PEPTIDIC INHIBITORS OF AMYLOID SELF- AND CROSS-ASSEMBLY
20250161408 · 2025-05-22
Inventors
Cpc classification
International classification
Abstract
The present invention relates to a peptide, and to a pharmaceutical composition and a heterocomplex comprising the peptide. Furthermore, the present invention relates to the peptide, the pharmaceutical composition, or the heterocomplex for use in a method of preventing or treating Alzheimer's disease and/or for use in a method of preventing or treating type 2 diabetes. The present invention further relates to the peptide, the pharmaceutical composition, or the heterocomplex for use in a method of diagnosing Alzheimer's disease and/or for use in a method of diagnosing type 2 diabetes. The present invention also relates to a kit for the in vitro or in vivo detection of amyloid fibrils or aggregates, or for the diagnosis of Alzheimer's disease and/or type 2 diabetes in a patient. Moreover, the present invention relates to the use of the peptide or of the heterocomplex in an in vitro assay for the detection of monomeric islet amyloid polypeptide (LAPP), monomeric A.sub.40 (.sub.42), amyloid fibrils, or amyloid aggregates.
Claims
1. A peptide having an amino acid sequence according to formula 1 TABLE-US-00013 (formula1) (X.sub.1).sub.m(X.sub.2).sub.n(X.sub.3).sub.p(X.sub.4).sub.qF.sub.aF.sub.bAEX.sub.5-X.sub.6X.sub.7X.sub.8-NKGAIIX.sub.9X.sub.10X.sub.11VG (G).sub.r(V).sub.s(V).sub.t wherein, m, n, p, q, r, s, and t are, independently at each occurrence, selected from 0 and 1; X.sub.1 is selected from glutamine, asparagine, alanine, glutamic acid, and aspartic acid; X.sub.2 is selected from lysine, modified lysine, alanine, modified alanine, ornithine, modified ornithine, diaminobutyric acid, diaminopropionic acid, arginine, and modified arginine; X.sub.3 is selected from leucine, N-methyl-leucine, alanine, N-methyl-alanine, isoleucine, N-methyl-isoleucine, valine, N-methyl-valine, glycine, N-methyl-glycine, norleucine, N-methyl-norleucine, phenylalanine, N-methyl-phenylalanine, tyrosine, and N-methyl-tyrosine; X.sub.4 is selected from leucine, N-methyl-leucine, alanine, N-methyl-alanine, isoleucine, N-methyl-isoleucine, valine, N-methyl-valine, glycine, N-methyl-glycine, norleucine, N-methyl-norleucine, phenylalanine, N-methyl-phenylalanine, tyrosine, and N-methyl-tyrosine; F.sub.a and F.sub.b are, independently at each occurrence, selected from phenylalanine, N-methyl-phenylalanine, halogenated phenylalanine, tyrosine, and N-methyl-tyrosine; A is, independently at each occurrence, selected from alanine, N-methyl-alanine, leucine, N-methyl-leucine, valine, N-methyl-valine, glycine, and N-methyl-glycine; E is selected from glutamic acid, N-methyl-glutamic acid, aspartic acid, N-methyl-aspartic acid, glutamine, N-methyl-glutamine, asparagine, N-methyl-asparagine, alanine, N-methyl-alanine, glycine, and N-methyl-glycine; X.sub.5 is selected from aspartic acid, glutamic acid, asparagine, aspartic acid, alanine, and glycine; X.sub.6, X.sub.7, and X.sub.8 are, independently at each occurrence, selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, valine, and modified forms thereof; N is selected from asparagine, glutamine and alanine; K is selected from lysine, modified lysine, alanine, norleucine, ornithine, modified ornithine, diaminobutyric acid, diaminopropionic acid, arginine, and modified arginine; G is, independently at each occurrence, selected from glycine and alanine; I is, independently at each occurrence, selected from isoleucine, leucine, norleucine, and valine; X.sub.9 is selected from glycine, alanine, and serine; X.sub.10 is selected from leucine, alanine, isoleucine, threonine, norleucine, and valine; X.sub.11 is selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine; and V is, independently at each occurrence, valine; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.
2. The peptide according to claim 1, having an amino acid sequence according to formula 2 TABLE-US-00014 (formula2) (X.sub.1).sub.m(X.sub.2).sub.n(L.sub.a).sub.p(V.sub.a).sub.qF.sub.aF.sub.bAEX.sub.5-X.sub.6X.sub.7X.sub.8-NKGAIIX.sub.9X.sub.10X.sub.11VG (G).sub.r(V).sub.s(V).sub.t wherein, m, n, p, q, r, s, and t are, independently at each occurrence, selected from 0 and 1; X.sub.1 is selected from glutamine, asparagine, alanine, glutamic acid, and aspartic acid; X.sub.2 is selected from lysine, modified lysine alanine, modified alanine, ornithine, modified ornithine, diaminobutyric acid, diaminopropionic acid, arginine, and modified arginine; L.sub.a is leucine or N-methyl-leucine; V.sub.a is valine or N-methyl-valine; F.sub.a and F.sub.b are, independently at each occurrence, selected from phenylalanine, N-methyl-phenylalanine, halogenated phenylalanine, tyrosine, and N-methyl-tyrosine; A is, independently at each occurrence, selected from alanine, N-methyl-alanine, leucine, N-methyl-leucine, valine, N-methyl-valine, glycine, and N-methyl-glycine; E is selected from glutamic acid, N-methyl-glutamic acid, aspartic acid, N-methyl-aspartic acid, glutamine, N-methyl-glutamine, asparagine, N-methyl-asparagine, alanine, N-methyl-alanine, glycine, and N-methyl-glycine ; X.sub.5 is selected from aspartic acid, glutamic acid, asparagine, aspartic acid, alanine, and glycine; X.sub.6, X.sub.7, and X.sub.8 are, independently at each occurrence, selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, valine, and modified forms thereof ; N is selected from asparagine, glutamine and alanine; K is selected from lysine, modified lysine , alanine, norleucine, ornithine, modified ornithine , diaminobutyric acid, diaminopropionic acid, arginine, and modified arginine ; G is, independently at each occurrence, selected from glycine and alanine ; I is, independently at each occurrence, selected from isoleucine, leucine, norleucine, and valine; X.sub.9 is selected from glycine, alanine, and serine ; X.sub.10 is selected from leucine, alanine, isoleucine, threonine, norleucine, and valine ; and X.sub.11 is selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine ; V is, independently at each occurrence, valine; wherein at least one of L.sub.a, V.sub.a, F.sub.a, and F.sub.b is an N-methylated amino acid; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.
3. The peptide according to claim 1, having an amino acid sequence according to any one of formulae 3-5 TABLE-US-00015 (formula3) QKL.sub.aV.sub.aF.sub.aF.sub.bAED-X.sub.6X.sub.7X.sub.8-NKGAIIGLNleVGGVV (formula4) F.sub.aF.sub.bAED-X.sub.6X.sub.7X.sub.8-NKGAIIGLNleVGGVV (formula5) QKL.sub.aV.sub.aF.sub.aF.sub.bAED-X.sub.6X.sub.7X.sub.8-NKGAIIGLNleVG wherein, Q is glutamine; K is, independently at each occurrence, lysine; L.sub.a is leucine or N-methyl-leucine; V.sub.a is valine or N-methyl-valine; F.sub.a and F.sub.b are, independently at each occurrence, phenylalanine or N-methyl-phenylalanine; A is, independently at each occurrence, alanine; E is glutamic acid; D is aspartic acid; X.sub.6, X.sub.7, and X.sub.8 are, independently at each occurrence, selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine; N is asparagine; G is, independently at each occurrence, glycine; I is, independently at each occurrence, isoleucine; L is leucine; Nle is norleucine; V is, independently at each occurrence, valine; wherein at least one of L.sub.a, V.sub.a, F.sub.a, and F.sub.b is an N-methylated amino acid; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.
4. The peptide according to claim 1, wherein X.sub.6, X.sub.7, and X.sub.8 are the same amino acid selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine.
5. The peptide according to claim 1, having an amino acid sequence according to any one of formulae 6-8, TABLE-US-00016 (formula6) QKL.sub.aV.sub.aF.sub.aF.sub.bAEDNleNleNleNKGAIIGLNleVGGVV, (formula7) QKL.sub.aV.sub.aF.sub.aF.sub.bAEDLLLNKGAIIGLNleVGGVV, (formula8) QKL.sub.aV.sub.aF.sub.aF.sub.bAEDFFFNKGAIIGLNleVGGVV, wherein, Q is glutamine; K is, independently at each occurrence, lysine; L.sub.a is leucine or N-methyl-leucine; V.sub.a is valine or N-methyl-valine; F.sub.a and F.sub.b are, independently at each occurrence, phenylalanine or N-methyl-phenylalanine; A is, independently at each occurrence, alanine; E is glutamic acid; D is aspartic acid; Ne is norleucine; N is asparagine; G is, independently at each occurrence, glycine; I is, independently at each occurrence, isoleucine; L is, independently at each occurrence, leucine; V is, independently at each occurrence, valine; F is, independently at each occurrence, phenylalanine; wherein at least one of L.sub.a, V.sub.a, F.sub.a, and F.sub.b is an N-methylated amino acid; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.
6. The peptide according to claim 2, wherein two or more of L.sub.a, V.sub.a, F.sub.a, and F.sub.b are an N-methylated amino acid.
7. The peptide according to claim 1, wherein said peptide has an amino acid sequence according to any one of formulae 9-17, TABLE-US-00017 (formula9) QK(N-methyl-L)V(N-methyl-F)FAEDNleNleNleNKGAII GLNleVGGVV, (formula10) QKL(N-methyl-V)F(N-methyl-F)AEDNleNleNleNKGAII GLNleVGGVV, (formula11) QK(N-methyl-L)V(N-methyl-F)FAEDLLLNKGAIIGLNleV GGVV, (formula12) QKL(N-methyl-V)F(N-methyl-F)AEDLLLNKGAIIGLNleV GGVV, (formula13) QK(N-methyl-L)V(N-methyl-F)FAEDFFFNKGAIIGLNleV GGVV, (formula14) QKL(N-methyl-V)F(N-methyl-F)AEDFFFNKGAIIGLNleV GGVV, (formula15) QK(N-methyl-L)(N-methyl-V)(N-methyl-F) (N-methyl-F)AEDFFFNKGAIIGLNleVGGVV, (formula16) QK(N-methyl-L)(N-methyl-V)(N-methyl-F) (N-methyl-F)AEDLLLNKGAIIGLNleVGGVV, (formula17) QK(N-methyl-L)(N-methyl-V)(N-methyl-F) (N-methyl-F)AEDNleNleNleNKGAIIGLNleVGGVV, wherein, Q is glutamine; K is, independently at each occurrence, lysine; L is, independently at each occurrence, leucine; V is, independently at each occurrence, valine; F is, independently at each occurrence, phenylalanine; A is, independently at each occurrence, alanine; E is glutamic acid; D is aspartic acid; Ne is, independently at each occurrence, norleucine; N is asparagine; G is, independently at each occurrence, glycine; I is, independently at each occurrence, isoleucine; and pharmaceutically acceptable salts, esters, solvates, polymorphs and modified forms thereof.
8. The peptide according to claim 1, wherein said peptide consists of a sequence according to any one of formulae 1-17.
9. A pharmaceutical composition comprising a peptide according to claim 1 and a pharmaceutically acceptable excipient.
10. A heterocomplex comprising a peptide according to claim 1, and amyloid- peptide and/or islet amyloid polypeptide.
11. A method selected from: A) a method of preventing or treating Alzheimer's disease and/or for preventing or treating type 2 diabetes, wherein said method comprises administering, to a subject in need of such prevention or treatment, the peptide according to claim 1, or a heterocomplex comprising a peptide according to claim 1 and amyloid- peptide and/or islet amyloid polypeptide; and B) a method of diagnosing Alzheimer's disease and/or diagnosing type 2 diabetes, wherein said method comprises administering, to a subject to be tested for Alzheimer's disease and/or type 2 diabetes an effective amount of the peptide according to claim 1, or a heterocomplex comprising a peptide according to claim 1 and amyloid- peptide and/or islet amyloid polypeptide.
12. (canceled)
13. The method according to claim 11, wherein said peptide is linked to, or administered together with, a suitable reporter molecule that allows detection of A40(42), islet amyloid polypeptide (IAPP), and/or amyloid aggregates or co-aggregates thereof by a suitable detection methodology and wherein said subject, after administration of said peptide, is subjected to said suitable detection methodology.
14. A kit for the in vitro or in vivo detection of amyloid fibrils or aggregates, or for the diagnosis of Alzheimer's disease and/or type 2 diabetes in a subject, said kit comprising the peptide according to claim 1, a pharmaceutical composition comprising the peptide according to claim 1, or a heterocomplex comprising a peptide according to claim 1 and amyloid- peptide and/or islet amyloid polypeptide , in a freeze-dried form in a suitable container, and a buffered solvent in a separate container for reconstitution of said peptide in solution.
15. A method for detecting monomeric islet amyloid polypeptide (IAPP), monomeric A40(42), amyloid fibrils, amyloid aggregates, and/or amyloid co-aggregates wherein said method comprises the use, in an in vitro assay, of the peptide according to claim 1, or of a heterocomplex comprising a peptide according to claim 1 and amyloid- peptide and/or islet amyloid polypeptide , in an in vitro assay.
16. The peptide according to claim 3, wherein X.sub.6, X.sub.7, and X.sub.8 are independently at each occurrence, selected from norleucine, leucine, and phenylalanine.
17. The peptide according to claim 4, wherein X.sub.6, X.sub.7, and X.sub.8 are the same amino acid selected from norleucine, leucine, and phenylalanine
18. The peptide according to claim 6, wherein L.sub.a and V.sub.a; L.sub.a and F.sub.a; L.sub.a and F.sub.b; V.sub.a and F.sub.a; V.sub.a and F.sub.b; F.sub.a and F.sub.b; L.sub.a, V.sub.a, and F.sub.a; L.sub.a, V.sub.a, and F.sub.b; L.sub.a, F.sub.a, and F.sub.b; V.sub.a, F.sub.a, and F.sub.b; or L.sub.a, V.sub.a, F.sub.a, and F.sub.b are N-methylated amino acids.
19. The peptide according to claim 18, wherein at least L.sub.a and F.sub.a, or at least V.sub.a and F.sub.b, are an N-methylated amino acid.
20. The method according to claim 13, wherein the suitable detection methodology is selected from positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI.
21. The method according to claim 15, wherein said in vitro assay is selected from an enzyme linked immunosorbent assay (ELISA), a radioimmuno assay (RIA), and a Dot/Slot blot assay.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0177] The present invention is now further described by reference to the following figures.
[0178] All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.
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[0186] Left, time course of synaptic transmission; meansSEM (n=7-8 for A42/ACM (1/10), n=8 for A42 (50 nM) and buffer controls, and n=36 for ACMs alone (500 nM)). Right, LTP values: averages from the last 10 min of recording; data, meansSEM (n, see above); ***P<0.001 versus A42 (one-way ANOVA & Bonferroni). b hf-A42/ACM are seeding incompetent. A42 (5 M) fibrillogenesis alone or seeded with fA42, hf-A342/Nle3-VF, or hf-A42-L3-VF (10%) determined by ThT binding (meansSD, 3 assays). c Degradation of hf-A42/Nle3-VF and fA42 by PK (37 C.) followed by dot blot; A42 quantification by A(1-17)-specific antibody. Representative membranes from 3 assays. d Thermolability of hf-A42/ACM versus fA42. TEM images of boiled fA42 (15 min) versus hf-A42/Nle3-VF (5 min); scale bars: 100 nm. e Phagocytosis of hf-A342/ACM versus fA42 by cultured murine BV2 microglia. Left and mid panels, representative microscopic images of cells after incubation (6 h, 37 C.) with TAMRA-fA42, hf-TAMRA-A42/Nle3-VF, and hf-TAMRA-A42/L3-VF (1 M); red dots indicated TAMRA-A42; scale bars, 100 m. Right panel, amounts of phagocytic cells (% of total). Data meansSD from 8-10 peptide preparations analyzed in 2 cell assays, each assay well analyzed in 3 fields of view; *P<0.05 (unpaired t-test). f Effects of ACMs on fIAPP-mediated cross-seeding of A42 fibrillogenesis (left panel) or cytotoxicity (right panel). Left panel, fibrillogenesis of A42 (10 M) or A42/ACM (1/2) mixtures following cross-seeding with fIAPP (20%) and of A42 w/o fIAPP seeds (10 M) determined by ThT binding (meansSD, n=4-8). Right panel, solutions (made as for left panel w/o ThT; 1.5 h aged) were added to PC12 cells; cell damage determined via MTT reduction (meansSD, 3 assays, n=3 each). g-j 2 M characterization of supramolecular co-assemblies in A42 solutions after cross-seeding with fIAPP (20%) in the absence (g,h) or presence of ACM (ij). g & h 2 M images of TAMRA-fIAPP-cross-seeded A42 containing HiLyte647-A42 (50%) (1.5 h; incubations as in f) show clusters of A42 assemblies bound to/branching out of fIAPP surfaces; yellow arrow, A42-fIAPP contact site; scale bars: 10 m (g) and 100 mm (h). i 2 M images of fibrillar co-assemblies in TAMRA-fIAPP-cross-seeded A42/Nle3-VF mixtures containing HiLyte647-A42/Fluos-Nle3-VF (50%) (1.5 h; incubations as in f); scale bars: 10 m. Upper panel, fIAPP covered by A42, Nle3-VF, and A42/Nle3-VF (co-)assemblies and surrounded by amorphous or round/elliptical co-assemblies (see also j). Lower panel, huge ternary nanofiber co-assembly. j 3D reconstruction of z-stacks/still images of fibrous co-assemblies shown in i/upper panel. White arrow and dashed line in image on the top indicate view of the section shown below; yellow arrows, round/elliptical co-assemblies; red arrow, fIAPP; blue & green arrows, A42 & Nle3-VF bound to fIAPP; encircled area indicates A42/Nle3-VF co-assembly bound to fIAPP. Scale bars, 10 m (top), 1 m (bottom).
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[0193] For comparison, effects of an aged fibrillar A40 (solution from b; at 20 M) are shown. Data are meansSD from 3 assays, n=3 each. d Nle3-VF oligomerization studied by far-UV CD spectroscopy. CD spectra at different peptide concentrations as indicated (aq. solution, pH 7.4) are shown. Loss of signal was indicative of oligomerization; however, no turbidity or precipitation was observed. e Self-assembly of Nle3-VF studied by fluorescence spectroscopic titrations. Emission spectra of Fluos-Nle3-VF (5 nM) alone and with various Nle3-VF amounts as indicated (Fluos-Nle3-VF/Nle3-VF) (pH 7.4); spectra are from one representative assay out of three. Inset, binding curve (data meansSD from 3 binding curves); determined app. K.sub.D=51.9 * 4.5 nM (mean * SD from 3 binding curves).
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[0209] Applied abbreviations of the analogs as follows: [0210] Abbreviation 17-40: stands for Nle3-VF analog w/o N-terminal segment QK [0211] Abbreviation 18-40: stands for Nle3-VF analog w/o N-terminal segment QKL [0212] Abbreviation 19-40: stands for Nle3-VF analog w/o N-terminal segment QKLV [0213] Abbreviation 20-40: stands for Nle3-VF analog w/o N-terminal segment QKLVF [0214] Abbreviation 21-40: stands for Nle3-VF analog w/o N-terminal segment QKLVFF
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[0216] Applied abbreviations of the analogs as follows: [0217] Abbreviation 15-38: stands for Nle3-VF analog w/o C-terminal segment VV [0218] Abbreviation 15-37: stands for Nle3-VF analog w/o C-terminal segment GVV [0219] Abbreviation 15-36: stands for Nle3-VF analog w/o C-terminal segment GGVV
[0220]
[0221] Effects of 4-fold N-methylated Nle3-VF analog termed Nle3-LVFF on IAPP amyloid self-assembly are shown. ThT binding assay of IAPP/peptide mixtures. Incubations were prepared in ThT buffer with 0.5% HFIP containing 16.5 M IAPP alone or its mixture with peptides (1:2) as indicated. Data are meansSD from 3 assays.
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[0225] In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.
EXAMPLES
Example 1: Methods
Peptides and Peptide Synthesis
[0226] IAPP, IAPP-GI, rat IAPP, and their N.sup.a-terminal fluorescein- or biotin-labeled analogs were synthesized by Fmoc-based solid phase synthesis (SPPS), subjected to air-oxidation, and purified by RP-HPLC. Their stock solutions were prepared in 1,1,3,3,3,3-hexafluoro-2-isopropanol (HFIP) (4 C.), filtered over 0.2 m filters (Millipore), and concentrations were determined by UV spectroscopy. TAMRA-IAPP was synthesized by overnight coupling of 5,6-carboxytetramethylrhodamine (TAMRA) (Novabiochem/Merck) to RINK-resin-bound IAPP using a 3-fold molar excess of 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (H BTU) and a 4.5 molar excess of N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF). TAMRA-IAPP cleavage from the resin and RP-HPLC purification were performed as for the other labeled IAPP analogs; stocks were made in HFIP (4 C.). A42 was synthesized on Tentagel R PHB resin (0.18 mmol/g; Rapp Polymere) by Fmoc-SPPS. Seed-free aqueous A42 stock solutions (10-20 M) were obtained by SEC performed. Briefly, HPLC-purified A42 was dissolved (1 mg/ml) in a solution of 5 M GdnHCl in 10 mM TRIS/HCl pH 6.0 and loaded onto a Superdex 75 10/300 GL column (eluent: 50 mM ammonium acetate pH 8.5, 0.5 ml/min). The monomeric A42 elution peak was collected on ice, stored at 4 C. and used within 1 week; peptide concentration was determined by UV spectroscopy. Fluorescein-isothiocyanate-p-Ala-labeled A42 (FITC-A42) and TAMRA-labeled A42 (TAMRA-A42) were from Bachem and HiLyte647-A42 from AnaSpec; their stocks were prepared in HFIP (4 C.).
[0227] All A(15-40) analogs comprising ACMs, non-inhibitors, and partial segments thereof (Tables 3 and 6) were synthesized using previously described standard Fmoc-SPPS protocols and in most cases WANG-resin (0.3-0.5 mmol/g; Iris Biotech); Tentagel R PHB resin was used for Nle3, R3, and G3-VF (0.16 mmol/g; Rapp Polymere). Briefly, double couplings were usually performed using 3-fold molar excess protected amino acid and HBTU and 4.5-fold molar excess of DIEA in DMF. For difficult couplings, we applied either 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophosphate (HATU), or 4-6-fold molar excess of protected amino acids, and/or triple couplings. N-terminal fluorescein-labeled A(15-40) analogs were synthesized by coupling peptide-resins with 5,6-carboxyfluorescein (Sigma-Aldrich) using 3-fold molar excess protected amino acid and HATU and 4.5-fold molar excess of DIEA (double couplings). N-terminal Atto647N-labeled Nle3-VF was synthesized by coupling peptide-resin with Atto647N (carboxy-derivative) (ATTO-TEC) using HATU. Peptide cleavage from the resin was performed with 95% TFA/H.sub.2O. All peptides were purified by RP-HPLC on Nucleosil 100 C18 (Grace) or Reprosil Gold 200 C18 columns (Dr. Maisch). Stock solutions were made in HFIP (4 C.); peptide concentrations were determined by peptide weight or by UV spectroscopy (fluorescently labeled analogs).
[0228] All synthetic peptides were characterized by matrix-assisted laser desorption ionization (MALDI-MS) or electrospray ionization (ESI-MS) mass spectrometry (Table 6).
Thioflavin T (ThT) Binding Assays
[0229] IAPP fibrillogenesis-related studies. Effects of the different peptides on IAPP fibrillogenesis including self- and cross-seeded fibrillogenesis were studied in combination with TEM and MTT reduction assays according to previously established ThT binding assay systems. At the indicated time points, aliquots of peptide incubations (made as described below) were gently mixed with the ThT solution (20 mM ThT in 0.05 M glycine/NaOH, pH 8.5, if not stated otherwise) in a 96-well black MTP (FluoroNune/Thermo Fisher Scientific). ThT binding was determined immediately by measuring fluorescence emission at 486 nm following excitation at 450 nm using a 2030 Multilabel Reader VictorX3 instrument (PerkinElmer Life Sciences). ThT binding of seeds/buffer were subtracted from the data in all assays related to seeding events; in all other cases raw or normalized data are shown if not stated otherwise. All IAPP-related incubations were performed at 20 C. except for the (cross-)seeded ones (RT). Preformed fAAPP were generally prepared by incubating IAPP (12 or 16.5 M) in ThT buffer for 3-9 days (20 C.), quantified by ThT binding, and verified by TEM.
[0230] Peptide incubations were performed as follows: for studying effects on IAPP fibrillogenesis, freshly made IAPP (16.5 M) and IAPP/peptide mixtures were incubated in 50 mM sodium phosphate buffer, pH 7.4, with 100 mM NaCl containing 0.5% HFIP (abbreviated ThT buffer) for up to 7 days. For studying effects on fIAPP-mediated seeding of IAPP fibrillogenesis, preformed fIAPP (10%) were added to freshly made IAPP (12 M) or IAPP/peptide-mixtures (1/2) in ThT buffer; solutions were incubated for several days as indicated. To determine the detection limit of the IAPP-related ThT binding assay, fIAPP were first made by incubating IAPP (16.5 M) in ThT buffer (7 days). Following fIAPP quantification by ThT binding and verification by TEM (
[0231] ACM fibrillogenesis-related studies. To study the fibrillogenic potential of ACMs, peptides and A40 (positive control) (100 M) were incubated in 10 mM aqueous sodium phosphate buffer, pH 7.4 (1% HFIP) for 4 days. ThT fluorescence was measured at 0 h and 4 days by mixing an aliquot with a ThT containing solution (121 M ThT, 0.05 M glycine/NaOH, pH 8.5); buffer values were subtracted from the data shown in
[0232] A42 fibrillogenesis-related studies. To study effects of the different peptides on A42 fibrillogenesis, synthetic A42 isolated from SEC (see Peptides & peptide synthesis) was used. Peptide incubations were performed in the presence of ThT in 96-well black MTPs (FluoroNunc, Thermo Fisher Scientific). Incubation conditions for all assays were (if not stated otherwise): A42 (5 M) alone or its mixture with the peptide (at the indicated molar ratios) in 45 mM ammonium acetate, pH 8.5, containing 10 M ThT (37 C.); MTPs were shaken (500 rpm; orbital shaker (CAT S20)) for the first 5 h of the fibrillogenesis. ThT fluorescence was measured with a 2030 Multilabel Reader VictorX3 instrument at the indicated time points as under IAPP-related assays. Values of seeds or buffer alone were subtracted from the data in self-/cross-seeding assays; all other data shown are raw data except for data in
[0233] For studying effects of ACMs on fA42-mediated seeding of A42, preformed fA42 (made by incubating A42 (5 M) as above but w/o ThT for 6 days (TEM,
Assessment of Cell Damage Via the MTT Reduction Assay
[0234] Studied on the effects of peptides on formation of cell damaging IAPP assemblies were performed in cultured RIN5fm cells in combination with the ThT binding assay and TEM. Briefly, cells were cultivated and platted in 96-well plates. Aliquots of solutions used for the ThT binding assays (see ThT binding assays) were diluted with cell medium at the indicated incubation time points (24 h or 7 days) and added to the cells at the indicated final concentrations. Following incubation with the cells for 20 h (37 C., humidified atmosphere, 5% CO.sub.2) cell damage was assessed by measuring cellular MTI reduction. IC.sub.50 values were determined. Briefly, IAPP (16.5 mM) was incubated with different molar ratios of the ACMs in ThT buffer (see under ThT binding assay) for 24 h and solutions added to the cells (IAPP, 100 nM); cell viability was assessed as above. To determine cell damaging effects of ACM-coated fIAPP, preformed fIAPP (16.5 M) was co-incubated with the ACM (33 mM) for 1 day as under ThT-binding assays. Solutions of ACM-coated fIAPP versus fIAPP alone were diluted with cell medium and incubated with the cells (fIAPP, 500 nM) as described above.
[0235] The studies on effects of ACMs on formation of cell-damaging A42 assemblies were performed in combination with the ThT binding assay and TEM using PC12 cells cultured and plated. Incubations of A42 alone and its mixtures were made in MTPs as described for the ThT binding assays (parallel to the incubations made for the ThT binding assay) but w/o ThT; 6 day-aged solutions (37 C.) were diluted with cell medium and added to the PC12 cells at the indicated final concentrations. Following incubation with the cells for 20 h (37 C., humidified atmosphere, 5% CO.sub.2), cell damage was assessed by measuring cellular MTT reduction. To determine IC.sub.50 values of the effects of ACMs, incubations of A42 (5 M) or its mixtures with various amounts of the ACMs were performed as for the ThT binding assay (37 C.) but w/o ThT in MTPs. 6 day-aged incubations were diluted with medium and added to the PC12 cells (A42, 1 mM) and cell damage was assessed following 20 h incubation with the cells as above. On note, anomalous concentration-dependence profiles were found for mixtures of A42 with L3-LF and Nle3-LF most likely due to aggregation; therefore, IC.sub.50 values were not determined.
[0236] To study effects of ACMs on fIAPP-mediating cross-seeding of formation of cell-damaging A42 assemblies, incubations were made as for the corresponding ThT binding assays but w/o ThT in MTPs. Solutions were aged for 1.5 h (37 C., shaking 500 rpm). Aliquots were diluted with cell medium, incubated with PC12 cells at the indicated final concentrations for 20 h and cellular MTT reduction was measured as above.
[0237] Effects of ACMs on PC12 cell viability were studied using the 4 day-aged solutions applied in the ThT binding assays which were performed to determine their amyloidogenic potential (see under ThT binding assays). Following incubation with the cells (at 20 mM) for 20 h, cell damage was assessed by MTT reduction; data were corrected for buffer effects. For comparison, effects of aged A40 were also studied and cytotoxicity was as expected.
Transmission Electron Microscopy (TEM)
[0238] Aliquots of solutions used for ThT binding, MTT reduction, or other assays were applied on formvar/carbon-coated grids at the indicated incubation time points. Grids were washed with ddH.sub.2O and stained using aqueous 2% (w/v) uranyl acetate. Examination of the grids was done with a JEOL 1400 Plus electron microscope (120 kV). For A42-related studies, solutions made as for the ThT binding assay but w/o containing ThT were used for TEM and the MTT reduction assays. Kinetics of evolution of IAPP homo- and IAPP/Nle3-VF hetero-fibrils from amorphous aggregates was followed by TEM in solutions made in 10 mM sodium phosphate buffer, pH 7.4 (
Immunogold-TEM
[0239] Immunogold-TEM was performed. Briefly, peptide solutions made as described for the corresponding ThT binding assays were applied onto the grids at the indicated incubation time points. Grids were blocked with 0.1% BSA in 1PBS. fIAPP was detected with a fibril-specific mouse anti-fIAPP antibody (Synaptic Systems; Cl. 91E7). Nle3-VF was revealed by a rabbit anti-A40 polyclonal antibody (Sigma-Aldrich) exhibiting 10-20% NSB to IAPP. The two antibodies (in 0.1% BSA in 1PBS; dilution 1/10) were deposited simultaneously onto the grid and incubated for 20 min. Following washing with 1PBS, grids were incubated (20 min) with secondary antibodies goat anti-rabbit gold-conjugate (10 nm) and goat anti-mouse gold-conjugate (5 nm) (Sigma-Aldrich) (in 0.1% BSA in 1PBS, dilution 1/10) as above. Following 1PBS and ddH.sub.2O washings, uranyl acetate staining and grid examination were performed as described under TEM. To quantify IAPP and Nle3-VF contents of fibrils, 5 and 10 nm gold particles were counted; antibody reactivity is expressed as % of total number of gold particles bound. Significance was analyzed by one-way ANOVA and Bonferroni's Multiple Comparison test.
Far-UV CD Spectroscopy
[0240] CD spectra were recorded using a Jasco 715 spectropolarimeter. CD spectra (average of 3 spectra) were measured between 195-250 nm, at 0.1 nm intervals, a response time of 1 see, and at RT. The spectrum of the buffer was always subtracted from the spectra of the peptide solutions. Peptide incubations related to ACM alone or ACM/IAPP interactions were performed. Briefly, to study peptide conformations and oligomerization propensities, CD spectra of freshly made solutions in 10 mM sodium phosphate buffer, pH 7.4, containing 1% HFIP were measured at 5 mM or at the indicated concentrations in concentration-dependence studies. For studying hf-IAPP/ACM, IAPP (16.5 M) was incubated with Nle3-VF or VGS-VF (33 M) in ThT buffer as for the ThT binding assay for 7 days and spectra were measured at the indicated time points. For comparison, spectra of IAPP, Nle3-VF and VGS-VF alone were also measured. For studying the structure of hf-A42/Nle3-VF, incubations were performed as for the ThT binding assays but in the absence of ThT. Briefly, A42 alone (5 M), Nle3-VF alone (5 M), and their mixtures (5 M each) in 45 mM ammonium acetate (pH 8.5) were incubated for 6 days at 37 C. and CD spectra were measured.
Fluorescence Spectroscopic Titration Assays
[0241] Fluorescence spectroscopic studies were performed with a Jasco FP-6500 fluorescence spectrophotometer. Briefly, excitation was at 492 nm and spectra were measured between 500 and 600 nm. All titrations were performed in freshly made solutions of synthetic N-terminal fluorescently labeled peptide (5 nM) and various amounts of unlabeled peptide in 10 mM sodium phosphate buffer, pH 7.4 (1% HFIP) within 2-5 min following solution preparation. Under these experimental conditions, freshly made solutions of Fluos-IAPP and FITC-A42 (5 nM) consist mostly of monomers and the same was found for Fluos-ACMs (5 nM) (
Cross-Linking, NuPAGE, and Western Blot (WB)
[0242] Hetero-complex cross-linking studies in combination with NuPAGE and WB were performed with a previously developed assay system used for the characterization of AP and IAPP homo- and hetero-assemblies. Briefly, for characterizing IAPP homo-/hetero-assemblies, IAPP (30 M), IAPP/ACM mixtures (1/2) and ACMs alone (60 M) were incubated in 10 mM sodium phosphate buffer (pH 7.4) for up to 7 days (20 C.). In the case of A42 homo-/hetero-assemblies, A42 (30 M) and A42/ACM mixtures (1/2) were incubated in 10 mM sodium phosphate buffer (pH 7.4) for up to 6 days. At the indicated time points (o h, 24 h, and 7 days (IAPP studies) or 0 h, 3 h, 24 h, and 6 days (A42 studies)) aliquots were cross-linked (2 min) with 25% aqueous glutaraldehyde (Sigma-Aldrich) and treated with a 2 M NaBH.sub.4 solution (in 0.1 M NaOH, 20 min). Following precipitation with trichloroacetic acid (10%) (4-C) and centrifugation (10 min, 12000 g), pellets were dissolved in reducing NuPAGE sample buffer, boiled (5 min, 95 C.) and subjected to NuPAGE gel electrophoresis as described using 4-12% Bis-Tris gels and MES running buffer (Thermo Fisher Scientific). Equal amounts of IAPP or A42 were loaded in all lanes. Peptides were transferred onto nitrocellulose membranes (XCell II Blot Module, Thermo Fisher Scientific). Membranes were blocked overnight (10 C.) with 5% milk in TBS-T (20 mM Tris/HCl, 150 mM NaCl and 0.05% Tween-20). To reveal homo-/hetero-assemblies, membranes were incubated (2 h) with one of the following primary antibodies (in 5% milk in TBS-T): rabbit polyclonal anti-IAPP (Peninsula; 1:1000) for IAPP-containing assemblies, rabbit polyclonal anti-A40 (Sigma-Aldrich; 1:2000) for ACM-containing assemblies, or mouse monoclonal anti-A(1-17) (6E10, BIOZOL; 1:2000) for A42-containing assemblies (no cross-reactivity with ACMs). Primary antibodies were combined with suitable peroxidase (POD)-coupled secondary antibodies (donkey anti-rabbit-POD (1:5000) or goat anti-mouse-POD (1:1000)) and homo-/hetero-assemblies were revealed with SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Membranes were stripped by incubating in stripping buffer (2% SDS, 100 mM b-mercaptoethanol, 50 mM TRIS, pH 6.8) for 20 min at 60 C. and at RT for 45 min. Prestained protein size markers ranging from 3.5 to 260 kDa (Invitrogen) were electrophoresed in the same gels.
Size Exclusion Chromatography (SEC)
[0243] SEC was performed with a Superdex 7510/300 GL column (GE Healthcare); flow rate was 0.5 ml/min and detection was at 214 nm. For IAPP-related studies, elution buffer was 50 mM sodium phosphate buffer, pH 7.4, containing 100 mM NaCl. IAPP (16.5 M) or IAPP/ACM (or IAPP/VGS-VF) mixtures (1/2) were incubated in ThT buffer as for the ThT binding assays and at the indicated incubation time points centrifuged (1 min, 20000 g) and loaded onto the column. For A42-related studies, elution buffer was 50 mM ammonium acetate, pH 8.5. A42 (5 M) or A42/ACM (1/1) mixtures were incubated under ThT binding assay conditions (w/o ThT) and loaded onto the column at indicated time points. The column was calibrated with proteins/peptides of known molecular weights.
ANS Binding Fluorescence Spectroscopy
[0244] ANS binding studies were performed with a Jasco FP-6500 fluorescence spectrophotometer. Briefly, excitation was at 355 nm and fluorescence emission spectra were recorded between 355 and 650 nm. Solutions of ANS alone (8 M) and its mixtures with IAPP (2 M) or IAPP/Nle3-VF (1/2) mixtures were freshly made in 10 mM sodium phosphate buffer, pH 7.4, containing 1% HFIP and spectra were recorded at the indicated time points.
Hetero-Complex Pull-Down Assays
[0245] Pull-down assays were performed using streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, Dynal). Briefly, solutions of Biotin-IAPP (16.5 M), Biotin-IAPP/Nle3-VF-mixtures (1/2), and Nle3-VF (33 M; control for non-specific binding (NSB) to beads) in 10 mM sodium phosphate buffer, pH 7.4 were aged for 7 days (20 C.) and subsequently incubated with the beads for 4 h at RT. Bead-bound complexes were isolated by magnetic affinity. Following washing, beads were boiled with reducing NuPAGE sample buffer (5 min, 95 C.) and supernatants subjected to NuPAGE electrophoresis and WB as described under Cross-linking, NuPAGE and WB. Equal amounts were loaded in all lanes; lane Nle3-VF (control), freshly dissolved Nle3-VF without incubation with the beads.
Confocal Laser Scanning Microscopy (CLSM) and 3D Stimulated Emission Depletion (STED) Imaging
[0246] IAPP or IAPP/ACM mixtures (1/2) containing 10% N-terminal fluorescently-labeled analogs TAMRA-IAPP and Atto647N-ACM (IAPP(total), 16.5 M; ACM(total), 33 mM) were incubated in 10 mM sodium phosphate buffer, pH 7.4 for 7 days (20 C.). Aliquots (30-40 l) were pipetted onto SuperFrost Plus adhesion slides (Thermo Fisher Scientific), air-dried, covered with a high precision covershlip (#1.5; Ibidi), and embedded using Prolong Diamond Antifade Mountant (Thermo Fisher Scientific). CLSM and STED were performed using a Leica SP8 STED 3 microscope (HC PL APO 93x/1.30 GLYC CORR STED objective) with a tunable white light laser source to excite fluorophores. Depletion power (660 nm (TAMRA), 775 nm (Atto647N)) and time-gated detection of excited light were chosen to minimize sample damage while optimizing xyz-resolutions. Images were collected in a sequential scanning mode (hybrid-diode detectors) to maximize signal collection while minimizing channel cross-talk (TAMRA: excitation 552 nm/emission 557-645 nm; Atto647N: excitation 646 nm/emission 651-700 nm). 3D reconstructions/fibril measurements were performed using Leica's LAS-X software package (v1.2). Datasets were deconvoluted using Leica's Lightning application.
Two-Photon Microscopy (2 M) and FLIM-FRET Studies
[0247] Solutions analyzed by 2 M or FLIM-FRET consisted of either N-terminal fluorescently-labeled peptides (100%) or mixtures of labeled with non-labeled peptides (when indicated) and were prepared as follows: for most IAPP-related studies, hf-IAPP/ACM were made by incubating TAMRA-IAPP (16.5 M) with synthetic N-terminal fluorescein- or Atto647N-labeled ACMs (33 M) in 10 mM sodium phosphate buffer, pH 7.4 (abbreviated 1 b) for 6-7 days (20 C.). For comparison, aged TAMRA-IAPP (16.5 M) was also examined and consisted mostly of fibrillar assemblies (TAMRA-fIAPP;
[0248] For 2 M and FLIM-FRET studies related to ACM-coated fIAPP, first fIAPP were made by incubating an IAPP/TAMRA-IAPP mixture (16.5 M IAPP(total) containing 10% TAMRA-IAPP) in 1 b for 48 h. fIAPP were then co-incubated with a mixture of Nle3-VF/Atto647-Nle3-VF (33 M Nle3-VF(total) for 1 day (20 C.)) to yield ACM-coated fIAPP. For 2 M and FLIM-FRET studies regarding the role of monomeric/prefibrillar IAPP on formation of IAPP/Nle3-VF nanofiber co-assemblies, aliquots from a freshly made mixture of Fluos-Nle3-VF (33 mM) with TAMRA-IAPP (1.65 mM) in 1 b (20 C.) were examined at the indicated incubation time points. For the corresponding studies on the role of fIAPP, preformed TAMRA-fIAPP seeds (3.3 mM); TAMRA-fIAPP was made by incubating TAMRA-IAPP (16.5 mM) in 1 b for 5 days (20 C.). For all A42-related studies, solutions consisted of 1/1 mixtures of unlabeled/labeled peptides (as indicated) and were prepared as for the ThT binding assays (w/o ThT) (45 mM ammonium acetate, pH 8.5, 37 C.; aging as indicated) as follows: for the studies on fA42 versus hf-A42/ACM, A42 solutions (A42(total) 5 M) consisted of 50% TAMRA-A42 and 50% A42 (6 day-aging); A42/ACM mixtures (1/2) contained, in addition to A42/TAMRA-A42 (1/1; A342(total), 5 M), ACM/Fluos-ACM (1/1; ACM(total), 10 M) (4-6 day-aging).
[0249] For studies on fIAPP-mediated cross-seeding of A42, the A42 alone solution (A42(total), 10 M) contained HiLyte647-A42 (50%); the A42/ACM (1/2) mixtures consisted of A42/HiLyte647-A42 (1/1) (A42(total), 10 M) and Nle3-VF/Fluos-Nle3-VF (1/1) (Nle3-VF(total), 20 M); solutions were aged for 1.5 h. TAMRA-fIAPP seeds were made by incubating TAMRA-IAPP (128 M) in ThT buffer for 6 days (20 C.)); their concentration in the cross-seeded solution was 2 M. Aliquots from the above solutions were applied onto SuperFrost Plus adhesion slides, air-dried, washed (A42-related studies) and embedded with Prolong Diamond Antifade Mountant as for CLSM and STED.
[0250] Samples were imaged on a two(multi)-photon Leica TCSPC SP8 DIVE FALCON LIGHTNING microscope (Leica) equipped with extended IR spectrum tunable laser (680-1300 nm) (New InSight X3, Spectra-Physics) and fixed IR laser (1045 nm), advanced Vario Beam Expander (VBE), ultra-high-speed resonance scanner (8 kHz), HC PL IRAPO 25/1.0 WATER objective, and FLIM-FRET modality. Images were collected in sequential scanning mode (hybrid-diode detectors; TAMRA: excitation 1100 nm/emission 560-630 nm; fluorescein (Fluos): excitation 920 nm/emission 480-550 nm; HiLyte647: excitation 1280 nm/emission 635-715 nm) and handled using Leica's LAS-X software package. Deconvolutions were performed using Huygens Professional or Leica's Lightning application.
[0251] For fluorescence lifetime imaging (FLIM), up to 1000 photons/pixel were captured (time-correlated single-photon counting (TCSPC) mode). Samples were prepared as described above. Fluorescence decays were fit using Leica's FALCON software applying multi-exponential models. Quality of fits was assessed by randomly distributed residuals/low Chi-square values. The number of components (n) used for fittings was manually fixed to values (n=2-4) that minimized Chi-square statistic. In control experiments, fluorescence lifetime of the donor fluorescent molecule (Fluos-Nle3-VF) (33 M, 1 b, aging 6 days) in absence of acceptor was acquired similarly (a multi-exponential model was applied). Amplitude-weighted average lifetime was calculated as =(.sub.i.sub.i)/.sub.i (.sub.i: amplitude of each lifetime .sub.i). FLIM-FRET efficiency was calculated by FRET eff=1(.sub.DA/.sub.D); (.sub.DA=lifetime donor in presence of acceptor; .sub.D=lifetime donor alone).
Dot Blot Assays to Assess Binding of ACMs to IAPP and A42Fibrils and Monomers
[0252] IAPP (128 M) and A42 (11 M) incubations were made as for ThT binding assays (A42 w/o ThT) and deposited onto nitrocellulose membranes (IAPP: 40 g, A42: 10 g) either directly following their preparation (for monomers) or after 2 days of aging (for fibrils; confirmed by ThT binding and TEM). After blocking (5% milk in TBS-T, 2 h, RT) and several washing steps (with TBS-T and ThT buffer), membranes were incubated with N-terminal fluorescein-labeled ACMs (Fluos-ACMs) at 0.2 M for IAPP-related membranes or 2 M for A42-related membranes; incubation was overnight (10 C.) in ThT buffer (containing 1% HFIP). To control for fibril autofluorescence, similar membranes were incubated in parallel with buffer only. Bound peptides were visualized using a LAS-400 mini instrument (Fujifilm) equipped with a suitable fluorescence filter.
Dot Blot Analysis for Quantification of Fibrils
[0253] Dot blot analysis was used to verify the presence of equal amounts of homo- and heteromeric fibrillar assemblies in the aliquots of solutions examined by the various different assays, e.g. the ThT binding assay, the MTT reduction assay, or the PK digestion assay. For example, in the case of the solutions used for ThT binding and MTT reduction assays, 7 day-aged IAPP (16.5 M) or IAPP/ACM (1/2) mixtures were prepared as described under ThT binding assays; TEM showed that fibrils were major species in both kinds of solutions (
Assessment of Fibril Thermostability by TEM and ThT Binding
[0254] Solutions consisting mainly of fIAPP, hf-IAPP/Nle3-VF, fA42 or hf-A42/Nle3-VF were prepared as described under ThT binding assays (fIAPP 16.5 M, 7 day-aged; hf-IAPP/Nle3-VF, IAPP 16.5 mM, Nle3-VF 33 M, 7 day-aged; fA42 5 M, 6 day-aged; hf-A42/Nle3-VF, 5 M each, 6 day-aged; A42 incubations w/o ThT) and boiled (95 C.) for 5 min except for fA42 which was boiled for 15 min. TEM grids were loaded, stained, and analyzed as under TEM. ThT binding of fIAPP and hf-IAPP/Nle3-VF solutions was assessed by mixing aliquots before or after boiling with a ThT solution as described under ThT binding assays; buffer values were subtracted from the data.
Proteinase K (PK)Fibril Digestion Assay in Combination with Dot Blot
[0255] The PK digestion assay was performed based on protocols by Ladiwala et al. and Cho et al. Briefly, PK stocks (100 g/ml) were prepared in 50 mM TRIS/HCl pH 8.0 containing 10 mM CaCl.sub.2; the final PK concentration in the assay was 0.5 g/ml. fIAPP (16.5 M) or hf-IAPP/ACM (1/2) were prepared by incubating the peptides in 10 mM sodium phosphate buffer, pH 7.4 for 7 days (20 C.); fibril formation was confirmed by ThT binding (fIAPP) and TEM. fA42 (5 M) and hf-A42/ACM (1/i) were made as described under ThT binding assays (w/o ThT; 7 day-aging). For IAPP-related assays, solutions were made by mixing 60 l of the fIAPP or hf-IAPP/ACM solutions with 0.3 l of the PK stock solution. For A42-related assays, solutions were made by mixing 200 l of fA42 or hf-A42/ACM solutions with 1 l PK stock. Solutions made as above but w/o PK were used as controls for 100% undigested fibrils. Solutions were incubated at 37 C. and at indicated time points aliquots were dotted onto nitrocellulose membranes and spots were quickly dried by air. Membranes were washed (TBS-T) and blocked (5% milk in TBS-T, overnight (10 C.)). The following primary antibodies were used for membrane development (2 h, in 5% milk in TBS-T (RT)): mouse anti-fIAPP (Synaptic Systems, Cl. 91E7; 1:500) for fIAPP; mouse anti-A(1-17) (6E10, BIOZOL; 1:2000) for A42); rabbit anti-A40 (Sigma-Aldrich; 1:2000) for ACMs. Primary antibodies were combined with goat anti-mouse-POD (1:1000) or donkey anti-rabbit-POD (1:5000); detection was as under Cross-linking, NuPAGE and WB.
Phagocytosis Assay
[0256] Phagocytosis of fIAPP and fA42 versus hf-IAPP/ACM and hf-A42/ACMs was studied in primary murine BMDMs and cultured murine BV2 microglia using TAMRA-IAPP and TAMRA-A42 and essentially following an established protocol. Briefly, BV2 cells were maintained in GlutaMAX-supplemented RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin on poly-L-ornithine-coated flasks. For the phagocytosis assay, BV2 cells were seeded into 24-well plates containing coverslips in serum-free RPMI1640-GlutaMAX and further incubated for 24 h (5% CO.sub.2, 37 C.) to reach 10000 cells/well. Primary BMDMs were obtained from bone-marrow monocytes isolated from wildtype C57BL/6 mice, plated in 24-well plates at a density of 10000cells/well, and differentiated with L929 cell-conditioned medium (RPMI1640, 10% FBS, 1% penicillin/streptomycin) for 7 days. Thereafter, cells were incubated with aged peptide solutions at 37 C. for 6 h. Peptide solutions were prepared as follows: for IAPP-related studies, TAMRA-fIAPP (16.5 M) and hf-TAMRA-IAPP/ACM (16.5 mM) were made by incubating peptides/peptide mixtures (1/2) in ThT buffer for 7 days as for the ThT binding assay. Solutions were diluted with cell medium and added to the cells at a final homo-/hetero-nanofiber (IAPP) concentration of 3.3 M. For A42-related cell uptake studies, TAMRA-fA42 (5 M) and hf-TAMRA-A42/ACM (5 M) were prepared by incubating the peptides/peptide mixtures (1/1) under ThT assay conditions (w/o ThT) for 6 days. Following centrifugation (20 min, 20000 g), pellets were resuspended in cell medium and incubated with the cells for 6 h at a final homo-/hetero-fibril concentration of 1 M. Of note, dot blot analysis and BCA showed that the main peptide fraction was present in the pellet.
[0257] To address the question whether addition of Nle3-VF or L3-VF to preformed A42 oligomers may affect their phagocytosis, preformed TAMRA-A42 oligomers (TAMRA-oA342) were prepared as for MTT reduction assays by incubating TAMRA-A42 (5 M) for 2 h under ThT binding assay conditions. For studying the effect of the ACMs, TAMRA-oA42 was mixed with Nle3-VF or L3-VF (1/1) and, following co-incubation for 2 h (under ThT binding assay conditions), mixtures were diluted with cell medium (TAMRA-A42, 1 M) and incubated with the BV2 cells for 6 h as described above. TAMRA-oA42 alone was incubated for 2 h as well and treated thereafter as its mixtures with the ACMs.
[0258] Following incubation of the cells with the peptides, supernatants were removed and cells on the coverslips were washed 5 times with ice-cold 1PBS, fixed with 4% paraformaldehyde, washed with 1PBS, permeabilized with 0.2% Triton-X 100, and rinsed three times with cold 1PBS. Coverslips were mounted with Vectashield Antifade mounting medium containing DAPI (Vector Laboratories). Images were acquired using a Leica DMi8 fluorescence microscope. The percentage of cells that had taken up peptides was calculated by dividing the number of BV2 cells or BMDMs that phagocytosed TAMRA-labeled peptide by the total cell count, multiplied by 100. Significance was analyzed by unpaired student's t-test.
Hippocampal Long-Term Potentiation (LTP) Measurements
[0259] LTP measurements were performed. Briefly, sagittal hippocampal slices (350 m) were obtained from C57BL/6N male mice (6-8 weeks) in ice-cold Ringer solution bubbled with a mixture of 95% O.sub.2 and 5% CO.sub.2 and according to protocols approved by the ethical committee on animal care and use of the government of Bavaria Germany. Extracellular recordings were performed using artificial cerebrospinal fluid (ACSF)-filled glass microelectrodes (2-3 MW) at RT. ACSF consisted of 125 mM NaCl, 2.5 mM KCl, 25 mM NaHCO, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 25 mM D-glucose, and 1.25 mM NaH.sub.2PO.sub.4 (pH 7.3) and was bubbled with 95% O.sub.2 and 5% CO.sub.2. Field excitatory postsynaptic potentials (fEPSPs) were evoked in the hippocampal CA1 dendritic region via two independent inputs by stimulating the Schaffer collateral commissural pathway (Scep). For LTP induction, high-frequency stimulation (HFS; 100 Hz/100 pulses) conditioning pulses were delivered to the same Seep inputs. Both stimulating electrodes were used to utilize the input specificity of LTP, thus allowing for the measurement of an internal control within the same slice. A42 (50 nM), A42/ACM mixtures (1/10) or ACMs alone (500 nM) were freshly dissolved in ACSF and applied 60-90 min before HFS. Responses were measured for 60 min after HFS. To study effects of ACMs on the LTP impairment mediated by pre-oligomerized A42, A42 (50 nM) was pre-incubated in ACSF for 24 h (30 C.) and then mixed with L3-VF or F3-LF (1/10) (in ACSF); pre-oligomerized A42 alone and A42/ACM mixtures were applied to the slices 90 min before HFS. fEPSP slope measurements (20-80% of peak amplitude) are presented as % fEPSP slope of baseline (the 20 min control period before tetanic stimulation was set to 100%). Data analysis by 1-way ANOVA and Bonferroni's multiple comparison test.
X-Ray Fiber Diffraction
[0260] fIAPP and hf-IAPP/Nle3-VF were made by aging IAPP (1024 M) or a mixture of IAPP (1024 M) and Nle3-VF (2048 M) in ddH.sub.2O for 3 days. A droplet of each solution was placed between glass rods supported by plasticine balls and allowed to dry (humidified atmosphere, 2-4 days, RT). X-ray diffraction data were collected at the facility Single-Crystal X-Ray Diffractometry of the TUM Catalysis Research Center (CRC) using a Bruker D8 Venture diffractometer equipped with a CPAD detector (Bruker Photon II), an IMS micro source with CuK radiation (=1.54178 ) and a Helios optic using the APEX3 software package (Version 2019-1.0, Bruker AXS Inc., Madison, Wisconsin, USA, 2019).
Example 2: Inhibitor Design and Concept Evaluation
[0261] For inhibitor design, A(15-40) was used as a template in the context of the fA40 fold; this features a -strand-loop--strand motif with A(12-22) and AR(30-40) forming the P-strands and A(23-29) the loop (
[0262] To evaluate the concept, 13 A(15-40) analogs containing various different loop tripeptide segments (LTS), comprising (Nle)3, (Leu)3, (Phe)3, (Arg)3, (Gly)3, or Val-Gly-Ser (control LTS) and one pair of two N-methylated residues were designed, synthesized and studied (
[0263] Peptide Nle3 was then used as a template to identify best-suited positions for N-methylations. The four Nle3 analogs Nle3-LF, Nle3-VF, Nle3-GI, and Nle3-GG were synthesized, each of them containing two N-methylations placed at specific residues either within the N-terminal region corresponding to A(15-23) (analogs Nle3-VF and Nle3-LF) or within the C-terminal region corresponding to A(27-40) (analogs Nle3-GI & Nle3-GG) (
[0264] To further evaluate the concept, we next synthesized and tested the four peptides L3-VF, L3-LF, F3-VF, and F3-LF containing loop tripeptides (Leu)3 or (Phe)3 and each of the two identified N-methylation patterns (
[0265] Far-UV CD spectroscopy revealed significant amounts of b-sheet structure in all six inhibitory ACMs whereas non-inhibitors VGS-VF and VGS-LF were less structured (
[0266] Together, the studies identified the six ACMs Nle3-VF, Nle3-LF, L3-VF, L3-LF, F3-VF, and F3-LF (
Example 3: ACMs Co-Assemble with IAPP into Amyloid-Like but Non-Toxic Nanofibers and their Diverse Highly Ordered Superstructures
[0267] To obtain insight into the inhibition mechanism, the inventors next studied ACM interactions and co-assemblies. First, fluorescence spectroscopic titrations of N-terminal fluorescein-labeled IAPP (Fluos-IAPP) (5 nM) with ACMs revealed high affinity interactions. In fact, most app. K.sub.Ds were <100 nM and in very good agreement with the determined IC.sub.50 values (
[0268] Far UV-CD spectroscopy revealed that IAPP/Nle3-VF co-assemblies exhibited a mixture of disordered and b-sheet structure (
[0269] Furthermore, anilino-naphthalene 8-sulfonate (ANS) binding studies indicated that IAPP/Nle3-VF co-assembly fully suppressed surface-exposure of hydrophobic clusters which occurs at early steps of IAPP amyloid self-assembly and is likely related to cytotoxic oligomer formation (
[0270] To characterize the morphology of the IAPP/ACM co-assemblies, solutions used for ThT binding and MTT reduction assays were examined with transmission electron microscopy (TEM). As expected, fibrillar assemblies were major species in aged IAPP and its mixtures with the non-inhibitor VGS-VF (
[0271] The ThT-invisible and non-cytotoxic fibrils found in the IAPP/ACM incubations (termed hf-IAPP/ACM) might thus be heteromeric. To obtain more evidence for this hypothesis, the inventors first applied immunogold TEM. In fact, aged IAPP/ACM mixtures contained fibrils which bound both the anti-IAPP and the anti-AP (anti-ACM) antibody (
[0272] Additional support was obtained by hetero-complex pull-down assays. Here, ThT-invisible fibrils present in aged mixtures of N-terminal biotin-labeled IAPP (Biotin-IAPP) with Nle3-VF were captured by streptavidin-coated magnetic beads and their components revealed by WB (
[0273] High-resolution advanced laser-scanning microscopy provided further unequivocal evidence for diverse supramolecular IAPP/ACM nanofiber co-assemblies (
[0274] Confocal laser scanning (CLSM), stimulated emission depletion (STED), and two-photon microscopy (2 M) visualization of aged IAPP/Nle3-VF mixtures containing N-terminal TAMRA-labeled IAPP (TAMRA-IAPP) and N-terminal Atto647-labeled Nle3-VF (Atto647N-Nle3-VF) or N-terminal fluorescein-labeled Nle3-VF (Fluos-Nle3-VF) revealed large amounts of mm-long heteromeric nanofiber bundles (
[0275] At this stage, detailed studies on the interaction of ACMs with fAPP were performed. Dot blots showed that ACMs and the non-inhibitor VGS-VF bind fIAPP. However, ACM/fIAPP co-assemblies (termed ACM-coated fIAPP) consisted of fIAPP bundles which were randomly covered by amorphous Nle3-VF aggregates and maintained the ThT binding and cytotoxic properties of fIAPP (
[0276] To learn more about the molecular architecture of the IAPP/ACM nanofibers, we used fluorescence lifetime imaging/Frster resonance energy transfer (FLIM-FRET). Pronounced FLIM-FRET events were observed in TAMRA-IAPP/Fluos-Nle3-VF nanofiber co-assemblies (
[0277] Together, these results suggested that the potent inhibitory effect of ACMs is mediated by nanomolar affinity co-assembly with IAPP monomers/prefibrillar species into amyloid-like but ThT-invisible and non-cytotoxic nanofibers and their diverse highly ordered superstructures.
Example 4: ACM/IAPP Nanofibers Evolve from Amorphous Co-Assemblies and IAPP May Act as a Template
[0278] The inventors next asked at which stage of the co-assembly process the fibrillar co-assemblies form. The cross-linking and SEC studies indicated that large hetero-assemblies were present already at the begin of the co-incubation (
[0279] Because ACMs were non-amyloidogenic in isolation but co-assembled with IAPP into amyloid-like nanofibers, the inventors hypothesized that the amyloidogenic character of IAPP could play a role. In fact, TEM showed that no fibrils formed in mixtures of Nle3-VF with the natively occurring (human) IAPP analog rat IAPP or the earlier designed double N-methylated IAPP analog IAPP-GI, which have high sequence identity to IAPP but are weakly or non-amyloidogenic (
Example 5: Additional Properties of Hf-IAPP/ACM
[0280] The inventors next studied whether the non-toxic hf-IAPP/ACM may differ from fIAPP regarding other properties as well. First, the inventors asked whether hf-IAPP/ACM can seed IAPP fibrillogenesis. However in contrast to fIAPP or fibrils present in IAPP/non-inhibitor mixtures, seed amounts of hf-IAPP/ACM were unable to accelerate IAPP fibrillogenesis as assessed by ThT binding (
[0281] Pathogenic amyloid fibrils are usually characterized by an extraordinary high stability. Therefore, the inventors compared the thermostabilities of IAPP/ACM nanofibers and fIAPP by using ThT binding and TEM. In contrast to fIAPP, hf-IAPP/Nle3-VF were fully converted into amorphous aggregates after heating to 95 C. for 5 min (
Example 6: Proposed Hypothetical Models of IAPP/ACM Nanofiber Co-Assembly
[0282] The structure of A/IAPP hetero-amyloids has not been yet elucidated. Based on suggested structures of fIAPP and fA40(42) and the polymorphic nature of self-/cross-amyloid assembly, various different interfaces could be involved in hf-IAPP/ACM formation (
Example 7: ACMs Inhibit A42 Amyloid Self-Assembly Via Co-Assembly into ThT-Invisible and Non-Toxic Nanofibers and their Diverse Superstructures
[0283] In analogy to other A-derived A inhibitors, ACMs also interfere with A amyloid self-assembly. In fact, ThT binding and MTT reduction assays in PC12 cells showed that all six ACMs (A42/ACM 1/i) effectively suppressed formation of A42 fibrils and cytotoxic assemblies (
[0284] Remarkably, TEM examination of the aged ThT-negative and non-cytotoxic A42/ACM mixtures revealed that they exclusively consisted of fibrils (
[0285] 2PM examination of aged A42/ACM mixtures containing N-terminal TAMRA-labeled A42 (TAMRA-A42) and Fluos-ACMs revealed diverse heteromeric fibrous superstructures. These comprised several m-long heteromeric nanofiber bundles with widths between 0.5-2 mm and related heterogeneous superstructures, i.e. ribbons, tapes, or nanotube-like ones with widths between 3-14 m (
[0286] ACM/A42 interactions and hetero-complexes were then studied by fluorescence spectroscopy, SEC, cross-linking in combination with NuPAGE and WB, and far-UV CD spectroscopy (
[0287] Together, the results suggested that the potent inhibitory effect of ACMs on A42 amyloid self-assembly is mediated by nanomolar affinity interactions of ACMs with A42 monomers/prefibrillar species which redirect them into long ThT-invisible and non-toxic hetero-nanofibers and their diverse mm-scaled superstructures. Further studies suggested that binding of ACMs to preformed fA42 does not result in this kind of co-assemblies (
Example 8: Additional Properties of A42/ACM Co-Assembly
[0288] Hippocampal synaptic plasticity is regarded as a key mediator of learning and memory processes; its damage by toxic A42 aggregates is a major responsible factor in AD pathogenesis. The inventors ex vivo electrophysiological studies in mouse brains revealed that in the presence of various different ACMs A42-mediated inhibition of hippocampal long-term potentiation (LTP) was fully ameliorated (
[0289] We then investigated whether hf-A42/ACM might differ from fA42 with respect to their seeding competence. In fact, the ThT binding assay showed that, in contrast to fA42, hf-A42/Nle3-VF and hf-Ap42/L3-VF were seeding-incompetent (
[0290] Together, these findings revealed that ThT-invisible and non-toxic hf-A42/ACM were seeding-incompetent and less thermostable than fA42 and that they became more efficiently degraded by PK and phagocytosed by BV2 microglia than fA42.
Example 9: Inhibition of fIAPP-Mediated Cross-Seeding of A42 Amyloid Self-Assembly by ACMs
[0291] Cross-seeding of A42 amyloid self-assembly by fIAPP accelerates A42 amyloid self-assembly and could link onset and pathogenesis of T2D with AD. In a simplified mechanistic scenario, fIAPP seeds will template formation of IAPP/A42 hetero-amyloids which will template further cytotoxic A42 self-/cross-assembly events. Thereby, polymorphic cross-interactions between amyloid core regions may play an important role.
[0292] Because ACMs contain the A amyloid core, bind with high affinity both IAPP and A42, incl. fIAPP and fA42, and inhibit their amyloid self-assembly, the inventors assumed that they might also interfere with cross-seeding of A42 by fIAPP. In fact, ACMs effectively suppressed fIAPP-mediated cross-seeding of A42 fibrillogenesis and cytotoxicity (
Example 10: Tables
TABLE-US-00007 TABLE 1 IC.sub.50 values of inhibitory effects of ACMs on IAPP-mediated cell damage.sup.[a] and app. K.sub.Ds of ACM/IAPP interactions.sup.[b][c]. IC.sub.50 (SD) app. K.sub.D (SD) ACM (nM).sup.[a] (nM).sup.[b][c] Nle3-VF 65.0 (5.2) 69.5 (1.4) Nle3-LF 82.1 (10.2) 55.4 (5.9) L3-VF 112.5 (8.1) 77.3 (2.9) L3-LF 133.2 (29.0) 143.2 (5.0) F3-VF 78.5 (13.6) 15.0 (1.9) F3-LF 41.7 (4.1) 37.6 (2.9) .sup.[a]IC.sub.50 values, means (SD) from 3 titration assays (n = 3 each) .sup.[b]Determined by titrations of N-terminal fluorescein-labeled IAPP (5 nM; pH 7.4) with ACMs .sup.[c]App. K.sub.Ds are means (SD) from 3 binding curves
TABLE-US-00008 TABLE 2 IC.sub.50 values of inhibitory effects of ACMs on A42-mediated cell damage.sup.[a] and app. K.sub.Ds of ACM/A42 interactions.sup.[b][c]. IC.sub.50 (SD) app. K.sub.D (SD) ACM (nM).sup.[a] (nM).sup.[b][c] Nle3-VF 367 (79) 14.5 (8.0) Nle3-LF n.d. 11.1 (6.0) L3-VF 261 (140) 38.0 (2.0) L3-LF n.d. 2.6 (1.4) F3-VF 1032 (297) 160.8 (12.9) F3-LF 262 (115) 430.6 (7.1) .sup.[a]IC.sub.50 values, means (SD) from 3 titration assays (n = 3 each); n.d.: not determined .sup.[b]Determined by titrations of N-terminal FITC-labelled A42 (5 nM; pH 7.4) with ACMs .sup.[c]App. K.sub.Ds, means (SD) from three binding curves
TABLE-US-00009 TABLE3 AminoacidsequencesandabbreviationsoftemplatesegmentA(15-40)(firstrow) andsynthesizedandtestedpeptidesi.e.the6amyloidinhibitors (ACMs;greybackground)andthenon-inhibitors.Thesequenceofsegment A(24-26)(highlighted)andappliedlooptripeptidesegments(LTS) areshownbythethree-lettercode;differentcolorsareusedforthe differentLTS;peptidesarearrangedingroupsaccordingtotheirLTS. N-methylatedaminoacidsareshowninbold.AllpeptideshaveafreeN-terminal aminogroup(NH.sub.2-)andareC-terminalcarboxylicacids(-COOH).Nle3-VF, Nle3-LF,L3-VF,L3-LF,F3-VF,andF3-LFarestronginhibitors. PeptideSequence Abbreviation QKLVFFAED-Val-Gly-Ser-NKGAIIGLMVGGVV A(15-40) QKLVFFAED-Nle-Nle-Nle-NKGAIIGLNleVGGVV Nle3 QKL(N-Me)VF(N-Me)FAED-Nle-Nle-Nle-NKGAIIGLNleVGGVV Nle3-VF QK(N-Me)LV(N-Me)FFAED-Nle-Nle-Nle-NKGAIIGLNleVGGVV Nle3-LF QKLVFFAED-Nle-Nle-Nle-NK(N-Me)GA(N-Me)IIGLNleVGGVV Nle3-GI QKLVFFAED-Nle-Nle-Nle-NK(N-Me)GAII(N-Me)GLNleVGGVV Nle3-GG QKLVFFAED-Leu-Leu-Leu-NKGAIIGLNleVGGVV L3 QKL(N-Me)VF(N-Me)FAED-Leu-Leu-Leu-NKGAIIGLNleVGGVV L3-VF QK(N-Me)LV(N-Me)FFAED-Leu-Leu-Leu-NKGAIIGLNleVGGVV L3-LF QKL(N-Me)VF(N-Me)FAED-Phe-Phe-Phe-NKGAIIGLNleVGGVV F3-VF QK(N-Me)LV(N-Me)FFAED-Phe-Phe-Phe-NKGAIIGLNleVGGVV F3-LF QKLVFFAED-Val-Gly-Ser-NKGAIIGLNleVGGVV VGS(A(15-40)) QKL(N-Me)VF(N-Me)FAED-Val-Gly-Ser-NKGAIIGLNleVGGVV VGS-VF QK(N-Me)LV(N-Me)FFAED-Val-Gly-Ser-NKGAIIGLNleVGGVV VGS-LF QKLVFFAED-Arg-ArgArg-NKGAIIGLNleVGGVV R3 QKL(N-Me)VF(N-Me)FAED-Arg-Arg-Arg-NKGAIIGLNleVGGVV R3-VF QK(N-Me)LV(N-Me)FFAED-Arg-Arg-Arg-NKGAIIGLNleVGGVV R3-LF QKLVFFAED-Gly-Gly-Gly-NKGAIIGLNleVGGVV G3 QKL(N-Me)VF(N-Me)FAED-Gly-Gly-Gly-NKGAIIGLNleVGGVV G3-VF
TABLE-US-00010 TABLE 4 Widths of fIAPP and fibrils found in aged IAPP, IAPP/ACM and IAPP/VGS-VF mixtures as determined by TEM. Peptide or Fibril width peptide mixture (nm).sup.[a] IAPP 9.7 (2.3) IAPP + Nle3-VF 8.3 (1.9) IAPP + L3-VF 7.7 (1.5) IAPP + F3-VF 8.7 (1.8) IAPP + Nle3-LF 6.1 (1.1) IAPP + L3-LF 7.9 (1.8) IAPP + F3-LF 6.6 (1.2) IAPP + VGS-VF 8.5 (2.0) .sup.[a]Measured in 7 days aged IAPP and IAPP/peptide mixtures (from FIG. 1d, f; TEM images FIG. 2f). Data are means (SD) from 21-47 fibrils
TABLE-US-00011 TABLE 5 Lengths and widths of fA42 and fibrils found in A42/ACM mixtures as determined by TEM. Peptide or Fibril length Fibril width peptide mixture (nm).sup.[a] (nm).sup.[a] A42 154 (58) 7.8 (1.6) A42 + Nle3-VF 472 (138) 7.3 (1.7) A42 + Nle3-LF 590 (268) 7.5 (1.1) A42 + L3-VF 458 (106) 6.9 (1.4) A42 + L3-LF 347 (72) 7.0 (2.2) A42 + F3-VF 354 (117) 6.9 (1.3) A42 + F3-LF 358 (118) 7.3 (1.5) .sup.[a]Measured in 6 days aged solutions of A42 and its mixtures (1/1) with ACMs (from FIG. 6b; TEM images FIG. 6d). Data are means (SD) from 15-23 (lengths) or 21-23 (widths) fibrils (see also bar diagram in FIG. 6d)
TABLE-US-00012 TABLE 6 Molecular weights (M) of synthesized peptides which were used in this study as determined by MALDI-TOF-MS or ESI-MS (*). [M + H].sup.+ [M + H].sup.+[a] or ([M + Na].sup.+) [M + Na].sup.+[b] Peptide calculated (g/mol) found (g/mol) Nle3 2726.61 (2748.59) 2748.71.sup.[b] Nle3-VF 2754.67 (2776.65) 2776.89.sup.[b] Nle3-LF 2754.67 (2776.65) 2776.83.sup.[b] Nle3-GI 2754.67 (2776.65) 2776.50.sup.[b] Nle3-GG 2754.67 (2776.65) 2777.13.sup.[b] L3 2726.61 (2748.59) 2727.00.sup.[a]* L3-VF 2754.67 (2776.65) 2776.99.sup.[b] L3-LF 2754.67 (2776.65) 2757.00.sup.[a]* F3-VF 2856.63 (2878.61) 2878.92.sup.[b] F3-LF 2856.63 (2878.61) 2878.73.sup.[b] VGS 2630.48 (2652.46) 2652.35.sup.[b] VGS-VF 2658.54 (2680.52) 2681.37.sup.[b] VGS-LF 2658.54 (2680.52) 2680.47.sup.[b] R3 2855.67 (2877.65) 2855.97.sup.[a] R3-VF 2883.73 (2905.71) 2884.80.sup.[a]* R3-LF 2883.73 (2905.71) 2883.99.sup.[a] G3 2558.43 (2580.41) 2580.42.sup.[b] G3-VF 2586.49 (2608.47) 2609.00.sup.[b] Nle3-VF(15-23).sup.[c] 1124.62 (1146.60) 1146.62.sup.[b] Nle3-VF(27-40).sup.[c] 1309.81 (1331.79) 1331.93.sup.[b] Nle3-VF(21-40).sup.[c] 1964.17 (1986.15) 1986.21.sup.[b] Fluos-Nle3-VF 3112.97 (3134.95) 3134.93.sup.[b] Fluos-Nle3-LF 3112.97 (3134.95) 3135.16.sup.[b] Fluos-L3-VF 3112.97 (3134.95) 3134.94.sup.[b] Fluos-L3-LF 3112.97 (3134.95) 3134.75.sup.[b] Fluos-F3-VF 3214.93 (3236.91) 3237.05.sup.[b] Fluos-F3-LF 3214.93 (3236.91) 3236.59.sup.[b] Fluos-VGS-VF 3016.84 (3038.82) 3038.84.sup.[b] A42 4512.28 (4534.26) 4512.83.sup.[a]* rat IAPP 3918.96 (3940.94) 3923.54.sup.[a] IAPP-GI 3929.92 (3951.90) 3930.47.sup.[a] IAPP 3901.86 (3923.84) 3902.41.sup.[a] Biotin-IAPP 4240.19 (4262.17) 4241.13.sup.[a] TAMRA-IAPP 4314.29 (4336.27) 4313.95.sup.[a] Fluos-IAPP 4260.16 (4282.14) 4260.48.sup.[a] Atto647N-Nle3-VF 3382.57 (3404.55) 3381.85.sup.[a] M, monoisotopic mass; .sup.[a][M + H].sup.+; .sup.[b][M + Na].sup.+; .sup.[c]numbering of residues/segments of partial A(15-40) or ACM analogs according to their sequence numbers in A40.
Example 11: Discussion
[0293] The inventors exploited A/IAPP cross-interactions to design A amyloid core mimics (ACMs) as inhibitors of amyloid self-assembly of both IAPP and A42. Collectively, the inventors identified six 26-residue peptides as effective amyloid inhibitors of both IAPP and A42. All six ACMs bound IAPP with nanomolar affinity and blocked its cytotoxic amyloid self-assembly with nanomolar IC.sub.50 values. In addition, all six ACMs bound A42 with nanomolar affinity and blocked its cytotoxic self-assembly, three of them with nanomolar IC.sub.50 values. Moreover, ex vivo electrophysiology in murine brains showed a full amelioration of A42-mediated damage of synaptic plasticity by ACMs. Importantly, ACMs also inhibited reciprocal cross-seeding of IAPP and A42 amyloid self-assembly. ACMs thus belong to the most effective inhibitors of in vitro amyloid self-assembly of IAPP, A42, or both polypeptides.
[0294] The inventors most remarkable finding was that ACMs, which were non-amyloidogenic in isolation, exerted their potent amyloid inhibitor function via a novel mechanism, i.e. by co-assembling with IAPP or A42 into amyloid-like but ThT-invisible and non-toxic nanofibers and their diverse highly ordered fibrous superstructures. The latter ones comprised large heteromeric nanofiber bundles and several mm-sized loops, ribbons, and nanotube-like superstructures. Furthermore, non-toxic ternary fibrous co-assemblies consisting of fIAPP, A42, and ACM formed when A42 cross-seeding by fIAPP was performed in the presence of ACMs.
[0295] Unexpectedly, IAPP(A42)/ACM nanofiber co-assembly was efficiently inhibited by the peptides of the invention. In fact, ACMs contained all three A hot segments for high affinity interactions with IAPP, A42, and themselves, and combined inbuilt -sheet extension blocking (N-methylations) with -sheet stabilization/extension enabling (LTS and A(21-40)) elements.
[0296] The identified IAPP/ACM nanofibers were indistinguishable from fIAPP by TEM and had the cross- amyloid core signature by XRD, but were less ordered than fIAPP according to CD spectroscopy. Our TEM, STED, 2 M, and FLIM-FRET studies suggested that nanofibers and basic parts of their fibrous superstructures consisted of laterally co-assembled, parallel arranged or intertwined/twisted, protofilament-like IAPP and ACM stacks. In addition, the inventors' studies showed that hf-IAPP/ACM evolve from large amorphous co-assemblies and suggested that IAPP monomers/prefibrillar species might template this process likely via hetero-dimers. Although the mechanistic steps are yet unclear, IAPP/ACM nanofiber co-assembly could proceed in analogy to proposed mechanisms of self- and co-assembly of IAPP and A.
[0297] The identified A42/ACM nanofibers had similar widths but 2-4 times greater lengths than fA42. The inventors' imaging results were consistent with both axial and lateral co-assembly, the former likely underlying hetero-nanofiber elongation.
[0298] Another notable finding of the inventors' study relates to the potentially beneficial properties of IAPP/ACM and A42/ACM nanofibers. These clearly distinguished them from pathogenic fIAPP and fA42 and, in addition to their non-toxic nature and seeding incompetence, comprised thermolability, proteolytic degradability, and a more efficient phagocytosis than fIAPP and fA42. Such features are reminiscent of labile/reversible functional amyloids, in which they may serve to control their formation/storage/disassembly related to their diverse biological functions. Examples are amyloids from certain secreted peptide hormones or from proteins forming reversible subcellular condensates. By contrast, most pathological amyloids are linked to cell damage and characterized by high stability and resistance to proteolysis. Furthermore, increasing evidence suggests that amyloid fold polymorphism underlies amyloid pathogenicity and functional diversity. The inventors' results suggest that, in addition to pathogenic IAPP/AP hetero-amyloids generated by fibril-mediated cross-seeding, potentially beneficial IAPP/AP hetero-amyloids, such as those mimicked by IAPP/ACM nanofibers, might also exist and this could be also the case for other cross-interacting amyloids. The structural characterization of IAPP/ACM nanofibers should help in identifying molecular factors that may diverge cytotoxic amyloid self-assembly into non-toxic and labile hetero-amyloids and enable the exploitation of amyloid fold versatility to designing effective anti-amyloid molecules.
[0299] In conclusion, the present invention offers a novel class of designed peptides as highly potent inhibitors of amyloid self-assembly and reciprocal cross-seeding of IAPP and A42, and as highly promising leads for effective anti-amyloid drugs in both T2D and AD. In addition, the identified nanofiber co-assemblies should guide the design of novel functional (hetero-)amyloid-based supramolecular nanomaterials for biomedical and biotechnological applications.
REFERENCES
[0300] [1] Yan, L. M. et al. Selectively N-Methylated Soluble IAPP Mimics as Potent IAPP Receptor Agonists and Nanomolar Inhibitors of Cytotoxic Self-Assembly of Both IAPP and Abeta40. Angew Chem Int Ed Engl 52 10378-10383, doi:10.1002/anie.201302840 (2013).
[0301] The features of the present invention disclosed in the specification, the claims, and/or in the accompanying figures may, both separately and in any combination thereof, be material for realizing the invention in various forms thereof.