The Hsp90 family of molecular chaperones is required for the maturation, activation, and/or stability of diverse proteins that play central roles in malignant progression. As a result of this diversity, Hsp90 inhibitors simultaneously antagonize a wide variety of oncogenic pathways and processes. Thus, Hsp90 inhibitors are widely envisioned to have great potential as anti-cancer drugs. The Hsp90 inhibitors that have progressed to clinical trials share a common mechanism of action, namely binding to the ATP binding site in Hsp90's N- terminal domain and inhibiting Hsp90 function in vivo. Unfortunately, N-terminal Hsp90 inhibitors also have the untoward effect of inducing the pro-survival heat shock response, which is acknowledged to undermine the clinical efficacies of N-terminal Hsp90 inhibitors. Small molecules that bind to Hsp90's C-terminus have also been identified that inhibit Hsp90 function in vitro, and show cytostatic/cytotoxic activity in cultured cancer cell lines, but do not appear to induce the heat shock response. Thus, targeting the C-terminal domain of Hsp90 may be a superior strategy for anti-cancer therapies based on the inhibition of Hsp90. However, the progression of C-terminal Hsp90 inhibitors as clinical agents is hindered by two significant knowledge gaps: (i) The structural basis of their binding to Hsp90 is unknown, but must be determined for the rational design and optimization of novel inhibitory compounds; and (ii) the physiological basis of their anti-proliferative / cytotoxic activities is poorly understood. To further the development of C-terminal Hsp90 inhibitors with clinical potential, we propose to: 1) determine the structure of Hsp90 complexed to C-terminal Hsp90 inhibitors; and 2) compare and contrast the impacts of C-terminal vs. N-terminal inhibitors on the proteomes of breast cancer cells cultured as 3D spheroids. The structure of a N-terminally truncated Hsp90 construct bound to Hsp90 C- terminal inhibitors will be determined by X-ray crystallography. The impact of N- and C-terminal Hsp90 inhibitors on breast cancer cell proteomes will be characterized by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC), in conjunction with multi-dimensional liquid chromatography and a state-of-the-art Fusion Tribrid mass spectrometer. The proposed studies will provide critical structural information regarding the binding of C-terminal inhibitors to Hsp90, culminating in the rational design of C-terminal inhibitors with improved affinities for Hsp90 and clinical potential. The mechanistic insights into the selective tumoricidal activities of N- and C-terminal Hsp90 inhibitors generated by the proteomic studies will also reveal tumor- specific features that might guide chemotherapeutic decisions, tumor cell responses that might be monitored during clinical treatment, and promising modalities for combinatorial treatments with both families of Hsp90 inhibitors.

R15CA219907

2017-07-01

2021-06-30