A high throughput substrate binding assay reveals hexachlorophene as an inhibitor of the ER-resident HSP70 chaperone GRP78

Abstract

Glucose regulated protein 78 (GRP78) is the ER resident 70 kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.

Function, Therapeutic Potential, and Inhibition of Hsp70 Chaperones

“…Currently, this site is under-probed, but we recently published that Hsp70 substrate peptide recognition may be less highly conserved between isoforms than would be predicted based on sequence homology. We conducted a pilot screen to find competitive inhibitors of peptide binding and were able to show that hexachlorophene was able to selectivity inhibit peptide binding in HspA5 as compared to DnaK . Hexachlorophene has been reported as a PAINS compound in a number of screens, but we believe that its selectivity in our assay may validate our hypothesis that Hsp70-client interactions are not conserved between isoforms.…”

“…We conducted a pilot screen to find competitive inhibitors of peptide binding and were able to show that hexachlorophene was able to selectivity inhibit peptide binding in HspA5 as compared to DnaK. 194 Hexachlorophene has been reported as a PAINS compound in a number of screens, but we believe that its selectivity in our assay may validate our hypothesis that Hsp70-client interactions are not conserved between isoforms. While more work in this area is needed and ongoing, we hypothesize that a characterization of the divergent peptide recognition at this position will reveal a strategy by which each Hsp70 can be selectively targeted in cells and in vivo.…”

A fluorogenic probe for TRPA1 channel imaging based on a molecular rotation mechanism

“…Based on the competitive binding theory, 4-Am may partially occupy the hydrophobic pocket of TRPA1 bound by DPCC , hindering the binding of DPCC to the channel. 45,46 This phenomenon enlightens us that agonists or antagonists of the TRPA1 channel can affect the binding of DPCC to TRPA1, suggesting that the DPCC probe may be a potential tool for screening TRPA1 regulators.…”

Visualizing TRPA1 in the Plasma Membrane for Rapidly Screening Optical Control Agonists via a Photochromic Ligand Based Fluorescent Probe

“…Competitive binding assay is one of effective methods to screen active compounds that act on the same sites of protein as known compounds. , Based on the competitive binding theory, we used A1CA to screen some azobenzene derivatives (AB1, AB2, and AB3) by measuring A1CA fluorescence with confocal microscopy. As shown in Figure , the fluorescent intensity of the three experimental groups (A1CA + AB1, A1CA + AB2, and A1CA + AB3) decreased differently comparing with A1CA group, which could be attributed to the resistance from these azobenzene derivatives which had resemble active groups that partly inhibited the binding of A1CA with TRPA1 channels, indicating that these azobenzene derivatives competed with A1CA for binding the same domain of TRPA1 channels.…”