans were plotted using the Origin 8 software (Originlab, Northhampton, MA), Ail spectra were smoothed using the Savitzky-Golay method with 5-12 points/window, and polynomial order 2.

FTIR Spectroscopy IR spectra were obtained using a Perkin-Eimer Spectrum 100 instrument equipped with a diamond attenuated total reflectance sample unit and an MCT detector. Peptide samples were pelleted and resitspended as a 1 -2 μΐ slurry. The slurry was applied to the diamond and dried to a film over a few minutes while following the disappearance of the broad liquid water band at -1636 cm ^"1 and the appearance of the protein amide T and amide II bands. The spectra were background-subtracted and comprised of 64 accumulations (4 cm ^-1 resolution; 1 cm/sec OPD velocity; strong apodization). Spectra shown here were recorded as soon as successively collected spectra (each recorded over 80 sec) stabilized, indicating little further evaporation of liquid water. This approach was taken to essentially eliminate spectral contributions from free liquid water without desiccating the peptide film any more than necessary. Second derivative spectra were calculated using the instrument software and 13 data points,

NMR Spectroscopy Peptides were prepared in 50 mM potassium phosphate buffer containing 1 00 mM KG pH 5,8. All NMR experiments were performed on Bruker Avance 600 and/or 500 MHz spectrometers equipped with cryogenic triple resonance probes. The sample temperature was kept at 25 "C, 4,4-dimet yl-4-silapentane- 1 -sulfonic acid (DSS) was used for proton chemical shift referencing whereas indirect referencing was used for carbon and nitrogen. The resonance assignments for peptides were carried out using ^-ί-Ή TOCSY and [Ή-Ή] NOESY spectra recorded in 90% H ₂ 0 and 10% ² H ₂ 0. The assignments thus obtained are translated onto the natural abundance 'H- ^1J ]s! HSQC and ¾-'¾ HSQC spectra. All spectra were processed with Topspin3.0 (Bruker) and analyzed using CARA

(http://cara.nmr.ch/doku.php) or Sparky (see web site cgl.ucsf.edu/home/sparky/); figures were made using CARA. Example 2. a-Shect Structure not Specific Amino Acid Sequences Dictate Inhibition Three a-sheet designs are addressed in Example 1 , but many more have been evaluated using these same assays and structural methods. Figure 5 contains a list of other designed a-sheet peptides, both linear and cyclic (specifically peptides #3-5 and #8-9), as well as other control, non-cc-sheet designs (peptides #17- 18, 20). In addition, Figure 5 contains 'control' designs aimed at reverse engineering the oil design, which is presented in more detail in Example 3. Figure 5 provides evidence that the primary amino acid sequence of the polypeptides does not per se dictate the inhibitory activity (here defined as inhibition of aggregation and/or binding of toxic oligomer). Instead it is the ability of the polypeptides to adopt a stable a-sheet structure that is operative, which is achieved through the recited polypeptide generic structure and the recited arrangement of alternating D/L amino acids. Random primary sequences of amino acids are active in the polypeptides of the invention; these same primary sequences are not active if the polypeptide is provided in an entirely L~ amino acid peptide or if the L/D amino acids are randomly distributed within the polypeptide such that they are not alternating (i.e. if they are not polypeptides according to the invention), As noted in Figure S:

« An all L-amino acid peptide (llas90, peptide #20, Control9) with the same amino acid sequence as ldas90 (peptide #1) does not form a-sheet and is inactive. The two peptides have 100% sequence identity and 100% composition match. o Thus, the primary amino acid sequence alone does not determine activity; it is the L/D patterning of the sequence that is critical.

All L/D alternating hairpin peptides investigated experimentally to date form a-sheet structure (see, for example, peptides #1- 14). They also all inhibit aggregation and/or bind the toxic oligomer. Some of the polypeptides are insoluble and the solution assay can't be performed, but they do bind the targeted toxic oligomer when they are immobilized to agarose beads to promote solubilization.

o Thus, this provides further evidence that the primary amino acid sequence per se is not determining structure and activity, the L/D patterning is. However, the sequence modulates the activity and solubility properties.

Both cyclic and linear designs with different sequences form a-sheet polypeptides, inhibit aggregation, and bind the toxic oligomer. (See peptides #! -#13.)

o ^' This provides further evidence that inhibitory behavior is determined by L/D patterning, not by primary amino acid sequence or the nature of the polypeptide turns.

Both charged N- and C-termini and neutral capped termini versions of ldas90 adopt a-sheet structure and demonstrate activity, (See peptides #1 and #7.) Note, however, that the charges can modulate the level of activity.

Next, consider all 14 designed peptides with the L/D turn template, they are all active. Some have issues with solubility but they all form a-sheet and are active amyloid inhibitors in solution and/or binding of the toxic oligomer. These designs are both linear and cyclic peptides designed to be hairpins. The sequence identity relative to ldas90 (peptide #1) ranges from 9-91% across these 14 designs with an average of 43%. Hence, these are different compounds and they show that conventional rales about composition and matter based on amino acid sequence don't hold for this situation. Instead all sequences tried in an L/D template form a-sheet and are active. Finally, there are 6 peptides with the same amino acid composition (100%

composition match) (peptides # 1, 6, 7, 12, 19 and 20) (Figure S and Table 1).

o Two of these have 100% sequence identity and have alternating L/D amino acids (idas90: #1 and #7) but one has charged termini and the other has capped termini (N-acetyiated and C-amidated). They both form α-sheet and are active in inhibiting aggregation and binding toxic oligomer. o The all L-amino acid version of ldas90 (llas90, or control9, peptide #20) has

100% sequence identity, but it doesn't form a-sheet and it's inactive, o Controls (peptide #6) has the ldas90 turn and the tails but the amino acids in the L/D strands were shuffled and randomized while maintaining the L/D patterning. This peptide adopts a-sheet structure and is active, o Control (peptide #12) has the ldas90 (peptide #1) GR tails (XI and X5) and the L/D patterning but all the positions aside from the tails are randomized.

The sequence composition is 100% and the sequence identity is 25%. This peptide forms a-sheet and is active,

o Controi2 (peptide #19) has 24% sequence identity with ldas90 (peptide #1 ) and all positions were randomized and the L/D patterning was not imposed.

This peptide does not form a-sheet and it is inactive.

Example 3, Reverse Engineering of al: Sequence Modifications and Effects on

Structural Stability and Inhibition

To determine the effects of sequence modifications on structure and function of the a- sheet, the al fdas90 peptide (peptide #1) was reverse engineered by successively modifying or eliminating stabilizing design features, including amino acid sequence of the strands, turns, and alternation of L- and D- amino acids. These "control" peptides' primary sequences, all applicable names, identity relative to al, and the modifications that gave rise to each peptide are listed in Table 1 and ordered in terms of decreasing sequence identity to idas90, al, in the L/D template group.

Moving down Table 1 , Controls (peptide #6) has the ldas90 L/D template, the ldas90 amino acid composition, the same relative chirality (i.e. the L/D patterning, or relative positions of the L and D residues in the sequence), and 65% sequence identity through scrambling of the amino acids in the two a-strands. Control6 (peptide #12) takes this a step further with scrambling of the strands and the intervening turn, resulting in 25% sequence identity while retaining 100% of the sequence composition of ldas90. Controls (peptide #1 1) has the alternating D/L template but with a completely random sequence, resulting in 16% sequence identity. Control4 (peptide #13) is the same, it retains the L/D patterning but has a completely random sequence, this time resulting in 9% sequence identity. Two non-L/D template controls are provided. Control2 (peptide #19) has the ldas90 (peptide #1) amino acid composition but everything is scrambled, resulting in 24% sequence identity. Finally, Control9 (or Has90, peptide #20) is a completely L-amino acid version of ldas90. It has 100% sequence identity with idas90 and 74% chirality in common with ldas90 (i.e. 74% represents the matching of the L-amino acids in the two peptides). The as90 and 1das90 peptides are identical in chemical terms aside from the stereochemistry.

The "control" designs in Table 1 were evaluated using circular dichroism (CD) and

Fourier Transform Infrared (FTIR) absorbance spectroscopy, carried out as described in Example 1. For hairpin peptides exhibiting stable a-sheet secondary structure, relatively flat CD spectra are expected, with some negative signal at 195-200 nm due to the turns (X3) and RG tails (XI and X5) (both of which include only L-amino acids). Characteristic "random coil" CD signal for L-amino acids occurs at 195-200 nm. α-sheet secondary structure is expected to give FTIR bands at 1680 cm ^{" 1} and 1640 cm ^{" 1} . Both CD and FTIR spectroscopy are well-established techniques for secondary structure elucidation. The results are summarized in Table 1; all peptides containing the L/D template form a-sheet (peptides #1, 6, 12, 1 1, and 13), while those lacking the alternating D/L amino acids do not (peptides #19, 20).

The "control" designs were tested as general inhibitors against the Amyloid β 1-42 peptide (Αβ) (panel A), the islet amylin polypeptide (LAPP, or amylin, which is implicated in type 2 diabetes) (panel B), and transthyretin (TTR) (panel C) amyloidosis (Figure 6).

Inhibitory activity was observed for all peptides featuring alternating L- and D- amino acids. As described in Example 1 , a Thio flavin T (ThT) fluorescence assay was used to monitor Αβ and IAPP aggregation. A Congo Red dye-binding assay was used to monitor TTR fibriliization. For inhibition against Αβ, peptides were co-incubated at 4-fold excess of Αβ at RT in neutral pH buffer containing ThT. Note that the results for al differ somewhat from what are included in Example 1 because the assay conditions were altered slightly to slo the process down to favor oiigomerization of Αβ (rather than fibril elongation) by lowering the temperature to 25 °C. The advantages of these systematic changes are evident in Figure 6: inhibitory activity is improved to the extent that al quenches Αβ aggregation at only a 4-fold excess. For inhibition against IAPP, peptides were co-incubated at 8-fold excess of IAPP at 37° C in neutral pH buffer containing 20 μΜ ThT. For inhibition against TTR, peptides were co-incubated at 10-fold excess of monomelic TTR. Note that several of the control s bind Congo Red so their inhibitory properties in solution against TTR could not be assessed (Controls 6, 3, 4 and 2), but the a-sheet designs (Controls 6, 3, and 4) do bind toxic oligomer when immobilized while Control 2 does not (Table 1 and Figure 5).

The data in Figure 6 were normalized to ThT fluorescence of the negative

(uninhibited) control (black squares), inhibition traces are ordered vertically according to sequence identity to al (see Table 1). Sigmoidal fits to data are included. Controls are identical throughout a single panel, and traces for inhibition samples are colored while the uninhibited aggregation profiles are in black. All of the alternating L/D designs inhibit aggregation (although Control 3 is weak) (Figure 6) and bind the toxic oligomer when immobilized (Table 1), while the non-L/D controls do not inhibit amyloidosis nor do they bind the oligomer.

• To investigate the effect of randomizing the amino acids within the two a-strands of ldas90 (peptide #1) a control was investigated in which the residues in the turn (X3) and at the ends of the peptide (X I and X5) were held constant and the amino acids in the strands were scrambled (peptide #6, Controls). The overall identity in amino acid composition between the polypeptides was 100% but the resulting primary amino acid sequence identity dropped to 65%, Despite having a random sequence in the strands, peptide #6 is a good inhibitor and it binds the toxic oligomer. In fact, this control with scrambled strands is as good as ldas90 (peptide #1) with capped ends and it is better than kias90 with charged ends (peptide #7).

• Control6 (peptide #12) takes the randomization of 3das90 a step further and maintains the GR ends (XI and X5) but randomizes the strands and turn (X3-X4-X5). The primary amino acid sequence identity drops to 25% while the composition remains at 100%. The scrambling of 75% of the sequence still yields an a-sheet structure and an active peptide: it inhibits aggregation and binds the toxic oligomer. Again this shows that the alternating L/D patterning is critical for activity of the polypeptides of the invention, not the primary amino acid sequence per se.

• Taking this a step further, Controls 3 and 4 (peptides #1 1 and #13) are totally random sequences on the L/D strand - turn - L/D strand template. These peptides have 16 and 9% sequence identity to klas90 (peptide #1), respectively. Both of these peptides adopt a-sheet structure and are active and prevent aggregation in solution and/or bind the toxic oligomer.

All of the L/D template designs are active but as the sequence is modified and the structure is destabilized with concomitant increasing random coil content (indicated by decreasing [9 _m m] at 195 nm), inhibition drops. Figure 7 illustrates the correlation between destabilization of the α-sheet structure, as measured through the increasing random coif signal, and drop in inhibitory potency as the sequence becomes increasingly randomized. All of the L/D template designs are active, even with random sequences through the stands, with some as active (for example Controls, peptide #6) as the parent design, ldas90 (peptide #1). Thus, the sequence per se does not determine inhibition and oligomer binding, the alternating L/D amino acids giving rise to the a-sheet stmcture direct inhibitory behavior. But, the sequence modulates the stability and some sequences lead to more stable a-sheets than others and more stable a-sheets yield more potent inhibitors of aggregation.