Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

Discovery of novel Hsp90 C-terminal domain inhibitors that disrupt co-chaperone binding.

Heat shock protein 90 (Hsp90) is an essential molecular chaperone that performs vital stress-related and housekeeping functions in cells and is a current therapeutic target for diseases such as cancers. Particularly, the development of Hsp90 C-terminal domain (CTD) inhibitors is highly desirable as inhibitors that target the N-terminal nucleotide-binding domain may cause unwanted biological effects. Herein, we report on the discovery of two drug-like novel Hsp90 CTD inhibitors by using virtual screening and intrinsic protein fluorescence quenching binding assays, paving the way for future development of new therapies that employ molecular chaperone inhibitors.

Identification of Isoform-Selective Ligands for the Middle Domain of Heat Shock Protein 90 (Hsp90).

The molecular chaperone heat shock protein 90 (Hsp90) is a current inhibition target for the treatment of diseases, including cancer. In humans, there are two major cytosolic isoforms of Hsp90 (Hsp90α and Hsp90ß). Hsp90α is inducible and Hsp90ß is constitutively expressed. Most Hsp90 inhibitors are pan-inhibitors that target both cytosolic isoforms of Hsp90. The development of isoform-selective inhibitors of Hsp90 may enable better clinical outcomes. Herein, by using virtual screening and binding studies, we report our work in the identification and characterisation of novel isoform-selective ligands for the middle domain of Hsp90ß. Our results pave the way for further development of isoform-selective Hsp90 inhibitors.

Chemical modulation of cytosolic BAX homodimer potentiates BAX activation and apoptosis.

The BCL-2 family protein BAX is a major regulator of physiological and pathological cell death. BAX predominantly resides in the cytosol in a quiescent state and upon stress, it undergoes conformational activation and mitochondrial translocation leading to mitochondrial outer membrane permeabilization, a critical event in apoptosis execution. Previous studies reported two inactive conformations of cytosolic BAX, a monomer and a dimer, however, it remains unclear how they regulate BAX. Here we show that, surprisingly, cancer cell lines express cytosolic inactive BAX dimers and/or monomers. Expression of inactive dimers, results in reduced BAX activation, translocation and apoptosis upon pro-apoptotic drug treatments. Using the inactive BAX dimer structure and a pharmacophore-based drug screen, we identify a small-molecule modulator, BDM19 that binds and activates cytosolic BAX dimers and prompts cells to apoptosis either alone or in combination with BCL-2/BCL-XL inhibitor Navitoclax. Our findings underscore the role of the cytosolic inactive BAX dimer in resistance to apoptosis and demonstrate a strategy to potentiate BAX-mediated apoptosis.