Examining the Potential of Targeting the HSP60 Chaperonin System as a Broadly Applicable Chemotherapeutic Strategy

Abstract

This study methodically examined our diversity set of GroEL and HSP60 inhibitors to identify lead candidates that exhibited the most potent and selective cytotoxicity to colon cancer cells over non-cancer cells in vitro. While several structurally distinct candidates were identified, we found that our nitrofuran and hydroxyquinoline-containing N-acylhydrazone series (NF-NAH and HQ-NAH, respectively) were among the most potent and selective. Subsequent screenings across an NCI panel of cancer cell lines of different origins revealed the superior efficacy of the NF-NAH and HQ-NAH series as chemotherapeutic candidates, in contrast to the ABK-based inhibitors we previously reported, which showed poor efficacy across the panel. Given the emerging evidence of the role of mis-localized HSP60 in cancer cell survival, this study also compared the structure and function of naive cHSP60 (the aberrant form presumed to be in the cytosol) with that of mHSP60 (the processed and mature form that is in mitochondria). Analytical size exclusion chromatography revealed cHSP60 is a more stable oligomer consisting of both single and double-ring complexes. Intriguingly, cryoEM analyses revealed that cHSP60 formed a unique face-to-face double-ring complex, as opposed to the structures of other double-ring GroEL and HSP60 chaperonins where their rings stack back-to-back with one another. Subsequent assays demonstrated similar ATPase activities for both mHSP60 and cHSP60, with stimulatory effects observed in the presence of HSP10 for both. Despite the apparent engagement of HSP10, cHSP60 was unable to refold the denatured MDH client protein efficiently, suggesting potential functional divergence in vivo. These enticing results offer novel insights into the physiological importance of the cHSP60 complex and its possible role in cancer progression. As our previous studies examined inhibitors that were developed as GroEL-targeting antibacterial candidates, and given the unique structural/functional differences of cHSP60 compared to GroEL and other chaperonins, including mHSP60, the findings from this study underscore the need for future to identify and optimize inhibitors specifically for targeting cHSP60 to enhance chemotherapeutic effectiveness.