Transportable, Chemical Genetic Methodology for the Small Molecule-Mediated Inhibition of Heat Shock Factor 1

Abstract

Proteostasis in the cytosol is governed by the heat shock response. The master regulator of the heat shock response, heat shock factor 1 (HSF1), and key chaperones whose levels are HSF1-regulated have emerged as high-profile targets for therapeutic applications ranging from protein misfolding-related disorders to cancer. Nonetheless, a generally applicable methodology to selectively and potently inhibit endogenous HSF1 in a small molecule-dependent manner in disease model systems remains elusive. Also problematic, the administration of even highly selective chaperone inhibitors often has the side effect of activating HSF1 and thereby inducing a compensatory heat shock response. Herein, we report a ligand-regulatable, dominant negative version of HSF1 that addresses these issues. Our approach, which required engineering a new dominant negative HSF1 variant, permits doseable inhibition of endogenous HSF1 with a selective small molecule in cell-based model systems of interest. The methodology allows us to uncouple the pleiotropic effects of chaperone inhibitors and environmental toxins from the concomitantly induced compensatory heat shock response. Integration of our method with techniques to activate HSF1 enables the creation of cell lines in which the cytosolic proteostasis network can be up- or down-regulated by orthogonal small molecules. Selective, small molecule-mediated inhibition of HSF1 has distinctive implications for the proteostasis of both chaperone-dependent globular proteins and aggregation-prone intrinsically disordered proteins. Altogether, this work provides critical methods for continued exploration of the biological roles of HSF1 and the therapeutic potential of heat shock response modulation.

Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis

“…Rather than using the anchor away technique that we used in yeast (Haruki et al, 2008), we propose using a conditional degradation system that has been previously established for small-molecule mediated degradation of HSF1 mutants expressed in mammalian cells on top of endogenous HSF1 (Moore et al, 2016;Ryno et al, 2014).…”

Multidimensional chemical control of CRISPR–Cas9

“…Previously, we applied destabilized domains (DDs) 10–12 to confer similar controls on transcription factors 13,14 . Briefly, structurally unstable protein domains derived from Escherichia coli dihydrofolate reductase (DHFR) 10 or the estrogen receptor (ER50) 11 are fused to a transcription factor.…”

“…These largely unfolded domains target the fusion protein for rapid proteasomal degradation. Small molecules that can bind and stabilize the DDs in their poorly populated folded state prevent proteasomal degradation of the fusion protein in a concentration-dependent manner, allowing transcription factor function 13,14 . We envisioned that such small-molecule-regulated DDs could be similarly deployed to establish Cas9 systems with multidimensional control of genome editing and transcriptional activities.…”

“…The pharmacologic chaperoning-based mechanism of DD stabilization can engender a broad range of dose control 13,14 . Indeed, as shown in Figure 1d, we were able to control the levels of TMP-mediated IL1RN mRNA induction in the DHFR-regulated PP7.…”

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“…Moreover, fusion of destabilized domains to dominant negative versions of the UPR transcription factors can permit small molecule-dependent inhibition of endogenous transcription factor activity (Shoulders et al 2013a). The advantages of destabilized domains for conferring small molecule control onto UPR and other stress-responsive transcription factors (Moore et al 2016) have led to their adoption by a number of research groups in studies of stress-responsive signaling.…”