Patent Application
20250161408
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 (IAPP), monomeric Aβ40(42), amyloid fibrils, or amyloid aggregates.
Amyloid self-assembly is linked to numerous devastating cell-degenerative diseases with Alzheimer's disease (AD) and type 2 diabetes (T2D) being two of the most prominent ones. The main component of amyloid plaques in AD brains is the 40(42)-residue peptide Aβ40(42), while pancreatic amyloid of T2D patients consists of fibrillar assemblies of the 37-residue IAPP. IAPP is secreted from pancreatic β-cells and functions as a neuroendocrine regulator of glucose homeostasis. However, formation of cytotoxic IAPP assemblies and amyloid fibrils mediates pancreatic β-cell degeneration in T2D.
Epidemiological studies suggest that T2D patients have an increased risk of AD and vice versa. In addition, increasing evidence suggests molecular and pathophysiological links between both diseases. Cross-interactions between Aβ and IAPP could be such molecular links. In fact, polymorphic Aβ/IAPP interactions are able to cross-seed or cross-suppress amyloid self-assembly depending on structures and self-assembly states of the interacting polypeptides. To this end, IAPP and Aβ fibrils act as reciprocal cross-seeds of amyloid self-assembly as shown by both in vitro and experimental in vivo studies. On the other hand, nanomolar affinity interactions between early pre-fibrillar and non-toxic IAPP and Aβ species redirect both polypeptides into initially non-fibrillar and non-toxic co-assemblies thus delaying amyloid self-assembly. Importantly, Aβ and IAPP were found to colocalize in AD- and T2D-related amyloid deposits both in humans and in mouse models. Aβ/IAPP cross-interactions and putative “hetero-amyloids” could thus be highly relevant to the pathogenesis of both diseases.
Based on the above, molecules targeting amyloid self-assembly and reciprocal cross-seeding effects of IAPP and Aβ are highly promising for anti-amyloid treatments in both AD and T2D. However, so far only few inhibitors of amyloid self-assembly of both polypeptides (termed “cross-amyloid” inhibitors) have been reported and none of them suppressed reciprocal AP/IAPP cross-seeding. Moreover, except for a recently approved anti-AP amyloid antibody, no anti-amyloid treatments for AD or T2D have yet reached the clinic.
Peptides derived from the IAPP amyloid core IAPP(8-28) as IAPP interaction surface mimics (ISMs) have been designed [1]. ISMs suppressed amyloid self-assembly of Aβ40(42) and/or IAPP by sequestering them into amorphous, non-toxic aggregates. However, none of the reported inhibitors of amyloid self-assembly of Aβ40(42) and/or IAPP has yet advanced to the clinic.
Conclusively, effective inhibitors of amyloid self-assembly are needed. Particularly, there is a need for new inhibitors of amyloid self-assembly of Aβ40(42) and/or IAPP. Furthermore, there remains the need for inhibitors of IAPP and Aβ cross-seeding. There is also a need to provide inhibitors of amyloid self- and cross-assembly that can be used to prevent, treat, and/or diagnose Alzheimer's disease and/or type 2 diabetes.
In the following, the elements of the invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine two or more of the explicitly described embodiments or which combine the one or more of the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
In a first aspect, the present invention relates to a peptide having an amino acid sequence according to formula 1
VG(G)
r
(V)
s
(V)
t
In one embodiment, the peptide according to the present invention has an amino acid sequence according to formula 2
VG(G)
r
(V)
s
(V)
t
In one embodiment, the peptide of the invention has an amino acid sequence according to any one of formulae 3-5
F
a
F
bAED-X6X7X8-NKGAIIGLNleVGGVV
In an embodiment of the peptide, X6, X7, and X8 are the same amino acid selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine, preferably selected from norleucine, leucine, and phenylalanine.
In one embodiment, the peptide of the present invention has an amino acid sequence according to any one of formulae 6-8,
In an embodiment, two or more of La, Va, Fa, and Fb are an N-methylated amino acid;
In an embodiment, said peptide has an amino acid sequence according to any one of formulae 9-17,
In an embodiment, said peptide consists of a sequence according to any one of formulae 1-17.
In a further aspect, the present invention also relates to a composition comprising a peptide according to the present invention, as defined herein, and a suitable solvent, such as water, and a buffer.
In a further aspect, the present invention also relates to a pharmaceutical composition comprising a peptide according to the present invention, as defined herein, and a pharmaceutically acceptable excipient.
In a further aspect, the present invention further relates to a heterocomplex comprising a peptide according to the present invention, as defined herein, and amyloid-β peptide and/or islet amyloid polypeptide.
In a further aspect, the present invention further relates to a peptide according to the present invention as defined herein, the pharmaceutical composition according to the present invention as defined herein, or the heterocomplex according to the present invention as defined herein, 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.
In an embodiment, said method comprises administering an effective amount of said peptide, of said composition, or of said heterocomplex to a subject in need thereof.
In a further aspect, the present invention further relates to a peptide according to the present invention as defined herein, the pharmaceutical composition according to the present invention as defined herein, or the heterocomplex according to the present invention as defined herein, for use in a method of diagnosing Alzheimer's disease and/or for use in a method of diagnosing type 2 diabetes;
In an embodiment, said method comprises administering an effective amount of said peptide, of said composition, or of said heterocomplex to a subject to be tested for Alzheimer's disease and/or type 2 diabetes.
In one embodiment of such peptide, pharmaceutical composition, or heterocomplex for use, said peptide is linked to or administered together with a suitable reporter molecule that allows detection of A1340(42), islet amyloid polypeptide (IAPP), and/or amyloid aggregates or co-aggregates thereof by a suitable detection methodology, such as positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI and wherein said subject, after administration of said peptide, is subjected to said suitable detection methodology, such as PET, NMR, MRI, PET-MRI.
In a yet a further aspect, the present invention 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 subject, said kit comprising the peptide according to the present invention as defined above, the pharmaceutical composition according to the present invention as defined above, or the heterocomplex according to the present invention as defined above, in a freeze-dried form in a suitable container, a buffered solvent in a separate container for reconstitution of said peptide in solution, and, optionally, means to dispense said peptide once reconstituted in solution, such as a syringe or pipette.
In a yet a further aspect, the present invention also relates to the use of the peptide according to the present invention as defined above or of the heterocomplex according to the present invention as defined above, in an in vitro assay, such as an enzyme linked immunosorbent assay (ELISA), a radioimmuno assay (RIA), or a Dot/Slot blot assay, for the detection of monomeric islet amyloid polypeptide (IAPP), monomeric Aβ40(42), amyloid fibrils, amyloid aggregates, and/or amyloid co-aggregates.
In a further aspect, the present invention relates to the use of a peptide according to the present invention as defined above, a pharmaceutical composition according to the present invention as defined above, or a heterocomplex according to the present invention as defined above, for the manufacture of a medicament for the treatment or diagnosis of Alzheimer's disease and/or of type 2 diabetes.
In yet a further aspect, the present invention also relates to a method of prevention, treatment, or diagnosis of Alzheimer's disease and/or type 2 diabetes, wherein said method comprises administering an effective amount of said peptide according to the present invention as defined above, said pharmaceutical composition according to the present invention as defined above, or said heterocomplex according to the present invention as defined above, to a subject in need thereof or to a subject to be tested.
Amyloid self-assembly is linked to numerous devastating cell-degenerative diseases. However, designing inhibitors of this pathogenic process remains a major challenge. Cross-interactions between amyloid-β peptide (Aβ) and islet amyloid polypeptide (IAPP), key polypeptides of Alzheimer's disease (AD) and type 2 diabetes (T2D), link AD with T2D pathogenesis. The present inventors herein show that peptides designed to mimic the amyloid core of Aβ (ACMs) are nanomolar cross-amyloid inhibitors of both IAPP and Aβ42 and effectively suppress reciprocal cross-seeding. Surprisingly, ACMs act by co-assembling with IAPP or Aβ42 into amyloid-like but non-toxic nanofibers and their highly ordered superstructures. Co-assembled nanofibers exhibit various beneficial features including thermolability, proteolytic degradability, and effective cellular clearance which are reminiscent of labile/reversible functional amyloids. ACMs thus provide potent anti-amyloid drugs in both T2D and AD while the supramolecular nanofiber co-assemblies inform the design of novel functional (hetero-)amyloid-based nanomaterials for biomedical/biotechnological applications.
The herein disclosed peptides are derived from the Aβ40 amyloid core Aβ(15-40) as A amyloid core mimics (ACMs) and inhibitors of amyloid self-assembly and cross-seeding interactions of IAPP and Aβ42. In one embodiment, the term “ACM” as used herein, relates to a peptide of the invention. The inhibitor design concept aimed at distorting the pathogenic fibril fold of A(15-40) and stabilize alternative, amyloid-like but non-amyloidogenic folds. The inventors aimed at yielding interaction surfaces with IAPP or Aβ42 and redirecting them into non-fibrillar and non-toxic aggregates. A series of conformationally constrained peptides was synthesized and studied. Unexpectedly, ACMs were non-amyloidogenic and non-cytotoxic, bound IAPP and Aβ42 with nanomolar affinity and fully blocked their cytotoxic amyloid self-assembly. Furthermore, ACMs effectively suppressed reciprocal cross-seeding effects. Surprisingly, ACMs exerted their inhibitory function by co-assembling with IAPP or Aβ42 into amyloid-like nanofibers and their diverse highly ordered superstructures. For their characterization, a spectrum of biophysical, biochemical, and advanced microscopy methods including CLSM, STED, 2 μM and FLIM-FRET was applied. In addition, in vitro and ex vivo cell-based assays were used. In strong contrast to IAPP or Aβ42 fibrils (fIAPP or fAβ42), co-assembled nanofibers were “ThT-invisible”, non-cytotoxic, and seeding-incompetent. Moreover, they were thermolabile, easily degradable by proteinase K (PK), and became efficiently phagocytosed in vitro by primary macrophages and cultured microglial cells. Conclusively, the peptides of the present invention are highly useful in the prevention and treatment of Alzheimer's disease and type 2 diabetes. ThT is a dye which typically stains pathogenic amyloid fibrils but not the new type of heterocomplexes, e.g. hetero-nanofibers, which are generated by interaction of ACMs with IAPP or Abeta42. The fact that the heterocomplexes do not bind ThT supports the observed differences in their properties and functions (e.g. the heterocomplexes being non-toxic, becoming easier proteolytically degraded, phagocytosed etc). The products of the interactions of ACMs with IAPP and Abeta42 have the advantage that they can be removed by proteolytic degradation, in contrast to IAPP and Abeta42 fibrils. Furthermore, the differential staining properties are useful for diagnostic assays.
The present invention provides highly effective inhibitors of amyloid self-assembly of Aβ40(42) and/or IAPP and amyloid cross-assembly. Surprisingly, the peptides of the invention inhibits not only Aβ40(42) and/or IAPP self-assembly, but also Aβ40(42) and IAPP cross-assembly. Thus, the present invention provides peptides, and compositions and heterocomplexes comprising these peptides, which are effective inhibitors of amyloid self- and cross-assembly that can be used to prevent, treat, and/or diagnose Alzheimer's disease and/or type 2 diabetes.
In one embodiment, the peptide of the invention has an amino acid sequence according to formula 1
(G)
r
(V)
s
(V)
t
A peptide “having” an amino acid sequence according to a formula may relate to a peptide comprising the amino acid sequence according to said formula and/or to a peptide consisting of the amino acid sequence according to said formula. In one embodiment, a peptide having an amino acid sequence according to a formula comprises or consists of the amino acid sequence according to said formula. In a preferred embodiment of the peptide according to formula 1, at least one of X3, X4, Fa, and Fb is an N-methylated amino acid, e.g. any one, two, three, or all of X3, X4, Fa, and Fb are an N-methylated amino acid. In one embodiment, two or more of X3, X4, Fa, and Fb are an N-methylated amino acid, for example X3 and Fa; X4 and Fb; or X3, X4, Fa, and Fb are an N-methylated amino acid. In a preferred embodiment, two or more of X3, X4, Fa, and Fb are an N-methylated amino acid. In one embodiment, X3 and X4; X3 and Fa; X3 and Fb; X3 and Fa; X3 and Fb; Fa and Fb; X3, X4, and Fa; X3, X4, and Fb; X3, Fa, and Fb; X4, Fa, and Fb; or X3, X4Fa, and Fb are N-methylated amino acids. In one embodiment, at least X3 and Fa, or at least X4 and Fb, are an N-methylated amino acid.
In a preferred embodiment of the peptide according to any one of formulae 2-8, at least one of La, Va, Fa, and Fb is an N-methylated amino acid, e.g. any one, two, three, or all of La, Va, Fa, and Fb are an N-methylated amino acid. In one embodiment, two or more of La, Va, Fa, and Fb are an N-methylated amino acid, for example La and Fa; Va and Fb; or La, Va, Fa, and Fb are an N-methylated amino acid. In a preferred embodiment, two or more of La, Va, Fa, and Fb are an N-methylated amino acid. In one embodiment, La and Va; La and Fa; La and Fb; La and Fa; La and Fb; Fa and Fb; La, Va, and Fa; La, Va, and Fb; La, Fa, and Fb; Va, Fa, and Fb; or La, Va, Fa, and Fb are N-methylated amino acids. In one embodiment, at least La and Fa, or at least Va and Fb, are an N-methylated amino acid.
In one embodiment of any of formulae 1-2, as defined herein, each of m, n, p, q, r, s, and t is any selected from 0 and 1. In one embodiment, the peptide of any of formulae 1-2, as defined herein comprises any combination of m, n, p, q, r, s, and t being independently selected from 0 and 1. In one embodiment of any of formulae 1-2, as defined herein, m, n, p, q, r, s, and t are all 0 or 1. In an alternative embodiment of any of formulae 1-2, as defined herein, m and t are 0 and n, p, q, s, and r are 1. In an alternative embodiment of any of formulae 1-2, as defined herein, m, n, s and t are 0 and p, q, and r are 1. In an alternative embodiment of any of formulae 1-2, as defined herein, m and n are 0 and s, t, p, q, and r are 1. Other combinations of m, n, p, q, r, s, and t being independently selected from 0 and 1 are understood by the person skilled in the art. The peptide of the invention can be provided with any combination of m, n, p, q, r, s, and t being independently selected from 0 and 1. In a preferred embodiment of any of formulae 1-2, as defined herein, m, n, p, and q are 0 and r, s, and t are 1, or m, n, p, and q are 1 and r, s, and t are 0. In a further preferred embodiment of any of formulae 1-2, as defined herein, m and n are 0 and p, q r, s, and t are 1; m, n and p are 0 and q, r, s, and t are 1; m, n, p and q are 0 and r, s, and t are 1; m, n, p, and q are 1 and r, s, and t are 0; m, n, p, q and r are 1 and s and t are 0; or m, n, p, q, r and s are 1 and t is 0.
In one embodiment, the peptide of the invention, particularly the peptide according to any of formulae 1-8 as defined herein, comprises a sequence FaFbAE, NKGAII, VG, and/or VGGVV, e.g. FaFbAE, NKGAII, and VGGVV; wherein Fa and Fb are, independently at each occurrence, selected from phenylalanine and N-methyl-phenylalanine; A is alanine; E is glutamic acid; N is asparagine; K is lysine; G is, independently at each occurrence, is glycine; I is, independently at each occurrence, isoleucine; and V is, independently at each occurrence, valine.
In one embodiment of the peptide according to any one of formulae 1-5 as defined herein, the substituents represented by X6, X7, and X8 are, independently at each occurrence, selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, valine, and modified forms thereof e.g. N-methylated forms thereof, preferably selected from norleucine, leucine, and phenylalanine. Modified forms of the respective amino acids are, e.g., N-methylated forms of the respective amino acids and/or D-amino acids. In an embodiment of the peptide, X6, X7, and X8 are any combination of amino acids selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine, preferably selected from norleucine, leucine, and phenylalanine; for example, three different amino acids (e.g. Nle-Leu-Phe), two amino acids of the same kind and one amino acid different therefrom (e.g. Nle-Nle-Phe), or three of the same amino acids (e.g. Nle-Nle-Nle). In a preferred embodiment of the peptide, X6, X7, and X8 are the same amino acid selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine, preferably selected from norleucine, leucine, and phenylalanine. In one embodiment, X6, X7, and X8 are an amino acid triplet comprising three amino acids of the same kind.
In one embodiment, the term “modified form”, as used herein in the context of amino acids, relates to a modified form of an amino acid in which the amino acid chain is derivatized (or a derivative of the amino acid side chain), e.g. an ester or an amino acid comprising a protecting group such as BOC, to a modified form of an amino acid in which the side chain of the amino acid comprises one or more substituents, to a D-amino acid of the respective amino acid, and/or to an N-methylated form or an N-alkylated form of the respective amino acid. In one embodiment, the amino acids defined for the amino acid residues of any one of formulae 1-17 can be present in a modified form of the amino acid, e.g. any of the substituents recited for X1, X2, X3, X4, Fa, Fb, A, E, X5, X6, X7, X8, N, K, G, I, X9, X10, X11, La, and Va can be a modified amino acid, e.g. an amino acid comprising a side chain with at least one substituent, a D-amino acid, and/or an N-methylated amino acid. For example, lysine may be present, as modified lysine e.g. Lys(Ac), Lys(N3), and Lys(biotin). When referring to modified amino acids by referring to an amino acid three-letter-code in combination with a modification specified in brackets, e.g. e.g. Lys(Ac), Lys(N3), Lys(biotin), e.g. Orn(Ac), and Arg(Me)2, such indication of the modification in brackets is meant to be understood as referring to a modification of the side chain of the amino acid represented by the three-letter-code. For example, Lys(Ac) is lysine with an acetylated sidechain, Lys(N3) is lysine with an azide functional group at the sidechain, Lys(biotin) is lysine with a biotinylated sidechain, Orn(Ac) is ornithine with an acetylated sidechain, and Arg(Me)2is arginine with the sidechain comprising two methyl groups. In one embodiment, the term “Lys(N3)” relates to N-epsilon-azido-lysine.
The amino acids mentioned in the appended claims, particularly any amino acid present in a peptide of the invention, may be present in a modified form of the respective amino acid, e.g. in an N-methylated form thereof, as an acetylated form thereof e.g. lysine in the form of Lys(Ac), as an biotinylated form thereof e.g. lysine in the form of Lys(biotin), and/or as a D-amino acid. In one embodiment, the peptide of the invention has an amino acid sequence as defined by any of formulae 1-17, as defined above.
In one embodiment of the peptide according to any one of formulae 1-2 as defined herein, X11 is selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine, preferably is norleucine. For example, a peptide according to any one of formulae 1-2 as defined herein, may comprise X11 being selected from norleucine, leucine, phenylalanine, methionine, tryptophan, tyrosine, threonine, alanine, isoleucine, and valine, preferably being norleucine; and may further comprise a sequence FaFbAE, NKGAII, VG, and/or VGGVV, e.g. FaFbAE, NKGAII, and VGGVV; wherein Fa and Fb are, independently at each occurrence, selected from phenylalanine and N-methyl-phenylalanine; A is alanine; E is glutamic acid; N is asparagine; K is lysine; G is, independently at each occurrence, is glycine; I is, independently at each occurrence, isoleucine; and V is, independently at each occurrence, valine; wherein, preferably, at least one of X3, X4, Fa, and Fb of formula 1 is an N-methylated amino acid and at least one of La, Va, Fa, and Fb of formula 2 is an N-methylated amino acid.
In one embodiment, the peptide of the invention is an amyloid inhibitory peptide that preferably binds to Aβ40(42) and/or to islet amyloid polypeptide (IAPP), or said peptide is a peptide that binds to Aβ40(42) and/or to islet amyloid polypeptide (IAPP), but is not necessarily an amyloid inhibitory peptide. It should be noted that in preferred embodiments, where reference is made to a “peptide” in general, such peptide may also be referred to as an “amyloid inhibitory peptide”. An “amyloid inhibitory peptide” is a peptide that functions as or can be used as an “amyloid inhibitor”. In one embodiment, the peptide of the invention comprises or consists of 19 to 28 amino acids, preferably 22 to 28 amino acids, more preferably comprises or consists of 26 amino acids.
In one embodiment, said peptide is linked to a reporter molecule that allows detection of Aβ40(42), islet amyloid polypeptide (IAPP) and/or amyloid aggregates or co-aggregates thereof by a detection methodology, such as positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI, preferably a dye or a quantum dot.
In one embodiment, the term “modified form”, as used herein in the context of a peptide, refers to modified forms of peptides known to the person skilled in the art, e.g. peptides having a protected N-terminus and/or C-terminus. In one embodiment, the N-terminus and/or the C-terminus of the peptides according to the present invention are protected. Suitable protecting groups are manifold and are known to a person skilled in the art. For example, the N-terminus may be acetylated or formylated, or there may be an even longer chain attached such as palmitoyl. The C-terminus could be protected via formation of an amide or carbonic acid ester.
In one embodiment, the C-termini of the peptide(s) according to the present invention, in particular the peptides according to any one formulae 1-17 according to the present invention, are in their free carboxy-form, are present in the form of an ester, and/or are protected by an amide. In one embodiment, the C-terminus is in its free carboxy-form; in another embodiment, it is in esterified form. In particularly preferred embodiments, the C-termini of the peptide(s) according to the present invention, in particular of the amyloid inhibitory peptides according to the present invention, more particularly of the peptides according to formulae 1-17 according to the present invention, are in their carboxy-form, and the respective N-termini of the peptides are in their NH2-form or NH3+-form. In one embodiment, the peptide of the invention is modified by an N-terminal acetylation, by formylation, by being coupled with a label e.g. fluorescent label or biotin label, by being coupled to any compound via a linker, and/or by C-terminal amidation.
Without wishing to be bound by any theory, the present inventors believe that the amyloid inhibitory effect of an amyloid inhibitory peptide is mediated by its binding to key amyloid polypeptides, in particular to Aβ40(42) and/or to islet amyloid polypeptide (IAPP). Hence, the term “amyloid inhibitory” as used herein in the context of a peptide, refers to the capability of such peptide to block or inhibit amyloid self-assembly, cross-assembly, and/or amyloidogenesis, preferably of the key amyloid polypeptides, in particular of Aβ40(42) and/or of islet amyloid polypeptide (IAPP). Amyloid inhibitory peptides in accordance with the present invention are useful as amyloid inhibitors, and may thus be used for therapeutic and/or diagnostic purposes, i.e. they may be used for treatment and/or diagnosis of diseases involving amyloid self-assembly, cross-assembly, or amyloidogenesis, in particular of Alzheimer's disease and/or of type 2 diabetes. In other embodiments, a peptide in accordance with the present invention may have the capability to bind to key amyloid polypeptides, but may not necessarily be capable of functioning as an amyloid inhibitor. Such peptides may herein also sometimes be referred to as “amyloid binding peptides”. They bind to key amyloid peptides, but are not capable of blocking or inhibiting amyloid self-assembly or amyloidogenesis. They may nevertheless be useful for detection purposes, in cases where detection of amyloid peptides may be desirable.
The present invention also relates to a pharmaceutical composition comprising a peptide according to the present invention and a pharmaceutically acceptable excipient. In such pharmaceutical composition, the peptide may occur as such, or it may be linked to other entities/molecules that endow the peptide with a specific functionality. For example, there may be a tag attached to increase blood-brain-barrier permeability, or it may be attached to a specific reporter molecule, such as a dye or a quantum dot, allowing the detection in diagnostic methods (preferably whilst retaining the therapeutic functionality of the peptide —“theranostic applications”). The use of quantum dots may be particularly useful in various imaging technologies, such as MRI, PET, PET-MRI, or specifically quantum-dot-based brain imaging methodologies. In certain embodiments, the peptide may be attached to a nanoparticle, to a suitable carrier molecule, to a targeting entity or other functional molecule.
Because the peptides according to the present invention are highly likely to be capable of passing the blood-brain-barrier, the present invention also relates to the use of a peptide according to the present invention, as defined herein, as a carrier for molecules, substances or compounds to pass the blood-brain-barrier. In certain embodiments, the molecule to pass the blood-brain-barrier is linked, preferably covalently linked, to a peptide according to the present invention as defined herein.
The peptides of the invention bind to Aβ40(42) and IAPP and inhibit amyloid self-assembly and cross-assembly, and are thus highly useful for the use in the prevention, treatment, and diagnosis of AD and/or T2D. In one embodiment, the term “treatment” or “treating”, as used herein, encompasses both prophylactic treatment and therapeutic treatment. In a preferred embodiment, it specifically refers to therapeutic treatment. The term “subject”, as used herein, relates to an individual, e.g. a human or an animal, preferably a human. In one embodiment, the subject is a patient having or being at risk of acquiring AD and/or T2D. The present inventors have managed to design peptidic inhibitors of amyloid self-assembly of both Aβ40(42) and IAPP, as well as of cross-assembly of Aβ40(42) and IAPP, which can be used in the prevention, treatment, and diagnosis of AD and/or T2D.
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 subject, said kit comprising the peptide according to the present invention as defined above, the pharmaceutical composition according to the present invention as defined above, or the heterocomplex according to the present invention as defined above, in a freeze-dried form in a suitable container, a buffered solvent in a separate container for reconstitution of said peptide in solution, and, optionally, means to dispense said peptide once reconstituted in solution, such as a syringe or pipette. Alternatively, the kit may contain the peptide according to the present invention as defined above, the pharmaceutical composition according to the present invention as defined above, or the heterocomplex according to the present invention as defined above, in an already reconstituted, ready-to-use form.
The present invention also relates to the use of the peptide according to the present invention as defined herein or of the heterocomplex according to the present invention as defined herein, in an in vitro assay, such as an enzyme linked immunosorbent assay (ELISA), a radioimmuno assay (RIA), or a Dot/Slot blot assay, for the detection of monomeric islet amyloid polypeptide (IAPP), monomeric A1340(42), amyloid fibrils, amyloid aggregates, and/or amyloid co-aggregates. Such use may, in certain embodiments, involve the analysis of blood, cerebrospinal fluid, or brain biopsies, and may also further involve the use of suitable reporter molecules to which the peptide may be attached or with which the peptide may be used together.
The present inventors have provided peptides which function as nanomolar inhibitors of amyloid self-assembly and cross-assembly of Aβ40(42) and IAPP, and which therefore have manifold applications. Moreover, the peptides bind with high affinity to Aβ40(42) and/or IAPP monomers and/or amyloid aggregates and co-aggregates thereof.
The term “heterocomplex”, as used herein, relates to a peptide of the present invention that is associated with an amyloid-β peptide and/or islet amyloid polypeptide, for example, a co-assembly comprising a peptide of the invention and amyloid-β peptide and/or islet amyloid polypeptide. In such heterocomplex, the peptide of the invention is associated with amyloid-β peptide and/or islet amyloid polypeptide by noncovalent binding. For example, a heterocomplex can be a heteromeric supramolecular nanofiber co-assembly comprising a peptide of the present invention, and amyloid-β peptide and/or islet amyloid polypeptide. Surprisingly, the heterocomplex of the invention, in contrast to fibrous IAPP or Abeta homo-assemblies, is non-cytotoxic, soluble, and does not act as an accelerator (seed) of amyloid formation of IAPP or Abeta. Furthermore, the heterocomplex of the invention is easily proteolytically degradable, and becomes easier recognized and phagocytosed by macrophages and microglia compared to fibrous IAPP or Abeta homo-assemblies. The peptides of the invention are highly advantageous in that they associate with IAPP and Abeta such that these do not convert into cytotoxic aggregates, co-aggregates, and amyloid. The peptides of the invention are advantageous in that they convert IAPP and Abeta into heterocomplexes having the above mentioned beneficial properties: no cytotoxic effect on cell viability, solubility, thermolability, proteolytic degradability, and effective cellular clearance. Furthermore, the purpose of the invention have the advantage that they convert IAPP and Abeta into heterocomplexes such that IAPP and Abeta are unable to act as seeds or cross-seeds of amyloid self-assembly. The fibrous co-assemblies, particularly the heterocomplexes of the invention, have thus a function; they are functional co-assemblies in that they act as a vehicle to prevent cytotoxic amyloid formation of IAPP and Abeta. By contrast, amyloid fibrils usually are badly soluble, cytotoxic, thermostable, proteolytically non-degradable, not efficiently phagocytosed by macrophages and microglia thereby contributing to cell- and neurodegeneration in AD and T2D, and act as accelerators/seeds of amyloid self-assembly and cross-seeds of IAPP and Abeta. In one embodiment, the terms “heterocomplex”, “hetero-assembly”, and “co-assembly” are used interchangeably. In one embodiment, the hetercomplex is a heteromeric nanofiber co-assembly, comprising a peptide of the present invention and an amyloid-β peptide and/or islet amyloid polypeptide.
In one embodiment, said heterocomplex is linked to a reporter molecule that allows detection of Aβ40(42), islet amyloid polypeptide (IAPP) and/or amyloid aggregates or co-aggregates thereof by a detection methodology, such as positron emission tomography (PET), nuclear magnetic resonance (NMR), magnetic resonance imaging (MRI), and PET-MRI, preferably a dye or a quantum dot.
In the present application, use is made of the one-letter-code for amino acid residues and the three-letter-code for amino acid residues. Hence, amino acid residues are typically designated herein by reference to their respective one-letter-code or three-letter-code. Accordingly, alanine is A or Ala; arginine is R or Arg; asparagine is N or Asn; aspartic acid is D or Asp; cysteine is C or Cys; glutamine is Q or Gln; glutamate is E or Glu; glycine is G or Gly; histidine is H or His; isoleucine is I or Ile; leucine is L or Leu; lysine is K or Lys; methionine is M or Met; phenylalanine is F or Phe; proline is P or Pro; serine is S or Ser; threonine is T or Thr; tryptophan is W or Trp; tyrosine is Y or Tyr; valine is V or Val; norleucine is Nle.
However, additionally in some instances, reference is made to a modified amino acid one-letter-code wherein some of the letters of the aforementioned “normal” one-letter-code are represented in italics. Such letters in italics refer to amino acid residues whose definition does not only comprise the respective amino acid usually represented by the one-letter-code as outlined in the preceding paragraph (e.g. F representing phenylalanine etc.), but which definition further comprises alternative amino acids, such as conservative substitutions of the respective amino acid represented by the one-letter-code (e.g. tyrosine being a conservative substitution of phenylalanine) and modified forms thereof (e.g. N-methyl-phenylalanine or halogenated phenylalanine). The amino acid residues represented by letters in italics are defined in the appended claims and should be understood to comprise all residues specified for the respective amino acid residue in the appended claims.
In one embodiment, the amino acids present in a peptide of the invention are L-amino acids or D-amino acids, or a mixture of L-amino acids and D-amino acids. In a peptide of the invention, some of the residues in the sequence may be L-amino acids and others may be D-amino acids. Sometimes, in this application, reference to amino acid sequences is made by reciting the individual residues as free amino acids, such as “glycine”, “glutamic acid”, etc., notwithstanding the fact that these residues appear in the respective amino acid sequence in their respective covalently linked form, i.e. with the individual residues linked by appropriate peptide bonds, i.e. amide bonds, between them.
The term “N-methyl” or “N-methylated”, as used herein, refers to a methyl group that is attached to the nitrogen in the amide bond between two amino acid residues. In one embodiment, the terms “N-methyl” and “N-methylated”, as used herein in the context of amino acids, are used interchangeably. In one embodiment, where an amino acid, e.g. phenylalanine, in an amino acid sequence is indicated as “N-methyl amino acid”, e.g. “N-methyl-phenylalanine”, this means that a methyl group is attached to the amide nitrogen forming the amide bond between phenylalanine and the amino acid that precedes phenylalanine in the amino acid sequence.
It should be noted that subscripts in the form of numbers, as used in the context of amino acids “X” in peptide sequences, are meant to denote the position of the individual residue within the sequence and not the total number of such residue in such position. As an example, the sequence X6X7X8 is meant to denote a part of formula 1 with three individual amino acid residues defined by X6, X7, and X8.
The terms “of the [present] invention”, “in accordance with the invention”, “according to the invention” and the like, as used herein are intended to refer to all aspects and embodiments of the invention described and/or claimed herein.
As used herein, the term “comprising” is to be construed as encompassing both “including” and “consisting of”, both meanings being specifically intended, and hence individually disclosed embodiments in accordance with the present invention. Where used herein, “and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±20%, ±15%, ±10%, and for example t5%. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect. Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.
The present invention is now further described by reference to the following figures.
All methods mentioned in the figure descriptions below were carried out as described in detail in the examples.
Left, time course of synaptic transmission; means±SEM (n=7-8 for Aβ42/ACM (1/10), n=8 for Aβ42 (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, means±SEM (n, see above); ***P<0.001 versus Aβ42 (one-way ANOVA & Bonferroni). b hf-Aβ42/ACM are seeding incompetent. Aβ42 (5 μM) fibrillogenesis alone or seeded with fAβ42, hf-A342/Nle3-VF, or hf-Aβ42-L3-VF (10%) determined by ThT binding (means±SD, 3 assays). c Degradation of hf-Aβ42/Nle3-VF and fAβ42 by PK (37° C.) followed by dot blot; Aβ42 quantification by Aβ(1-17)-specific antibody. Representative membranes from 3 assays. d Thermolability of hf-Aβ42/ACM versus fAβ42. TEM images of boiled fAβ42 (15 min) versus hf-Aβ42/Nle3-VF (5 min); scale bars: 100 nm. e Phagocytosis of hf-A342/ACM versus fAβ42 by cultured murine BV2 microglia. Left and mid panels, representative microscopic images of cells after incubation (6 h, 37° C.) with TAMRA-fAβ42, hf-TAMRA-Aβ42/Nle3-VF, and hf-TAMRA-Aβ42/L3-VF (1 μM); red dots indicated TAMRA-Aβ42; scale bars, 100 μm. Right panel, amounts of phagocytic cells (% of total). Data means±SD 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 Aβ42 fibrillogenesis (left panel) or cytotoxicity (right panel). Left panel, fibrillogenesis of Aβ42 (10 μM) or Aβ42/ACM (1/2) mixtures following cross-seeding with fIAPP (20%) and of Aβ42 w/o fIAPP seeds (10 μM) determined by ThT binding (means±SD, 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 (means±SD, 3 assays, n=3 each). g-j 2 μM characterization of supramolecular co-assemblies in Aβ42 solutions after cross-seeding with fIAPP (20%) in the absence (g,h) or presence of ACM (ij). g & h 2 μM images of TAMRA-fIAPP-cross-seeded Aβ42 containing HiLyte647-Aβ42 (50%) (1.5 h; incubations as in f) show clusters of Aβ42 assemblies bound to/branching out of fIAPP surfaces; yellow arrow, Aβ42-fIAPP “contact site”; scale bars: 10 μm (g) and 100 mm (h). i 2 μM images of fibrillar co-assemblies in TAMRA-fIAPP-cross-seeded Aβ42/Nle3-VF mixtures containing HiLyte647-Aβ42/Fluos-Nle3-VF (50%) (1.5 h; incubations as in f); scale bars: 10 μm. Upper panel, fIAPP covered by Aβ42, Nle3-VF, and Aβ42/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, Aβ42 & Nle3-VF bound to fIAPP; encircled area indicates Aβ42/Nle3-VF co-assembly bound to fIAPP. Scale bars, 10 μm (top), 1 μm (bottom).
For comparison, effects of an aged fibrillar Aβ40 (solution from b; at 20 μM) are shown. Data are means±SD 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 means±SD from 3 binding curves); determined app. KD=51.9 * 4.5 nM (mean * SD from 3 binding curves).
Applied abbreviations of the analogs as follows:
Applied abbreviations of the analogs as follows:
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 μM IAPP alone or its mixture with peptides (1:2) as indicated. Data are means±SD from 3 assays.
In the following, reference is made to the examples, which are given to illustrate, not to limit the present invention.
IAPP, IAPP-GI, rat IAPP, and their Na-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 μm 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.). Aβ42 was synthesized on Tentagel R PHB resin (0.18 mmol/g; Rapp Polymere) by Fmoc-SPPS. Seed-free aqueous Aβ42 stock solutions (10-20 μM) were obtained by SEC performed. Briefly, HPLC-purified Aβ42 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 Aβ42 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 Aβ42 (FITC-Aβ42) and TAMRA-labeled Aβ42 (TAMRA-Aβ42) were from Bachem and HiLyte647-Aβ42 from AnaSpec; their stocks were prepared in HFIP (4° C.).
All Aβ(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 Aβ(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/H2O. 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).
All synthetic peptides were characterized by matrix-assisted laser desorption ionization (MALDI-MS) or electrospray ionization (ESI-MS) mass spectrometry (Table 6).
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 μM) in ThT buffer for 3-9 days (20° C.), quantified by ThT binding, and verified by TEM.
Peptide incubations were performed as follows: for studying effects on IAPP fibrillogenesis, freshly made IAPP (16.5 μM) 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 μM) 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 μM) in ThT buffer (7 days). Following fIAPP quantification by ThT binding and verification by TEM (
ACM fibrillogenesis-related studies. To study the fibrillogenic potential of ACMs, peptides and Aβ40 (positive control) (100 μM) 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 μM ThT, 0.05 M glycine/NaOH, pH 8.5); buffer values were subtracted from the data shown in
Aβ42 fibrillogenesis-related studies. To study effects of the different peptides on Aβ42 fibrillogenesis, synthetic Aβ42 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): Aβ42 (5 μM) alone or its mixture with the peptide (at the indicated molar ratios) in 45 mM ammonium acetate, pH 8.5, containing 10 μM 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
For studying effects of ACMs on fAβ42-mediated seeding of Aβ42, preformed fAβ42 (made by incubating Aβ42 (5 μM) as above but w/o ThT for 6 days (TEM,
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% CO2) cell damage was assessed by measuring cellular MTI reduction. IC50 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 μM) 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.
The studies on effects of ACMs on formation of cell-damaging Aβ42 assemblies were performed in combination with the ThT binding assay and TEM using PC12 cells cultured and plated. Incubations of Aβ42 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% CO2), cell damage was assessed by measuring cellular MTT reduction. To determine IC50 values of the effects of ACMs, incubations of Aβ42 (5 μM) 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 (Aβ42, 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 Aβ42 with L3-LF and Nle3-LF most likely due to aggregation; therefore, IC50 values were not determined.
To study effects of ACMs on fIAPP-mediating cross-seeding of formation of cell-damaging Aβ42 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.
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 Aβ40 were also studied and cytotoxicity was as expected.
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 ddH2O 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 Aβ42-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 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 1×PBS. fIAPP was detected with a fibril-specific mouse anti-fIAPP antibody (Synaptic Systems; Cl. 91E7). Nle3-VF was revealed by a rabbit anti-Aβ40 polyclonal antibody (Sigma-Aldrich) exhibiting 10-20% NSB to IAPP. The two antibodies (in 0.1% BSA in 1×PBS; dilution 1/10) were deposited simultaneously onto the grid and incubated for 20 min. Following washing with 1×PBS, 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 1×PBS, dilution 1/10) as above. Following 1×PBS and ddH2O 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.
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 μM) was incubated with Nle3-VF or VGS-VF (33 μM) 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-Aβ42/Nle3-VF, incubations were performed as for the ThT binding assays but in the absence of ThT. Briefly, Aβ42 alone (5 μM), Nle3-VF alone (5 μM), and their mixtures (5 μM each) in 45 mM ammonium acetate (pH 8.5) were incubated for 6 days at 37° C. and CD spectra were measured.
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-Aβ42 (5 nM) consist mostly of monomers and the same was found for Fluos-ACMs (5 nM) (
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 μM), IAPP/ACM mixtures (1/2) and ACMs alone (60 μM) were incubated in 10 mM sodium phosphate buffer (pH 7.4) for up to 7 days (20° C.). In the case of Aβ42 homo-/hetero-assemblies, Aβ42 (30 μM) and Aβ42/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 (Aβ42 studies)) aliquots were cross-linked (2 min) with 25% aqueous glutaraldehyde (Sigma-Aldrich) and treated with a 2 M NaBH4 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 Aβ42 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-Aβ40 (Sigma-Aldrich; 1:2000) for ACM-containing assemblies, or mouse monoclonal anti-Aβ(1-17) (6E10, BIOZOL; 1:2000) for Aβ42-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.
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 μM) 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 Aβ42-related studies, elution buffer was 50 mM ammonium acetate, pH 8.5. Aβ42 (5 μM) or Aβ42/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 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 μM) and its mixtures with IAPP (2 μM) 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.
Pull-down assays were performed using streptavidin-coupled magnetic beads (Dynabeads M-280 Streptavidin, Dynal). Briefly, solutions of Biotin-IAPP (16.5 μM), 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.
IAPP or IAPP/ACM mixtures (1/2) containing 10% N-terminal fluorescently-labeled analogs TAMRA-IAPP and Atto647N-ACM (IAPP(total), 16.5 μM; ACM(total), 33 mM) were incubated in 10 mM sodium phosphate buffer, pH 7.4 for 7 days (20° C.). Aliquots (30-40 μl) 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.
Solutions analyzed by 2 μM 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 μM) with synthetic N-terminal fluorescein- or Atto647N-labeled ACMs (33 μM) 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 μM) was also examined and consisted mostly of fibrillar assemblies (TAMRA-fIAPP;
For 2 μM and FLIM-FRET studies related to ACM-coated fIAPP, first fIAPP were made by incubating an IAPP/TAMRA-IAPP mixture (16.5 μM 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 μM Nle3-VF(total) for 1 day (20° C.)) to yield ACM-coated fIAPP. For 2 μM 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 Aβ42-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 fAβ42 versus hf-Aβ42/ACM, Aβ42 solutions (Aβ42(total) 5 μM) consisted of 50% TAMRA-Aβ42 and 50% Aβ42 (6 day-aging); Aβ42/ACM mixtures (1/2) contained, in addition to Aβ42/TAMRA-Aβ42 (1/1; A342(total), 5 μM), ACM/Fluos-ACM (1/1; ACM(total), 10 μM) (4-6 day-aging).
For studies on fIAPP-mediated cross-seeding of Aβ42, the Aβ42 alone solution (Aβ42(total), 10 μM) contained HiLyte647-Aβ42 (50%); the Aβ42/ACM (1/2) mixtures consisted of Aβ42/HiLyte647-Aβ42 (1/1) (Aβ42(total), 10 μM) and Nle3-VF/Fluos-Nle3-VF (1/1) (Nle3-VF(total), 20 μM); solutions were aged for 1.5 h. TAMRA-fIAPP seeds were made by incubating TAMRA-IAPP (128 μM) in ThT buffer for 6 days (20° C.)); their concentration in the cross-seeded solution was 2 μM. Aliquots from the above solutions were applied onto SuperFrost Plus adhesion slides, air-dried, washed (Aβ42-related studies) and embedded with Prolong Diamond Antifade Mountant as for CLSM and STED.
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.
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 μM, 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 τ=Σ(αiτi)/Σαi (αi: amplitude of each lifetime τi). FLIM-FRET efficiency was calculated by FRET eff=1−(τDA/τD); (τDA=lifetime donor in presence of acceptor; τD=lifetime donor alone).
IAPP (128 μM) and Aβ42 (11 μM) incubations were made as for ThT binding assays (Aβ42 w/o ThT) and deposited onto nitrocellulose membranes (IAPP: 40 μg, Aβ42: 10 μg) 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 μM for IAPP-related membranes or 2 μM for Aβ42-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 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 μM) 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 (
Solutions consisting mainly of fIAPP, hf-IAPP/Nle3-VF, fAβ42 or hf-Aβ42/Nle3-VF were prepared as described under “ThT binding assays” (fIAPP 16.5 μM, 7 day-aged; hf-IAPP/Nle3-VF, IAPP 16.5 mM, Nle3-VF 33 μM, 7 day-aged; fAβ42 5 μM, 6 day-aged; hf-Aβ42/Nle3-VF, 5 μM each, 6 day-aged; Aβ42 incubations w/o ThT) and boiled (95° C.) for 5 min except for fAβ42 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
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 CaCl2; the final PK concentration in the assay was 0.5 μg/ml. fIAPP (16.5 μM) 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. fAβ42 (5 μM) and hf-Aβ42/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 μl of the fIAPP or hf-IAPP/ACM solutions with 0.3 μl of the PK stock solution. For Aβ42-related assays, solutions were made by mixing 200 μl of fAβ42 or hf-Aβ42/ACM solutions with 1 μl 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-Aβ(1-17) (6E10, BIOZOL; 1:2000) for Aβ42); rabbit anti-Aβ40 (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 of fIAPP and fAβ42 versus hf-IAPP/ACM and hf-Aβ42/ACMs was studied in primary murine BMDMs and cultured murine BV2 microglia using TAMRA-IAPP and TAMRA-Aβ42 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% CO2, 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 μM) 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 μM. For Aβ42-related cell uptake studies, TAMRA-fAβ42 (5 μM) and hf-TAMRA-Aβ42/ACM (5 μM) 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 μM. Of note, dot blot analysis and BCA showed that the main peptide fraction was present in the pellet.
To address the question whether addition of Nle3-VF or L3-VF to preformed Aβ42 oligomers may affect their phagocytosis, preformed TAMRA-Aβ42 oligomers (TAMRA-oA342) were prepared as for MTT reduction assays by incubating TAMRA-Aβ42 (5 μM) for 2 h under ThT binding assay conditions. For studying the effect of the ACMs, TAMRA-oAβ42 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-Aβ42, 1 μM) and incubated with the BV2 cells for 6 h as described above. TAMRA-oAβ42 alone was incubated for 2 h as well and treated thereafter as its mixtures with the ACMs.
Following incubation of the cells with the peptides, supernatants were removed and cells on the coverslips were washed 5 times with ice-cold 1×PBS, fixed with 4% paraformaldehyde, washed with 1×PBS, permeabilized with 0.2% Triton-X 100, and rinsed three times with cold 1×PBS. 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.
LTP measurements were performed. Briefly, sagittal hippocampal slices (350 μm) were obtained from C57BL/6N male mice (6-8 weeks) in ice-cold Ringer solution bubbled with a mixture of 95% O2 and 5% CO2 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 CaCl2, 1 mM MgCl2, 25 mM D-glucose, and 1.25 mM NaH2PO4 (pH 7.3) and was bubbled with 95% O2 and 5% CO2. 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. Aβ42 (50 nM), Aβ42/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 Aβ42, Aβ42 (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 Aβ42 alone and Aβ42/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.
fIAPP and hf-IAPP/Nle3-VF were made by aging IAPP (1024 μM) or a mixture of IAPP (1024 μM) and Nle3-VF (2048 μM) in ddH2O 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 CuKα radiation (λ=1.54178 Å) and a Helios optic using the APEX3 software package (Version 2019-1.0, Bruker AXS Inc., Madison, Wisconsin, USA, 2019).
For inhibitor design, Aβ(15-40) was used as a template in the context of the fAβ40 fold; this features a β-strand-loop-β-strand motif with Aβ(12-22) and AR(30-40) forming the P-strands and Aβ(23-29) the loop (
To evaluate the concept, 13 Aβ(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 (
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 Aβ(15-23) (analogs Nle3-VF and Nle3-LF) or within the C-terminal region corresponding to Aβ(27-40) (analogs Nle3-GI & Nle3-GG) (
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 (
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 (
Together, the studies identified the six ACMs Nle3-VF, Nle3-LF, L3-VF, L3-LF, F3-VF, and F3-LF (
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. KDs were <100 nM and in very good agreement with the determined IC50 values (
Far UV-CD spectroscopy revealed that IAPP/Nle3-VF co-assemblies exhibited a mixture of disordered and b-sheet structure (
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 (
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 (
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 (
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 (
High-resolution advanced laser-scanning microscopy provided further unequivocal evidence for diverse supramolecular IAPP/ACM nanofiber co-assemblies (
Confocal laser scanning (CLSM), stimulated emission depletion (STED), and two-photon microscopy (2 μM) 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 (
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 (
To learn more about the molecular architecture of the IAPP/ACM nanofibers, we used fluorescence lifetime imaging/Förster resonance energy transfer (FLIM-FRET). Pronounced FLIM-FRET events were observed in TAMRA-IAPP/Fluos-Nle3-VF nanofiber co-assemblies (
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.
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 (
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 (
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 (
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 (
The structure of Aβ/IAPP hetero-amyloids has not been yet elucidated. Based on suggested structures of fIAPP and fAβ40(42) and the polymorphic nature of self-/cross-amyloid assembly, various different interfaces could be involved in hf-IAPP/ACM formation (
In analogy to other Aβ-derived Aβ inhibitors, ACMs also interfere with Aβ amyloid self-assembly. In fact, ThT binding and MTT reduction assays in PC12 cells showed that all six ACMs (Aβ42/ACM 1/i) effectively suppressed formation of Aβ42 fibrils and cytotoxic assemblies (
Remarkably, TEM examination of the aged “ThT-negative” and non-cytotoxic Aβ42/ACM mixtures revealed that they exclusively consisted of fibrils (
2PM examination of aged Aβ42/ACM mixtures containing N-terminal TAMRA-labeled Aβ42 (TAMRA-Aβ42) and Fluos-ACMs revealed diverse heteromeric fibrous superstructures. These comprised several μm-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 μm (
ACM/Aβ42 interactions and hetero-complexes were then studied by fluorescence spectroscopy, SEC, cross-linking in combination with NuPAGE and WB, and far-UV CD spectroscopy (
Together, the results suggested that the potent inhibitory effect of ACMs on Aβ42 amyloid self-assembly is mediated by nanomolar affinity interactions of ACMs with Aβ42 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 fAβ42 does not result in this kind of co-assemblies (
Hippocampal synaptic plasticity is regarded as a key mediator of learning and memory processes; its damage by toxic Aβ42 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 Aβ42-mediated inhibition of hippocampal long-term potentiation (LTP) was fully ameliorated (
We then investigated whether hf-Aβ42/ACM might differ from fAβ42 with respect to their seeding competence. In fact, the ThT binding assay showed that, in contrast to fAβ42, hf-Aβ42/Nle3-VF and hf-Ap42/L3-VF were seeding-incompetent (
Together, these findings revealed that ThT-invisible and non-toxic hf-Aβ42/ACM were seeding-incompetent and less thermostable than fAβ42 and that they became more efficiently degraded by PK and phagocytosed by BV2 microglia than fAβ42.
Cross-seeding of Aβ42 amyloid self-assembly by fIAPP accelerates Aβ42 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/Aβ42 hetero-amyloids which will template further cytotoxic Aβ42 self-/cross-assembly events. Thereby, polymorphic cross-interactions between amyloid core regions may play an important role.
Because ACMs contain the Aβ amyloid core, bind with high affinity both IAPP and Aβ42, incl. fIAPP and fAβ42, and inhibit their amyloid self-assembly, the inventors assumed that they might also interfere with cross-seeding of Aβ42 by fIAPP. In fact, ACMs effectively suppressed fIAPP-mediated cross-seeding of Aβ42 fibrillogenesis and cytotoxicity (
[a]IC50 values, means (±SD) from 3 titration assays (n = 3 each)
[b]Determined by titrations of N-terminal fluorescein-labeled IAPP (5 nM; pH 7.4) with ACMs
[c]App. KDs are means (±SD) from 3 binding curves
[a]IC50 values, means (±SD) from 3 titration assays (n = 3 each); n.d.: not determined
[b]Determined by titrations of N-terminal FITC-labelled Aβ42 (5 nM; pH 7.4) with ACMs
[c]App. KDs, means (±SD) from three binding curves
[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
[a]Measured in 6 days aged solutions of Aβ42 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)
The inventors exploited Aβ/IAPP cross-interactions to design A amyloid core mimics (ACMs) as inhibitors of amyloid self-assembly of both IAPP and Aβ42. Collectively, the inventors identified six 26-residue peptides as effective amyloid inhibitors of both IAPP and Aβ42. All six ACMs bound IAPP with nanomolar affinity and blocked its cytotoxic amyloid self-assembly with nanomolar IC50 values. In addition, all six ACMs bound Aβ42 with nanomolar affinity and blocked its cytotoxic self-assembly, three of them with nanomolar IC50 values. Moreover, ex vivo electrophysiology in murine brains showed a full amelioration of Aβ42-mediated damage of synaptic plasticity by ACMs. Importantly, ACMs also inhibited reciprocal cross-seeding of IAPP and Aβ42 amyloid self-assembly. ACMs thus belong to the most effective inhibitors of in vitro amyloid self-assembly of IAPP, Aβ42, or both polypeptides.
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 Aβ42 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, Aβ42, and ACM formed when Aβ42 cross-seeding by fIAPP was performed in the presence of ACMs.
Unexpectedly, IAPP(Aβ42)/ACM nanofiber co-assembly was efficiently inhibited by the peptides of the invention. In fact, ACMs contained all three Aβ hot segments for high affinity interactions with IAPP, Aβ42, and themselves, and combined inbuilt β-sheet extension blocking (N-methylations) with β-sheet stabilization/extension enabling (LTS and Aβ(21-40)) elements.
The identified IAPP/ACM nanofibers were indistinguishable from fIAPP by TEM and had the cross-β amyloid core signature by XRD, but were less ordered than fIAPP according to CD spectroscopy. Our TEM, STED, 2 μM, 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 Aβ.
The identified Aβ42/ACM nanofibers had similar widths but 2-4 times greater lengths than fAβ42. The inventors' imaging results were consistent with both axial and lateral co-assembly, the former likely underlying hetero-nanofiber elongation.
Another notable finding of the inventors' study relates to the potentially beneficial properties of IAPP/ACM and Aβ42/ACM nanofibers. These clearly distinguished them from pathogenic fIAPP and fAβ42 and, in addition to their non-toxic nature and seeding incompetence, comprised thermolability, proteolytic degradability, and a more efficient phagocytosis than fIAPP and fAβ42. 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.
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 Aβ42, 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.
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22158021.0 | Feb 2022 | EP | regional |
This application is a National Stage Application of International Application Number PCT/EP2023/054273, filed Feb. 21, 2023; which claims priority to European Patent Application No. 22158021.0, filed Feb. 22, 2022, both of which are incorporated herein by reference in their entirety. The Sequence Listing for this application is labeled “SeqList-14 Aug24.xml”, which was created on Aug. 14, 2024 and is 147,997 bytes. The entire content is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/054273 | 2/21/2023 | WO |
