Structural Basis for the Inhibition of HSP70 and DnaK Chaperones by Small-Molecule Targeting of a C-Terminal Allosteric Pocket

Maintaining protein homeostasis (proteostasis) is central to the survival of all cells, and altered protein quality control is characteristic of many human diseases. Critical components in this regulatory network are the mammalian stress-inducible heat shock protein-70 (inducible HSP70, also called HSPA1A or HSP72), as well as its evolutionarily conserved bacterial orthologue, DnaK.1−5 These molecular chaperones coordinate key processes needed to maintain protein quality, especially under conditions of increased cellular stress. Their activities include protein folding, protein transport across membranes, modulating protein–protein interactions, and preventing a buildup of toxic protein aggregates. These molecular chaperones protect against proteotoxic stress, and not surprisingly, therefore, they are key survival proteins, especially for tumor cells. HSP70 and DnaK are part of an evolutionarily conserved family of 70 kDa heat shock proteins.1−5 The proteins have an approximately 44 kDa N-terminal nucleotide binding domain (NBD), followed by a conserved flexible linker, and an approximately 25 kDa C-terminal substrate binding domain (SBD). Each major domain contains several dynamic subdomains. These molecular chaperones transiently interact with a multitude of diverse substrates, or clients, by binding exposed hydrophobic regions of partially folded or unfolded proteins. ATP binding induces conformational changes in the NBD subdomains, promotes interdomain docking between the SBD and NBD, and promotes high on–off rates for the substrate.6−9 In contrast, when ADP is bound to the chaperone, the NBD and SBD are more loosely held together by the linker region.3,10 Allosteric communication between the NBD and SBD is critical to protein function; cycles of nucleotide binding and hydrolysis correlate with the binding and release of substrate, all of which are mediated by conformational changes in protein subdomains. The basic features of this allostery have been investigated for some time, often using Escherichia coli (E. coli) DnaK as a model. However, many key questions remain about the molecular details of this process, including whether inhibitors of these chaperones can interrupt this allosteric cycle. Cancer cells are subject to an enhanced stress environment, and this promotes protein misfolding. Additionally, many cancer cells contain oncoproteins that contain missense mutations that can alter protein stability and conformation; thus, cancer cells are believed to be particularly dependent on the activities of HSP70 to maintain proteostasis. In support of this premise, HSP70 is constitutively expressed at elevated levels in most cancers, and silencing or inhibition of HSP70 has been found to be cytotoxic to many tumor but not normal cells.2,5,11,12 The preferential cytotoxicity of HSP70 inhibition for cancer cells is believed to relate to the modest to undetectable expression of this protein in normal cells, suggesting that there is a therapeutic index that can be exploited for preferential targeting of tumors. Indeed, HSP70 and DnaK have emerged as highly attractive targets for the development of new treatments for many human diseases, including microbial infections, neurodegenerative pathologies, and other disorders of protein folding. Despite a great deal of interest in the translational potential of small molecules that target these chaperones, identifying and characterizing effective modulators for basic research and therapeutic use has proven to be challenging. In particular, the dynamic nature and conformational flexibility of these molecular chaperones have complicated efforts to generate molecular data, which would inform structure-based design of improved inhibitors.12−14 As a consequence, few selective inhibitors have been identified or are well-characterized at the structural and biophysical levels.11−18 We previously reported that the small molecule, 2-phenylethynesulfonamide (PES), and a chlorinated derivative (PES-Cl), interact with HSP70 and DnaK, and are cytotoxic to tumor cells in a manner that requires HSP70.19−21 Unlike most other HSP70 inhibitors characterized to date, we identified the interaction site for PES and its derivatives as the C-terminal substrate binding domain of HSP70. This is in contrast with most other HSP70 inhibitors, which interact with the N-terminal nucleotide-binding domain.13,18,27 The SBD is less conserved among HSP70 family members, and our data indicate that PES and its derivatives do not interact with the organelle-specific members of this family, GRP75 and GRP78 (BiP), which are required for the viability of all cells.5,19 We have shown that these small molecules effectively inhibit HSP70 activities; importantly, this has now been confirmed by several other groups.19−26 In this study, we carried out structure–activity relationship analyses to identify inhibitors with enhanced cytotoxicity and to define critical moieties that are required for the ability to inhibit HSP70 and DnaK chaperone activities. We report the identification of a new small molecule, triphenyl(phenylethynyl)phosphonium bromide (herein referred to as PET-16) that interacts with both HSP70 and DnaK, and alters in vivo function. We have successfully cocrystallized PET-16 with purified DnaK. Notably, our X-ray crystallographic data on PET-16 in complex with the C-terminal domain of DnaK, together with data from isothermal titration calorimetry and mutagenesis studies, now provide a model for how these compounds act as inhibitors of HSP70 as well as DnaK activity, by binding to a conserved region in these proteins and impeding substrate binding. These findings should facilitate efforts to further probe the physiologic functions of these molecular chaperones and support efforts to optimize potency and efficacy in developing HSP70 and DnaK modulators for therapeutic use.