Design of Allosteric Stimulators of the Hsp90 ATPase as New Anticancer Leads

Abstract

We rationally designed allosteric compounds that stimulate Hsp90 ATPase activity and show anticancer potencies in the low micromolar to nanomolar range. In parallel, we clarified their mode of action and developed a quantitative model that links the dynamic ligand-protein cross-talk to observed cellular and in vitro activities. Our results support the potential of using dynamicsbased approaches to develop original mechanism-based cancer therapeutics.Heat Shock Protein 90 (Hsp90) is a chaperone that controls the folding of more than 200 client proteins and constitutes a central node in many signaling pathways [1] . Overexpression and dysregulation of Hsp90 have been linked with cancer and neurodegeneration. It is thus not surprising that this chaperone has become an important drug-target: in principle its inhibition can result in the simultaneous degradation of multiple clients associated with different pathological hallmarks [2] . Inhibitors targeting the N-terminal ATP-binding site have been developed and some reached clinical trials [3] . However, they all showed problems due to the induction of the HSF1 mediated heat shock response and of Hsp70 overexpression, leading to drug resistance and toxicity [3a-c, 4] .A viable alternative to interfering with Hsp90 is represented by allosteric ligands, which perturb the chaperone by targeting sites alternative to the ATP-site. Novobiocin was shown to inhibit Hsp90 by binding the C-terminal region without inducing the heat shock response. Based on this observation, a number of new derivatives with promising activities against a variety of cancers were developed [5] .Correspondence to: Giorgio Colombo. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptIn this context, we have developed a method for the identification of allosteric pockets via the analysis of residue-pair distance fluctuations in the structural ensemble around the active state of the chaperone. Such analysis unveiled an allosteric pocket at 65Å from the active site, located at the MD:CTD border in a region overlapping with the client binding site [6] . This facilitated the design of modulators showing promising anticancer activities and a novel molecular mechanism of perturbation of Hsp90 functions: the ligands in fact proved to be activators of closure kinetics and ATPase of the chaperone in vitro, induce cancer cell death, and interfere with client maturation. We developed a first Quantitative-Structure-DynamicsActivity-Relationship (QSDAR) model correlating the structures of an initial set of modulators to observed activation effects [6c] .Here, based on this initial model, we report the rational design of new allosteric ligands, reaching low micromolar to nanomolar anticancer activities, which supports their potential in the development of anticancer therapeutics. On the computational side, we further develop a model to evaluate the potency of allosteric modulators by taking into account the dynamic cross-talk that exists between the protein and the ligand.The conform...

Designing Molecular Spanners to Throw in the Protein Networks

“…In the context of allosteric perturbation of PPIs, we characterized the effects of allosteric Hsp90 activators on its interactions in vitro, and on the stability of a number of its clients in cellular models: consistent with observations from other groups, administration of these accelerators induced a marked decrease in levels and viability of stringent client proteins of Hsp90 (for whose production Hsp90 is essential). This supports the concept that acceleration of conformational dynamics may in fact represent a new way of perturbing the chaperoning mechanisms that underlie cell viability: indeed, some of our allosteric compounds inhibit the proliferative potential of tumor cells, including those resistant to Hsp90 ATP‐competitive inhibitors …”

“…Comparative analyses of allosteric activators (Figure , left) and inhibitors (Figure , right) showed that the ligands induced different dynamics states and conformational ensembles in the protein, responsible for different functional responses . One of the Hsp90 activators is illustrated in Scheme (compound 26 ) …”

Allosteric Regulation of Hsp90α’s Activity by Small Molecules Targeting the Middle Domain of the Chaperone

“…Therefore, developing non-ATP competitive compounds targeting Hsp90 deserves to be tested. In fact, quite a few of research works published during past several years indicate that the allosteric modulators binding to either the N-terminal domain or the interface region in between the middle domain and the C-terminal domain of Hsp90 might serve as new potential therapeutic opportunities (Bassanini et al, 2018;D'Annessa et al, 2017;Ferraro et al, 2019;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2016;Zierer et al, 2014). Different from the working mode of the canonical ATP competitive inhibitors of Hsp90, the allosteric modulators target the non-canonical binding sites of the chaperone and induce activation of its ATPase activity (D'Annessa et al, 2017;Roe et al, 2018;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2014).…”

“…In fact, quite a few of research works published during past several years indicate that the allosteric modulators binding to either the N-terminal domain or the interface region in between the middle domain and the C-terminal domain of Hsp90 might serve as new potential therapeutic opportunities (Bassanini et al, 2018;D'Annessa et al, 2017;Ferraro et al, 2019;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2016;Zierer et al, 2014). Different from the working mode of the canonical ATP competitive inhibitors of Hsp90, the allosteric modulators target the non-canonical binding sites of the chaperone and induce activation of its ATPase activity (D'Annessa et al, 2017;Roe et al, 2018;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2014). Meanwhile, although presenting accelerating effect on Hsp90's ATP hydrolysis activity, the application of these allosteric modulators would down-regulate the chaperoning function of Hsp90 for client proteins and cause cytotoxicity (D'Annessa et al, 2017;Ferraro et al, 2019;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2014).…”

“…Multiple exogenous small molecules targeting the different structural domains of Hsp90 have been developed. They regulate Hsp90's function mainly via five ways: ATP competitive inhibitors, which block the access of ATP to the Hsp90's NTD (Prodromou, 2009;Roe et al, 1999;Sidera and Patsavoudi, 2014;Verma et al, 2016); inhibitors binding to the Hsp90's NTD and interfering with the interactions between Hsp90 and co-chaperones such as p23 and Cdc37 (Li et al, 2009(Li et al, , 2018Verma et al, 2016); compounds interacting with Hsp90's CTD and inhibiting its dimerization (Garg et al, 2016;Verma et al, 2016); allosteric activators binding to either the N-terminal domain or the interface region in between the middle domain and the C-terminal domain of Hsp90 (Bassanini et al, 2018;D'Annessa et al, 2017;Ferraro et al, 2019;Sattin et al, 2015;Yokoyama et al, 2015;Zierer et al, 2016;Zierer et al, 2014); and small molecules covalently bonding to the cysteine residue in the middle domain of Hsp90 (Li et al, 2016;Nakamoto et al, 2018;Zhang et al, 2018). Among the reported chemical compounds targeting Hsp90, ATP competitive inhibitors form a dominant group, and quite a few compounds from this group are undergoing clinical trials for the treatment of cancer (Nabi et al, 2018;Sidera and Patsavoudi, 2014;Tatokoro et al, 2015).…”