Peptides modulating conformational changes in secreted chaperones: From in silico design to preclinical proof of concept

Abstract

Blocking conformational changes in biologically active proteins holds therapeutic promise. Inspired by the susceptibility of viral entry to inhibition by synthetic peptides that block the formation of helixhelix interactions in viral envelope proteins, we developed a computational approach for predicting interacting helices. Using this approach, which combines correlated mutations analysis and Fourier transform, we designed peptides that target gp96 and clusterin, 2 secreted chaperones known to shift between inactive and active conformations. In human blood mononuclear cells, the gp96-derived peptide inhibited the production of TNF␣, IL-1␤, IL-6, and IL-8 induced by endotoxin by >80%. When injected into mice, the peptide reduced circulating levels of endotoxin-induced TNF␣, IL-6, and IFN␥ by >50%. The clusterin-derived peptide arrested proliferation of several neoplastic cell lines, and significantly enhanced the cytostatic activity of taxol in vitro and in a xenograft model of lung cancer. Also, the predicted mode of action of the active peptides was experimentally verified. Both peptides bound to their parent proteins, and their biological activity was abolished in the presence of the peptides corresponding to the counterpart helices. These data demonstrate a previously uncharacterized method for rational design of protein antagonists.cancer ͉ contact map prediction ͉ cytokines ͉ inflammation ͉ helix C onformational changes in proteins have an essential role in regulating activity. Natural and synthetic molecules that modulate such changes are of considerable biological importance. Such molecules include allosteric effectors that alter the rate of enzymecatalyzed reactions (1), molecules that shift the oligomerization equilibrium of proteins (2), and molecules that interfere with transmembrane helix-helix associations (3).Conformational changes that have been extensively studied are those that take place during viral-induced membrane fusion. This process is required for the propagation of enveloped viruses, and is facilitated by viral encoded type 1 integral membrane proteins (4, 5). Viral entry of enveloped viruses depends on a conformational change involving the formation of helix-helix interactions, i.e., 2 alpha helices that do not interact in the native (nonfusogenic) state, but do interact with each other when the protein folds into its fusion-active (fusogenic) form. Remarkably, synthetic peptides corresponding to some of these helical segments have an antiviral activity (6 -8), of which one has been developed for the treatment of HIV-1 infection (9, 10).We hypothesized that such a mode of inhibition could be also applied on nonviral proteins. The aim of the present study was to develop a computational tool for the detection of intramolecular helix-helix interactions and to use this tool for detecting such interacting helices in proteins of interest. This study focuses on secreted chaperones due to their biological and therapeutic relevance (11-14), and because conformational changes are known to take p...

The Molecular Chaperone gp96/GRP94 Interacts with Toll-like Receptors and Integrins via Its C-terminal Hydrophobic Domain

“…A recent study reported that a peptide sequence targeting part of the charged linker region of gp96 was able to inhibit endotoxic shock when administered in vivo (36). It is noteworthy that CBD-targeted peptide is able to significantly inhibit gp96-integrin interaction.…”

Chaperone gp96-independent Inhibition of Endotoxin Response by Chaperone-based Peptide Inhibitors

“…Therefore, gp96 appears to be an attractive drug target for inflammation, sepsis, and autoimmune disease. In a recent study utilizing in silico methods, a peptide inhibitor of gp96 was designed by targeting the N-terminal helix-loop-helix sequence, and the authors demonstrated that this peptide could effectively inhibit LPS responses both in vitro and in vivo (24). The proposed mechanism of action was that this peptide mimics the sequence of the helix, therefore disrupting the helix-helix interaction and the chaperoning function of gp96.…”

“…As proposed by the Kliger et al (24) study, the design of PIER1 peptide is based on a potential helix-helix interaction between PIER1 and the target sequence in the HSP that requires a conformation-dependent interaction. To examine this possibility, we designed another peptide, PIER2, by substituting the middle two residues in the helix of PIER1, Val and Arg, with two Pro residues.…”

“…Other TLR Ligands-We synthesized a 37-mer peptide corresponding to residues 444 -480 of gp96 based on the study by Kliger et al (24). For consistency in this study, we named this peptide PIER1.…”

The Molecular Chaperone Apolipoprotein J/Clusterin as a Sensor of Oxidative Stress: Implications in Therapeutic Approaches - A Mini-Review

“…This possibility is currently being exploited in clinical trials [81] by the successful paradigm of OGX-011, a 2'-methoxyethyl-modified phosphorothioate antisense oligonucleotide that is complementary to CLU mRNA; work in progress aims to finalize plans for two phase III clinical studies. Additional translational applications in relation to CLU biology may include peptides that bind and inhibit CLU in vivo [82] or molecular imaging of tumors by using CLU-binding fluorescent peptides [83]. …”