Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 °C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4 mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.     

Type IV collagen prevents amyloid beta-protein fibril formation

The potential of targeting through molecular therapeutics the underlying amyloid beta-protein (A beta) fibrillogenesis causing the initiation and progression of Alzheimer's disease (AD) offers an opportunity to improve the disease. Type IV collagen (collagen IV) is localized in senile plaques in patients with AD. By using thioflavin T fluorescence spectroscopy and electron microscopy, we found that collagen IV inhibited A beta1-40 (A beta40) fibril formation. The critical concentration of collagen IV for this inhibition was 5 microg/mL. Circular dichroism data indicate that collagen IV prevents formation of a beta-structured aggregate of A beta40. These studies demonstrated that collagen IV is apparently a potent inhibitor of A beta fibril formation.     

Peptides homologous to the amyloid protein of Alzheimer's disease containing a glutamine for glutamic acid substitution have accelerated amyloid fibril formation.

beta-Amyloid (A beta) deposition in fibril form is the central event in a number of diseases, including Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis - Dutch type (HCHWA-D). A beta is produced by degradation of a larger amyloid precursor protein (APP). Recently a mutation in the APP gene has been found in HCHWA-D causing a glutamine for glutamic acid substitution at residue 22 of A beta. The influence of this mutation on fibrillogenesis is not known, although it is clear that affected patients have accelerated cerebrovascular amyloid deposition, with disease symptoms early in life. We report the in vitro demonstration of accelerated fibril formation in a 28 residue synthetic peptide homologous to the Dutch variant A beta. Furthermore, in eight residue peptides homologous to A beta the presence of the mutation is necessary for fibril formation. These findings provide a mechanism for accelerated amyloid formation in the Dutch variant of APP.     

Examining the inhibitory actions of copolypeptides against amyloid fibrillogenesis of bovine insulin

Amyloid fibrillogenesis has been involved in at least 40 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been demonstrated to fibrillate in vitro. With bovine insulin as a model, the research presented here examines the influence of two simple, unstructured d,l-lysine-co-glycine (d,l-lys-co-gly) and d,l-lysine-co-L-phenylalanine (d,l-lys-co-phe) copolypeptides, on the in vitro fibril formation process of bovine insulin at pH 2.0 and 55 °C. Our results showed that amyloid fibrillogenesis of insulin may be suppressed by both copolypeptides in a concentration-dependent fashion. In addition, the copolypeptides with higher molar fractions of glycine or l-phenylalanine residue, which are considered to possess higher hydrophobic interacting capacities, demonstrated the superior inhibitory potency against insulin fibril formation. Our findings suggest that the association of insulin and copolypeptides, which is likely dominated by hydrophobic interactions and hydrogen bonding, may mitigate the extent of insulin fibrillogenesis. We believe the results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the unstructured polypeptides and amyloid-forming proteins.     

The sulfate moieties of glycosaminoglycans are critical for the enhancement of beta-amyloid protein fibril formation

Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind beta-amyloid protein (Abeta) 1-40 and 1-42, but are also potent enhancers of Abeta fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of Abeta fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on Abeta 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of Abeta fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of Abeta 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of Abeta amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) Abeta amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of Abeta amyloid deposition in Alzheimer's disease brain may also participate in the enhancement of Abeta amyloidosis.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH

Beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit in the musculo-skeletal system in patients undergoing long-term hemodialysis. How beta(2)m self-assembles in vivo is not understood, since the monomeric wild-type protein is incapable of forming fibrils in isolation in vitro at neutral pH, while elongation of fibril-seeds made from recombinant protein has only been achieved at low pH or at neutral pH in the presence of detergents or cosolvents. Here we describe a systematic study of the effect of 11 physiologically relevant factors on beta(2)m fibrillogenesis at pH 7.0 without denaturants. By comparing the results obtained for the wild-type protein with those of two variants (DeltaN6 and V37A), the role of protein stability in fibrillogenesis is explored. We show that DeltaN6 forms low yields of amyloid-like fibrils at pH 7.0 in the absence of seeds, suggesting that this species could initiate fibrillogenesis in vivo. By contrast, high yields of amyloid-like fibrils are observed for all proteins when assembly is seeded with fibril-seeds formed from recombinant protein at pH 2.5 stabilized by the addition of heparin, serum amyloid P component (SAP), apolipoprotein E (apoE), uremic serum, or synovial fluid. The results suggest that the conditions within the synovium facilitate fibrillogenesis of beta(2)m and show that different physiological factors may act synergistically to promote fibril formation. By comparing the behavior of wild-type beta(2)m with that of DeltaN6 and V37A, we show that the physiologically relevant factors enhance fibrillogenesis by stabilizing fibril-seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers.     

Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide

Phospholipids are known to influence fibril formation of amyloid beta (Aβ) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phospholipid, enhances Aβ(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Aβ(1-40) fibril formation, and appeared to be inhibitory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of Aβ(1-40) and Aβ(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Aβ(1-42), but not of Aβ(1-40). Tris-tricine gradient SDS/PAGE revealed that LPC increased the formation of higher-molecular-weight species of Aβ(1-42), including trimers and tetramers. LPC had no such effect on Aβ(1-40), and thus may specifically influence the oligomerization and nucleation processes of Aβ(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Aβ(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Aβ(1-42) fibrils, the main component of senile plaques in Alzheimer's disease patients, and may be involved in Alzheimer's disease pathology.     

Fibril formation by primate, rodent, and Dutch-hemorrhagic analogues of Alzheimer amyloid beta-protein.

Deposition of extraneuronal fibrils that assemble from the 39-43 residue beta/A4 amyloid protein is one of the earliest histopathological features of Alzheimer's disease. We have used negative-stain electron microscopy, Fourier-transform infrared (FT-IR) spectroscopy, and fiber X-ray diffraction to examine the structure and properties of synthetic peptides corresponding to residues 1-40 of the beta/A4 protein of primate [Pm(1-40); human and monkey], rodent [Ro(1-40); with Arg5-->Gly, Tyr10-->Phe, and His13-->Arg], and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) [Du(1-40); with Glu22-->Gln]. As controls, we examined a reverse primate sequence [Pm*(40-1)] and an extensively substituted primate peptide [C(1-40); with Glu3-->Arg, Arg5-->Glu, Asp7-->Val, His13-->Lys, Lys16-->His, Val18-->Asp, Phe19-->Ser, Phe20-->Tyr, Ser26-->Pro, Ala30-->Val, Ile31-->Ala, Met35-->norLeu, Gly38-->Ile, Val39-->Ala, and Val40-->Gly]. The assembly of these peptides was studied to understand the relationship between species-dependent amyloid formation and beta/A4 sequence and the effect of a naturally occurring point mutation of fibrillogenesis. The three N-terminal amino acid differences between Pm(1-40) and Ro(1-40) had virtually no effect on the morphology or organization of the fibrils formed by these peptides, indicating that the lack of amyloid deposits in rodent brain is not due directly to specific changes in its beta/A4 sequence. beta-Sheet and fibril formation, judged by FT-IR, was maximal within the pH range 5-8 for Pm(1-40), pH 5-10.5 for Du(1-40), and pH 2.5-8 for Ro(1-40).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of amyloid fibrillogenesis and toxicity by a peptide chaperone

Aggregation of proteins in tissues is associated with several diseases, including Alzheimer's disease. It is characterized by the accumulation of amyloid beta peptide (Abeta) in the extracellular spaces of the brain cells, resulting in neuronal death and other pathological changes. alpha-Crystallin, a small heat-shock protein in lens, and a peptide chaperone having the functional site sequence DFVIFLDVKHFSPEDLTVK of alphaA-crystallin may inhibit Abeta fibrillogenesis and toxicity. The peptide chaperone (mini-alphaA-crystallin), having an Abeta interacting domain and a complex solubilizing domain, was shown in previous studies to prevent aggregation of several proteins under denaturing conditions. In this in vitro study, using transmission electron microscopy and thioflavin T binding assay, we show that mini-alphaA-crystallin arrests the fibril formation of Abeta peptides. Mini-alphaA-crystallin also suppresses the toxic action of Abeta on rat pheochromocytoma (PC 12) cells. The wide chaperoning capability of the peptide and its ability to inhibit amyloid fibril formation and suppress toxicity suggest that mini-alphaA-crystallin may serve as a universal chaperone in controlling diseases of protein aggregation, including Alzheimer's disease.     

The ratio of monomeric to aggregated forms of Abeta40 and Abeta42 is an important determinant of amyloid-beta aggregation, fibrillogenesis, and toxicity

Aggregation and fibril formation of amyloid-beta (Abeta) peptides Abeta40 and Abeta42 are central events in the pathogenesis of Alzheimer disease. Previous studies have established the ratio of Abeta40 to Abeta42 as an important factor in determining the fibrillogenesis, toxicity, and pathological distribution of Abeta. To better understand the molecular basis underlying the pathologic consequences associated with alterations in the ratio of Abeta40 to Abeta42, we probed the concentration- and ratio-dependent interactions between well defined states of the two peptides at different stages of aggregation along the amyloid formation pathway. We report that monomeric Abeta40 alters the kinetic stability, solubility, and morphological properties of Abeta42 aggregates and prevents their conversion into mature fibrils. Abeta40, at approximately equimolar ratios (Abeta40/Abeta42 approximately 0.5-1), inhibits (> 50%) fibril formation by monomeric Abeta42, whereas inhibition of protofibrillar Abeta42 fibrillogenesis is achieved at lower, substoichiometric ratios (Abeta40/Abeta42 approximately 0.1). The inhibitory effect of Abeta40 on Abeta42 fibrillogenesis is reversed by the introduction of excess Abeta42 monomer. Additionally, monomeric Abeta42 and Abeta40 are constantly recycled and compete for binding to the ends of protofibrillar and fibrillar Abeta aggregates. Whereas the fibrillogenesis of both monomeric species can be seeded by fibrils composed of either peptide, Abeta42 protofibrils selectively seed the fibrillogenesis of monomeric Abeta42 but not monomeric Abeta40. Finally, we also show that the amyloidogenic propensities of different individual and mixed Abeta species correlates with their relative neuronal toxicities. These findings, which highlight specific points in the amyloid peptide equilibrium that are highly sensitive to the ratio of Abeta40 to Abeta42, carry important implications for the pathogenesis and current therapeutic strategies of Alzheimer disease.     

Turn nucleation perturbs amyloid β self-assembly and cytotoxicity

The accumulation of senile plaques composed of amyloid β (Aβ) fibrils is a hallmark of Alzheimer's disease, although prefibrillar oligomeric species are believed to be the primary neurotoxic congeners in the pathogenesis of Alzheimer's disease. Uncertainty regarding the mechanistic relationship between Aβ oligomer and fibril formation and the cytotoxicity of these aggregate species persists. β-Turn formation has been proposed to be a potential rate-limiting step during Aβ fibrillogenesis. The effect of turn nucleation on Aβ self-assembly was probed by systematically replacing amino acid pairs in the putative turn region of Aβ (residues 24-27) with d-ProGly ((D)PG), an effective turn-nucleating motif. The kinetic, thermodynamic, and cytotoxic effects of these mutations were characterized. It was found that turn formation dramatically accelerated Aβ fibril self-assembly dependent on the site of turn nucleation. The cytotoxicity of the three (D)PG-containing Aβ variants was significantly lower than that of wild-type Aβ40, presumably due to decreased oligomer populations as a function of a more rapid progression to mature fibrils; oligomer populations were not eliminated, however, suggesting that turn formation is also a feature of oligomer structures. These results indicate that turn nucleation is a critical step in Aβ40 fibril formation.     

NBD-labeled phospholipid accelerates apolipoprotein C-II amyloid fibril formation but is not incorporated into mature fibrils

Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by submicellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by submicellar lipids using the fluorescently labeled, short-chain phospholipid 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxyglycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately 2-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril-forming concentrations of apoC-II indicated NBD-lyso-12-PC binds rapidly, on the millisecond time scale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-lyso-12-PC binds to both apoC-II monomers and tetramers at approximately five sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-lyso-12-PC were devoid of lipid, indicating a purely catalytic role for submicellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules in controlling protein folding and fibril assembly pathways.     

Sodium louroyl sarcosinate (sarkosyl) modulate amyloid fibril formation in hen egg white lysozyme (HEWL) at alkaline pH: a molecular insight study

Amyloid fibril formation is responsible for several neurodegenerative diseases and are formed when native proteins misfold and stick together with different interactive forces. In the present study, we have determined the mode of interaction of the anionic surfactant sarkosyl with hen egg white lysozyme (HEWL) [EC No. 3.2.1.17] at two pHs (9.0 and 13.0) and investigated its impact on fibrillogenesis. Our data suggested that sarkosyl is promoting amyloid fibril formation in HEWL at the concentration range between 0.9 and 3.0 mM and no amyloid fibril formation was observed in the concentration range of 3.0–20.0 mM at pH 9.0. The results were confirmed by several biophysical and computational techniques, such as turbidity measurement, dynamic light scattering, Raleigh scattering, ThT fluorescence, intrinsic fluorescence, far-UV CD and atomic force microscopy. Sarkosyl was unable to induce aggregation in HEWL at pH 13.0 as confirmed by turbidity and RLS measurements. HEWL forms larger amyloid fibrils in the presence of 1.6 mM of sarkosyl. The spectroscopic, microscopic and molecular docking data suggest that the negatively charged carboxylate group and 12-carbon hydrophobic tail of sarkosyl stimulate amyloid fibril formation in HEWL via electrostatic and hydrophobic interaction. This study leads to new insight into the process of suppression of fibrillogenesis in HEWL which can be prevented by designing ligands that can retard the electrostatic and hydrophobic interaction between sarkosyl and HEWL.     

Optimum amyloid fibril formation of a peptide fragment suggests the amyloidogenic preference of beta2-microglobulin under physiological conditions

Beta(2)-microglobulin (beta(2)m) is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Although full-length beta(2)m readily forms amyloid fibrils in vitro by seed-dependent extension with a maximum at pH 2.5, fibril formation under physiological conditions as detected in patients has been difficult to reproduce. A 22-residue K3 peptide of beta(2)m, Ser(20)-Lys(41), obtained by digestion with Acromobacter protease I, forms amyloid fibrils without seeding. To obtain further insight into the mechanism of fibril formation, we studied the pH dependence of fibril formation of the K3 peptide and its morphology using a ThT fluorescence assay and electron microscopy, respectively. K3 peptide formed amyloid fibrils over a wide range of pH values with an optimum around pH 7 and contrasted with the pH profile of the seed-dependent extension reaction of full-length beta(2)m. This suggests that once the rigid native-fold of beta(2)m is unfolded and additional factors triggering the nucleation process are provided, full-length beta(2)m discloses an intrinsic potential to form amyloid fibrils at neutral pH. The fibril formation was strongly promoted by dimerization of K3 through Cys(25). The morphology of the fibrils varied depending on the fibril formation conditions and the presence or absence of a disulfide bond. Various fibrils had the potential to seed fibril formation of full-length beta(2)m accompanied with a characteristic lag phase, suggesting that the internal structures are similar.     

Cellular membrane composition defines A beta-lipid interactions.

Alzheimer's disease pathology has demonstrated amyloid plaque formation associated with plasma membranes and the presence of intracellular amyloid-beta (A beta) accumulation in specific vesicular compartments. This suggests that lipid composition in different compartments may play a role in A beta aggregation. To test this hypothesis, we have isolated cellular membranes from human brain to evaluate A beta 40/42-lipid interactions. Plasma, endosomal, lysosomal, and Golgi membranes were isolated using sucrose gradients. Electron microscopy demonstrated that A beta fibrillogenesis is accelerated in the presence of plasma and endosomal and lysosomal membranes with plasma membranes inducing an enhanced surface organization. Alternatively, interaction of A beta with Golgi membranes fails to progress to fibril formation, suggesting that A beta-Golgi head group interaction stabilizes A beta. Fluorescence spectroscopy using the environment-sensitive probes 1,6-diphenyl-1,3,5-hexatriene, laurdan, N-epsilon-dansyl-L-lysine, and merocyanine 540 demonstrated variations in the inherent lipid properties at the level of the fatty acyl chains, glycerol backbone, and head groups, respectively. Addition of A beta 40/42 to the plasma and endosomal and lysosomal membranes decreases the fluidity not only of the fatty acyl chains but also the head group space, consistent with A beta insertion into the bilayer. In contrast, the Golgi bilayer fluidity is increased by A beta 40/42 binding which appears to result from lipid head group interactions and the production of interfacial packing defects.     

Role of the N and C-terminal strands of beta 2-microglobulin in amyloid formation at neutral pH

Beta 2-microglobulin (beta(2)m) is known to form amyloid fibrils de novo in vitro under acidic conditions (below pH 4.8). Fibril formation at neutral pH, however, has only been observed by deletion of the N-terminal six residues; by the addition of pre-assembled seeds; or in the presence of Cu(2+). Based on these observations, and other structural data, models for fibril formation of beta(2)m have been proposed that involve the fraying of the N and C-terminal beta-strands and the consequent loss of edge strand protective features. Here, we examine the role of the N and C-terminal strands in the initiation of fibrillogenesis of beta(2)m by creating point mutations in strands A and G and comparing the properties of the resulting proteins with variants containing similar mutations elsewhere in the protein. We show that truncation of buried hydrophobic side-chains in strands A and G promotes rapid fibril formation at neutral pH, even in unseeded reactions, and increases the rate of fibril formation under acidic conditions. By contrast, similar mutations created in the remaining seven beta-strands of the native protein have little effect on the rate or pH dependence of fibril formation. The data are consistent with the view that perturbation of the N and C-terminal edge strands is an important feature in the generation of assembly-competent states of beta(2)m.     

New class of inhibitors of amyloid-beta fibril formation. Implications for the mechanism of pathogenesis in Alzheimer's disease

The amyloid hypothesis suggests that the process of amyloid-beta protein (Abeta) fibrillogenesis is responsible for triggering a cascade of physiological events that contribute directly to the initiation and progression of Alzheimer's disease. Consequently, preventing this process might provide a viable therapeutic strategy for slowing and/or preventing the progression of this devastating disease. A promising strategy to achieve prevention of this disease is to discover compounds that inhibit Abeta polymerization and deposition. Herein, we describe a new class of small molecules that inhibit Abeta aggregation, which is based on the chemical structure of apomorphine. These molecules were found to interfere with Abeta1-40 fibrillization as determined by transmission electron microscopy, Thioflavin T fluorescence and velocity sedimentation analytical ultracentrifugation studies. Using electron microscopy, time-dependent studies demonstrate that apomorphine and its derivatives promote the oligomerization of Abeta but inhibit its fibrillization. Preliminary structural activity studies demonstrate that the 10,11-dihydroxy substitutions of the D-ring of apomorphine are required for the inhibitory effectiveness of these aporphines, and methylation of these hydroxyl groups reduces their inhibitory potency. The ability of these small molecules to inhibit Abeta amyloid fibril formation appears to be linked to their tendency to undergo rapid autoxidation, suggesting that autoxidation product(s) acts directly or indirectly on Abeta and inhibits its fibrillization. The inhibitory properties of the compounds presented suggest a new class of small molecules that could serve as a scaffold for the design of more efficient inhibitors of Abeta amyloidogenesis in vivo.     

Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone

It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p-benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p-benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p-benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.