Identification and characterization of key kinetic intermediates in amyloid beta-protein fibrillogenesis

Amyloid beta-protein (Abeta) assembly into toxic oligomeric and fibrillar structures is a seminal event in Alzheimer's disease, therefore blocking this process could have significant therapeutic benefit. A rigorous mechanistic understanding of Abeta assembly would facilitate the targeting and design of fibrillogenesis inhibitors. Prior studies have shown that Abeta fibrillogenesis involves conformational changes leading to the formation of extended beta-sheets and that an alpha-helix-containing intermediate may be involved. However, the significance of this intermediate has been a matter of debate. We report here that the formation of an oligomeric, alpha-helix-containing assembly is a key step in Abeta fibrillogenesis. The generality of this phenomenon was supported by conformational studies of 18 different Abeta peptides, including wild-type Abeta(1-40) and Abeta(1-42), biologically relevant truncated and chemically modified Abeta peptides, and Abeta peptides causing familial forms of cerebral amyloid angiopathy. Without exception, fibrillogenesis of these peptides involved an oligomeric alpha-helix-containing intermediate and the kinetics of formation of the intermediate and of fibrils was temporally correlated. The kinetics varied depending on amino acid sequence and the extent of peptide N- and C-terminal truncation. The pH dependence of helix formation suggested that Asp and His exerted significant control over this process and over fibrillogenesis in general. Consistent with this idea, Abeta peptides containing Asp-->Asn or His-->Gln substitutions showed altered fibrillogenesis kinetics. These data emphasize the importance of the dynamic interplay between Abeta monomer conformation and oligomerization state in controlling fibrillogenesis kinetics.     

Dissecting the assembly of Abeta16-22 amyloid peptides into antiparallel beta sheets

Multiple long molecular dynamics simulations are used to probe the oligomerization mechanism of Abeta(16-22) (KLVFFAE) peptides. The peptides, in the monomeric form, adopt either compact random-coil or extended beta strand-like structures. The assembly of the low-energy oligomers, in which the peptides form antiparallel beta sheets, occurs by multiple pathways with the formation of an obligatory alpha-helical intermediate. This observation and the experimental results on fibrillogenesis of Abeta(1-40) and Abeta(1-42) peptides suggest that the assembly mechanism (random coil --> alpha helix --> beta strand) is universal for this class of peptides. In Abeta(16-22) oligomers both interpeptide hydrophobic and electrostatic interactions are critical in the formation of the antiparallel beta sheet structure. Mutations of either hydrophobic or charged residues destabilize the oligomer, which implies that the 16-22 fragments of Arctic (E22G), Dutch (E22Q), and Italian (E22K) mutants are unlikely to form ordered fibrils.     

Identification of peptide inhibitors of transforming growth factor beta 1 using a phage-displayed peptide library

Pathologies such as liver fibrosis and scleroderma are characterized by harmful levels of transforming growth factor beta 1 (TGFbeta1). These levels could be neutralized if inhibitors of this cytokine were available. With this aim we searched for peptides with binding affinity for TGFbeta1 using a phage-displayed random 15-mer peptide library. Some peptides thus identified blocked activity of TGFbeta1 in vitro, as measured by their capacity to restore growth of Mv-1-Lu cells in presence of added TGFbeta1. Also, they inhibited TGFbeta1-dependent expression of collagen type I mRNA in liver of mice orally insulted with CCl(4). Intraperitoneal administration of 50 microg of peptide P17 (the most active 15-mer peptide, also referred to as P17(1-15)) inhibited expression of collagen type I mRNA by almost 100%. Interestingly, titration experiments showed that P17(1-12) (a peptide encompassing the first 12 amino acids of P17) was approximately four times more active than P17. These results suggest that both peptides, as well as others reported here, may be of therapeutic interest in processes requiring control of undesired high levels of TGFbeta1.     

Identification of peptide inhibitors of Pseudomonas aeruginosa exotoxin A function using a yeast two-hybrid approach

The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.     

Fibronectin peptides derived from two distinct regions stimulate adipocyte differentiation by preventing fibronectin matrix assembly

Here, we show that fibronectin (FN) peptides derived from two distinct regions promote the insulin-induced adipocyte differentiation of ST-13 cells by preventing FN fibrillogenesis. ST-13 cells formed numerous FN fibrils under nonadipogenic conditions, whereas this FN fibrillogenesis was suppressed by adipose induction with insulin. The insulin-induced adipocyte differentiation was promoted by an amino-terminal 24-kDa fragment of FN, accompanied by further suppression of FN fibrillogenesis. The 24 K fragment prevented FN matrix assembly by direct incorporation into the FN matrix. Like the 24 K fragment, a peptide from the 14th type III repeat, termed FNIII14, which suppressed the integrin alpha 5 beta 1-mediated adhesion of ST-13 cells to FN, accelerated the adipocyte differentiation by preventing FN fibrillogenesis without direct incorporation into the FN matrix. FNIII14 induced the conformation change of beta1 integrins of K562 cells from active to resting, as judged by FACS analysis using a monoclonal antibody AG89 directed to an active beta1 integrin-dependent epitope. Binding of a (125)I-labeled FN fragment containing the RGD cell adhesive site to ST-13 cell surface was dissociated by FNIII14, with a concomitant binding of FNIII14 itself to the cell surface. The affinity labeling of ST-13 cells using biotinylated FNIII14 showed that FNIII14 specifically bound to a nonintegrin membrane protein with M(r) of around 50 kDa. Thus, the results indicated that prevention of FN fibrillogenesis by the 24 K Fib 1 fragment and FNIII14 caused the promotion of adipocyte differentiation of ST-13 cells and that the former was due to the direct incorporation into the FN matrix and that the latter might be interpreted by negative regulation of FN receptor alpha 5 beta 1 activity.     

Detection of beta-amyloid peptide aggregation using DNA electrophoresis

DNA could readily associate with the aggregated forms of the beta-amyloid peptides beta(1-40) and beta(25-35), giving rise to a shift in the electrophoretic mobility of DNA. As a result, DNA was retained at the top of a 1% agarose gel. In contrast, the electrophoretic mobility of DNA was little influenced by the monomeric forms of beta(1-40) and beta(25-30). DNA from different sources such as lambda phage, Escherichia coli plasmid, and human gene showed similar results. However, the electrophoretic mobility of RNA was shifted by the monomeric beta(1-40) and beta(25-35) as well as by the aggregated beta(1-40) and beta(25-35). The association of DNA with the aggregated beta-amyloid peptides could occur at pH 4-9. The inhibitory action of hemin on beta-amyloid aggregation could be confirmed using the DNA mobility shift assay. These results indicate that the DNA mobility shift assay is useful for kinetic study of beta-amyloid aggregation as well as for testing of agents that might modulate beta-amyloid aggregation.     

以表位模拟肽为亲和靶标的肉毒毒素受体结合抑制剂的筛选及鉴定

以设计合成的A型肉毒毒素表位模拟肽为亲和靶标,对噬菌体随机肽库进行筛选,寻找能与A型肉毒毒素表位模拟肽特异结合并能拮抗毒素毒性效应的分子,通过ELISA鉴定阳性克隆,并对鉴定的阳性克隆进行特异性分析及DNA测序。氨基酸序列同源性分析发现,针对P4、P5表位模拟肽获得了两条特异的结合序列,并通过动物保护实验在噬菌体展示肽水平对特异结合分子的毒素毒性拮抗效应进行了初步研究,初步证明结合肽对A型肉毒毒素有一定的保护作用。     

Potent cyclic monomeric and dimeric peptide inhibitors of VLA-4 (alpha4beta1 integrin)-mediated cell adhesion based on the Ile-Leu-Asp-Val tetrapeptide.

Potent monomeric and dimeric cyclic peptide very late antigen-4 (VLA-4) inhibitors have been designed based on a tetrapeptide (Ile-Leu-Asp-Val) sequence present in a 25-amino acid peptide (CS-1) reported in the literature. The peptides, synthesized by the SPPS techniques, were evaluated in the in vitro cell adhesion assays and in the in vivo inflammation models. The N- to C-terminal cyclic peptides such as cyclo(Ile-Leu-Asp-Val-NH-(CH2)2-S-(CH2)2-CO) (28) and cyclo(MeIle-Leu-Asp-Val-D-Ala-D-Ala) (31), monomeric and dimeric peptides containing piperazine (Pip) or homopiperazine (hPip) residues as linking groups, e.g. cyclo(MeIle-Leu-Asp-Val-Pip-CH2CO-NH-(CH2)2-S-CH2-CO) (49) and cyclo(MeIle-Leu-Asp-Val hPip-CH2CO-MeIle-Leu-Asp-Val-hPip-CH2CO) (58) and cyclic peptides containing an amide bond between the side chain amino group of an amino acid such as Lys and the C-terminal Val carboxyl group, e.g. Ac-cyclo(D-Lys-D-Ile-Leu-Asp-Val) (62) and beta-Ala-cyclo(D-Lys-D-Leu-Leu-Asp-Val) (68) were more potent than CS-1 in inhibiting the adhesion of the VLA-4-expressing MOLT-4 cells to fibronectin. The more potent compounds were highly selective and did not affect U937 cell adhesion to fibronectin (VLA-5), PMA-differentiated U937 cell adhesion to intercellular cell adhesion molecule- 1-expressing Chinese hamster ovary cells (LFA-1) and ADP-induced platelet aggregation (GPIIb/IIIa). A number of the more potent compounds inhibited ovalbumin-induced delayed type hypersensitivity in mice and some were 100-300 times more potent (ED50 = 0.003-0.009 mg/kg/day, s.c.) than CS-1. Two peptides, Ac-cyclo(D-Lys D-Ile-Leu-Asp-Val) (62) and cyclo(CH2CO-Ile-Leu-Asp-Val-Pip-CH2CO-Ile-Leu-Asp-Val-Pip) (55), were formulated in poly(DL-lactide-co-glycolide) depots and the release profile was investigated in vitro over a 30-day period.     

Disassembly of preformed amyloid beta fibrils by small organofluorine molecules

A potential therapeutic approach for Alzheimer’s disease is to reduce the amount of toxic amyloid-beta oligomers and fibrillar amyloid plaques. In order to contribute to this approach the ability of small organofluorine compounds that were previously reported as successful inhibitors of fibrillogenesis to destabilize preformed fibrils of the amyloid-beta peptide was studied. These organofluorine molecules including chiral compounds were tested in vitro using standard methods based on Thioflavin-T (THT) fluorescence spectroscopy, atomic force microscopy (AFM) and Fourier-transform infrared spectroscopy (FTIR). It was observed that 5′-halogen substituted 3,3,3-trifluoromethyl-2-hydroxyl-(indol-3-yl)-propionic acid esters showed significant activity in the disassembly of the preformed fibrils. Since the same compounds were identified as strong fibrillogenesis inhibitors as well, this dual action makes them promising candidates for further drug development.     

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.     

Black tea theaflavins inhibit formation of toxic amyloid-β and α-synuclein fibrils

Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Here, we show that theaflavins (TF1, TF2a, TF2b, and TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-β (Aβ) and α-synuclein (αS) fibrillogenesis. Their mechanism of action was compared to that of two established inhibitors of amyloid formation, (-)-epigallocatechin gallate (EGCG) and congo red (CR). All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3, EGCG, and CR revealed that all three bind to two regions of the Aβ peptide, amino acids 12-23 and 24-36, albeit with different specificities. However, their mechanisms of amyloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of Aβ and αS into nontoxic, spherical aggregates that are incompetent in seeding amyloid formation and remodel Aβ fibrils into nontoxic aggregates. When compared to EGCG, TF3 was less susceptible to air oxidation and had an increased efficacy under oxidizing conditions. These findings suggest that theaflavins might be used to remove toxic amyloid deposits.     

Synthesis and evaluation of novel 1-(2-acylhydrazinocarbonyl)-cycloalkyl carboxamides as interleukin-1beta converting enzyme (ICE) inhibitors

Novel 1-(2-acylhydrazinocarbonyl)cycloalkyl carboxamides were designed as peptidomimetic inhibitors of interleukin-1beta converting enzyme (ICE). A short synthesis was developed and moderately potent ICE inhibitors were identified (IC(50) values <100 nM). Most of the synthesized examples were selective for ICE versus the related cysteine proteases caspase-3 and caspase-8, although several dual-acting inhibitors of ICE and caspase-8 were identified. Several of the more potent ICE inhibitors were also shown to inhibit IL-1beta production in a whole cell assay (IC(50) < 500 nM).     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

In vitro peptide binding to soluble empty class I major histocompatibility complex molecules isolated from transfected Drosophila melanogaster cells.

A soluble form of a mouse class I major histocompatibility antigen (H-2Kb) has been expressed in transfected Drosophila melanogaster cells. These molecules were efficiently secreted (up to 4 mg/liter) as noncovalent heterodimers and purified to homogeneity from cell supernatants. The isolated soluble Kb molecules were devoid of endogenous peptides. Using these molecules, we have characterized the Kb heavy chain-beta 2-microglobulin (beta 2m) assembly as well as peptide binding in vitro. In detergent-free solution the heavy chains readily re-assembled with beta 2m even in the absence of peptides. Kinetic analyses showed that the peptide binding is rapid and reversible and dependent on the heavy chains being assembled with beta 2m. Likewise, peptide dissociated from Kb molecules without the displacement of beta 2m. Equilibrium binding experiments using various peptides confirmed that octapeptides bind to Kb molecules with the highest affinity and form the most stable complexes. However, in contrast to earlier studies, the amino-terminal positioning of peptide to Kb molecules was more crucial than the carboxyl-terminal positioning and amidation of the peptide carboxylate did not affect the binding. Soluble Kb molecules could selectively bind allele-specific peptides among a mixture of randomly synthesized octapeptides in vitro; however, no dominant residue was observed at the carboxyl terminus of bound peptides. This suggests that the previously observed hydrophobic residues at the carboxyl terminus of peptides may reflect the specificity of enzyme(s) or protein(s) involved in peptide processing in vivo.     

Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-amyloid(40) fibrils by short beta-amyloid congeners containing N-methyl amino acids at alternate residues.

A potential goal in the prevention or therapy of Alzheimer's disease is to decrease or eliminate neuritic plaques composed of fibrillar beta-amyloid (Abeta). In this paper we describe N-methyl amino acid containing congeners of the hydrophobic "core domain" of Abeta that inhibit the fibrillogenesis of full-length Abeta. These peptides also disassemble preformed fibrils of full-length Abeta. A key feature of the inhibitor peptides is that they contain N-methyl amino acids in alternating positions of the sequence. The most potent of these inhibitors, termed Abeta16-22m, has the sequence NH(2)-K(Me-L)V(Me-F)F(Me-A)E-CONH(2). In contrast, a peptide, NH(2)-KL(Me-V)(Me-F)(Me-F)(Me-A)-E-CONH(2), with N-methyl amino acids in consecutive order, is not a fibrillogenesis inhibitor. Another peptide containing alternating N-methyl amino acids but based on the sequence of a different fibril-forming protein, the human prion protein, is also not an inhibitor of Abeta40 fibrillogenesis. The nonmethylated version of the inhibitor peptide, NH(2)-KLVFFAE-CONH(2) (Abeta16-22), is a weak fibrillogenesis inhibitor. Perhaps contrary to expectations, the Abeta16-22m peptide is highly soluble in aqueous media, and concentrations in excess of 40 mg/mL can be obtained in buffers of physiological pH and ionic strength, compared to only 2 mg/mL for Abeta16-22. Analytical ultracentrifugation demonstrates that Abeta16-22m is monomeric in buffer solution. Whereas Abeta16-22 is susceptible to cleavage by chymotrypsin, the methylated inhibitor peptide Abeta16-22m is completely resistant to this protease. Circular dichroic spectroscopy of Abeta16-22m indicates that this peptide is a beta-strand, albeit with an unusual minimum at 226 nm. In summary, the inhibitor motif is that of alternating N-methyl and nonmethylated amino acids in a sequence critical for Abeta40 fibrillogenesis. These inhibitors appear to act by binding to growth sites of Abeta nuclei and/or fibrils and preventing the propagation of the network of hydrogen bonds that is essential for the formation of an extended beta-sheet fibril.     

All or none fibrillogenesis of a prion peptide.

Amyloid proteins and peptides comprise a diverse group of molecules that vary both in size and amino-acid sequence, yet assemble into amyloid fibrils that have a common core structure. Kinetic studies of amyloid fibrillogenesis have revealed that certain amyloid proteins form oligomeric intermediates prior to fibril formation. We have investigated fibril formation with a peptide corresponding to residues 195-213 of the human prion protein. Through a combination of kinetic and equilibrium studies, we have found that the fibrillogenesis of this peptide proceeds as an all-or-none reaction where oligomeric intermediates are not stably populated. This variation in whether oligomeric intermediates are stably populated during fibril formation indicates that amyloid proteins assemble into a common fibrillar structure; however, they do so through different pathways.     

Molecular modeling, structure--activity relationships and functional antagonism studies of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketones as a novel class of dopamine transporter inhibitors.

We previously disclosed the discovery of 4-hydroxy-1-methyl-4-(4-methylphenyl)-3-piperidyl 4-methylphenyl ketone (3) as a novel class of dopamine transporter (DAT) inhibitors and showed that (+/-)-3 has a significant functional antagonism against cocaine in vitro. Our previous preliminary structure-activity relationship study led to identification of a more potent DAT inhibitor [(+/-)-4] but this compound failed to show any significant functional antagonism. To search for more potent analogues than 3 but still displaying significant functional antagonism, further SARs, molecular modeling studies and in vitro pharmacological evaluation of this novel class of DAT inhibitors were performed. Sixteen new analogues were synthesized in racemic form and evaluated as DAT inhibitors. It was found that seven new analogues are reasonably potent DAT inhibitors with K(i) values of 0.041--0.30 and 0.052--0.16 microM in [(3)H]mazindol binding and inhibition of DA reuptake. Chiral isomers of several potent DAT inhibitors were obtained through chiral HPLC separation and evaluated as inhibitors at all the three monoamine transporter sites. In general, the (-)-isomer is more active than the (+)-isomer in inhibition of DA reuptake and all the (-)-isomers are selective inhibitors at the DAT site. Evaluation of cocaine's effect on dopamine uptake in the presence and absence of (+)-3 and (-)-3 showed that (-)-3 is responsible for the functional antagonism obtained with the original lead (+/-)-3. Out of the new compounds synthesized, analogue (+/-)-20, which is 8- and 3-fold more potent than (+/-)-3 in binding and inhibition of DA reuptake, appeared to have improved functional antagonism as compared to (+/-)-3.     

Interfacial peptide inhibitors of HIV-1 integrase activity and dimerization

Peptides derived from the interfacial region of dimeric HIV-1 integrase were evaluated as inhibitors of integrase's 3'-endonuclease activity. Three peptides were found to be moderately potent inhibitors with IC(50) values in the low micromolar range. The mode of inhibition was probed through protein crosslinking experiments. Active interfacial peptides were found to inhibit crosslinking of the dimeric form of integrase. Interfacial peptides that were poor inhibitors had no effect on integrase crosslinking.     

Biochemical and cell-based assays for characterization of BACE-1 inhibitors

The deposition of beta-amyloid peptides (A beta42 and A beta40) in neuritic plaques is one of the hallmarks of Alzheimer's disease (AD). A beta peptides are derived from sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. BACE-1 has been shown to be the major beta-secretase and is a primary therapeutic target for AD. In this article, two novel assays for the characterization of BACE-1 inhibitors are reported. The first is a sensitive 96-well HPLC biochemical assay that uses a unique substrate containing an optimized peptide cleavage sequence, NFEV, spanning from the P2-P2' positions This substrate was processed by BACE-1 approximately 10 times more efficiently than was the widely used substrate containing the Swedish (NLDA) sequence. As a result, the concentration of the enzyme required for the assay can be as low as 100 pM, permitting the evaluation of inhibitors with subnanomolar potency. The assay has also been applied to related aspartyl proteases such as cathepsin D (Cat D) and BACE-2. The second assay is a homogeneous electrochemiluminescence assay for the evaluation of BACE-1 inhibition in cultured cells that assesses the level of secreted amyloid EV40_NF from HEK293T cells stably transfected with APP containing the novel NFEV sequence. To illustrate the use of these assays, the properties of a potent, cell-active BACE-1 inhibitor are described.