Structure-Based Design of Non-Natural Amino Acid Inhibitors of Amyloid Fibrillation

Many globular and natively disordered proteins can convert into amyloid fibers. These fibers are associated with numerous pathologies1 as well as with normal cellular functions2,3, and frequently form during protein denaturation4,5. Inhibitors of pathological amyloid fibers could serve as leads for therapeutics, provided the inhibitors were specific enough to avoid interfering with normal processes. Here we show that computer-aided, structure-based design can yield highly specific peptide inhibitors of amyloid formation. Using known atomic structures of segments of amyloid fibers as templates, we have designed and characterized an all D-amino acid inhibitor of fibrillation of the tau protein found in Alzheimer’s disease, and a non-natural L-amino acid inhibitor of an amyloid fiber that enhances sexual transmission of HIV. Our results indicate that peptides from structure-based designs can disrupt the fibrillation of full-length proteins, including those like tau that lack fully ordered native structures.We thank M.I. Ivanova, J. Corn, T. Kortemme, D. Anderson, M.R. Sawaya, M. Phillips, S. Sambashivan, J. Park, M. Landau, Q. Zhang, R. Clubb, F. Guo, T. Yeates, J. Nowick, J. Zheng, and M.J. Thompson for discussions, HHMI, NIH, NSF, the GATES foundation, and the Joint Center for Translational Medicine for support, R. Peterson for help with NMR experiments, E. Mandelkow for providing tau constructs, R. Riek for providing amyloid beta, J. Stroud for amyloid beta preparation. Support for JK was from the Damon Runyon Cancer Research Foundation, for HWC by the Ruth L. Kirschstein National Research Service Award, for JM from the programme for junior-professors by the ministry of science, Baden-Württemberg, and for SAS by a UCLA-IGERT bioinformatics traineeship

Macrocyclic β-Sheet Peptides That Inhibit the Aggregation of a Tau-Protein-Derived Hexapeptide

This paper describes studies of a series of macrocyclic β-sheet peptides 1 that inhibit the aggregation of a tau-protein-derived peptide. The macrocyclic β-sheet peptides comprise a pentapeptide "upper" strand, two δ-linked ornithine turn units, and a "lower" strand comprising two additional residues and the β-sheet peptidomimetic template "Hao". The tau-derived peptide Ac-VQIVYK-NH(2) (AcPHF6) aggregates in solution through β-sheet interactions to form straight and twisted filaments similar to those formed by tau protein in Alzheimer's neurofibrillary tangles. Macrocycles 1 containing the pentapeptide VQIVY in the "upper" strand delay and suppress the onset of aggregation of the AcPHF6 peptide. Inhibition is particularly pronounced in macrocycles 1a, 1d, and 1f, in which the two residues in the "lower" strand provide a pattern of hydrophobicity and hydrophilicity that matches that of the pentapeptide "upper" strand. Inhibition varies strongly with the concentration of these macrocycles, suggesting that it is cooperative. Macrocycle 1b containing the pentapeptide QIVYK shows little inhibition, suggesting the possibility of a preferred direction of growth of AcPHF6 β-sheets. On the basis of these studies, a model is proposed in which the AcPHF6 amyloid grows as a layered pair of β-sheets and in which growth is blocked by a pair of macrocycles that cap the growing paired hydrogen-bonding edges. This model provides a provocative and appealing target for future inhibitor design

Comprehensive N-methyl scanning of a potent peptide inhibitor of malaria invasion into erythrocytes leads to pharmacokinetic optimization of the molecule

Apical membrane antigen-1 is a protein found in the merozoite stage of the malaria parasite Plasmodium falciparum and has been shown to be critical in the invasion of host erythrocytes. Using a random peptide library displayed on the surface of phage, a 20-residue peptide, R1 (VFAEFLPLFSKFGSRMHILK), was identified which specifically recognized and bound to P. falciparum AMA-1. Moreover, the peptide was found to competitively inhibit parasite invasion of red blood cells (RBC). In this study we report the synthesis and properties of the R1 peptide modified by comprehensive N-methyl scanning along the backbone of the peptide. The native R1 and eighteen R1 analogues containing single N-methyl substitutions were synthesized by manual solid-phase Fmoc peptide chemistry. The set of N-methylated peptides was assessed for relative binding affinity with Pf AMA-1 and RBC invasion inhibitory ability. N-methylation in positions 1, 8, and 14 in the R1 peptide produced analogues exhibiting stronger binding characteristics to Pf AMA-1. A tri N-methylated peptide was more than 10 times more active in the inhibition of RBC invasion assay. This peptide also exhibited enhanced serum stability which may be partially responsible for the increase in binding and bioactivity. We have therefore shown that modifications to a biologically active peptide can dramatically enhance activity and potentially provide a lead to a peptide therapeutic molecule with optimized pharmacokinetic properties.Denis Scanlon, Karen S. Harris, Andrew M. Coley, John A. Karas, Joanne L. Casey, Andrew B. Hughes, Michael Fole

Retro-inversal of intracellular selected ?-amyloid-interacting peptides: implications for a novel Alzheimer's disease treatment

The aggregation of ?-amyloid (A?) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind A?1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving A?-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease

The Natural Product Betulinic Acid Rapidly Promotes Amyloid-β Fibril Formation at the Expense of Soluble Oligomers

[Image: see text] The biochemical hallmarks of Alzheimer’s disease (AD) are the aggregates of amyloid-β (Aβ) peptide that deposit in brains of AD patients as senile plaques. The monomeric Aβ undergoes aggregation in a nucleation-dependent manner to form insoluble fibrils. Emerging evidence suggests that the low-molecular-weight aggregates called “soluble oligomers” are the primary neurotoxic agents as opposed to the fibrils. Needless to say, developing Aβ aggregation inhibitors is imperative for a meaningful progress toward AD therapy. In this report, we have explored the in vitro interactions between a natural product called betulinic acid (BA) and Aβ42 peptide. BA has found its therapeutic use in several human pathologies including cancer, HIV-related AIDS, and nervous system disorders. The results from this study indicate that BA rapidly promotes the formation of Aβ42 fibrils and, in doing so, partly circumvents the formation of potentially neurotoxic soluble oligomers. Furthermore, the promotion of fibrils by BA seems to be specific for the fibril formation “on-pathway”, and it fails to interact with aggregates that are formed outside this obligatory pathway. The unique ability of BA to promote fibrils at the expense of oligomers along with its well-known pharmacological properties make BA a potential therapeutic agent for AD