Hsp90 Inhibitors

Our Recent publications in the Hsp90 Field

Using NMR to identify binding regions for N and C-terminal Hsp90 inhibitors using Hsp90 domains Jeanette R. McConnell, H. Jane Dyson*, and Shelli R. McAlpine* RSC Med.Chem. V12, p410-415 2021 DOI.: 10.1039/D0MD00387E

We present the first NMR study of the interaction between heat shock protein 90 (Hsp90) and amino (N)-terminal inhibitors 17-AAG, and AUY922, and carboxy (C)-terminal modulators SM253, and LB51. We show that the two ATP mimics, 17-AAG and AUY922, bind deeply within the ATP binding pocket of the N-terminal domain, consistent with the crystal structures. In contrast, SM253, a C-terminal Hsp90 modulator, binds to the linker region between the N and middle domains. We also show that C-terminal inhibitor LB51 binds to the C-terminus with a more significant spectroscopic change than previously reported using NMR binding studies of C-terminal inhibitors novobiocin and silybin. These data provide key insights into how the allosteric inhibitor SM253 controls the C-terminal co-chaperones and confirms the binding domain of LB51.

C-terminal Hsp90 inhibitors block the HIF-1 hypoxic response by degrading HIF-1a through the oxygen-dependent degradation pathway Nalin Kataria, Chloe-Anne Martinez, Bernadette Kerr, Samantha S. Zaiter, Monica Morgan, Shelli R. McAlpine and Kristina M Cook* Cell. Physiol. Biochem V53, p480-495 2019 DOI: 10.33594/000000152.

Hypoxia Inducible Factor-1α (HIF-1α) is involved in cancer progression and is stabilized by the chaperone HSP90 (Heat Shock Protein 90), preventing degradation. Previously identified HSP90 inhibitors bind to the N-terminal pocket of HSP90, which blocks binding to HIF-1α and induces HIF-1α degradation. N-terminal inhibitors have failed in the clinic as single therapy treatments partially because they induce a heat shock response. SM molecules are HSP90 inhibitors that bind to the C-terminus of HSP90 and do not induce a heat shock response. The effects of these C-terminal inhibitors on HIF-1α are unreported. Results: We show that SM compounds decrease HIF-1α target expression at the mRNA and protein level under hypoxia in colorectal, breast and prostate cancer cells, leading to cell death, without inducing a heat shock response. Surprisingly, we found that when the C-terminal of HSP90 is inhibited, HIF-1α degradation occurs through the proteasome and prolyl hydroxylases in an oxygen-dependent manner even in very low levels of oxygen (tumor hypoxia levels). RACK1 was not required for proteasomal degradation of HIF-1α. Conclusion: Our results suggest that by targeting the C-terminus of HSP90 we can exploit the prolyl hydroxylase and proteasome pathway to induce HIF-1α degradation in hypoxic tumors.

Protein-protein inhibitors designed de-novo to target the C-terminus of Hsp90 block co-chaperone activity Marwa N. Rahimi and Shelli R. McAlpine* Chem. Commun. V55, p846-849, 2019 DOI: 10.1039/C8CC07576J

Protein-protein interactions control all cellular functions. Presented is the first de-novo designed protein-protein inhibitor that targets the C-terminus of Heat shock protein 90 (Hsp90) and blocks co-chaperones from binding. Compound LB76, which was created from an Hsp90 co-chaperone, selectively pulls down Hsp90 from cell lysates, binds to Hsp90’s C-terminal domain, and blocks the interactions between Hsp90 and TPR-containing co-chaperones. Through these interactions, LB76 inhibits the protein-folding function of Hsp90. Blocking these protein-protein interactions betweeen Hsp90 and C-terminal co-chaperones regulate the cell’s entire protein-folding machinery.

Hsp90 mediates membrane deformation and exosome release Elsa Lauwers,* Yu-Chung Wang, Rodrigo Gallardo, Rob Van der Kant, Emiel Michiels, Jef Swerts, Pieter Baatsen, Samantha S. Zaiter, Shelli R. McAlpine, Natalia V. Gounko, Frederic Rousseau, Joost Schymkowitz, and Patrik Verstreken* Molecular Cell V71, p689-702, e9 2018 DOI: 10.1016/j.molcel.2018.07.016

Hsp90 is an essential chaperone that guards proteome integrity and amounts to 2% of cellular protein. We now ﬁnd that Hsp90 also has the ability to directly interact with and deform membranes via an evolutionarily conserved amphipathic helix. Using a new cell-free system and in vivo measurements, we show this amphipathic helix allows exosome release by promoting the fusion of multivesicular bodies (MVBs) with the plasma membrane. We dissect the relationship between Hsp90 conformation and mem-brane-deforming function and show that mutations and drugs that stabilize the open Hsp90 dimer expose the helix and allow MVB fusion, while these effects are blocked by the closed state. Hence, we structurally separated the Hsp90 membrane-deform-ing function from its well-characterized chaperone activity, and we show that this previously unrecognized function is required for exosome release.

Synthesis and structure-activity relationships of inhibitors that target the C-terminal MEEVD on Heat shock protein 90 (Hsp90) Marwa N. Rahimi, Laura K. Buckton, Samantha S. Zaiter, Jessica Kho, Vickie Chan, Aldwin Guo, Jenane Konesan, SuHyeon Kwon, Lok K. O. Lam, Michael F. Lawler, Michael Leong, Gabriel D. Moldovan, David A. Neale, Gillian Thornton, and Shelli R. McAlpine* ACS Med. Chem. Lett. V9 p73-77 2018

Herein we describe the synthesis and structure-activity relationships of cyclic peptides designed to target heat shock protein 90 (Hsp90). Generating 19 compounds and evaluating their binding affinity reveals that increasing electrostatic interactions allows the compounds to bind more effectively with Hsp90 compared to the lead structure. Exchanging specific residues for lysine improves binding affinity for Hsp90, indicating some residues are not critical for interacting with the target, whereas others are essential. Replacing l- for d- amino acids produced compounds with decreased binding affinity compared to the parent structure, confirming the importance of conformation and identifying key residues most important for binding. Thus, a specific conformation and electrostatic interactions are required in order for these inhibitors to bind to Hsp90.

Redefining the phenotype of Heat shock protein 90 (Hsp90) inhibitors

Yao Wang, Yen Chin Koay, and Shelli R. McAlpine*Chem. Eur. J. V23 2010-2013 2017

The phenotypes produced when cells are treated with the Hsp90 inhibitors AUY922 or 17-AAG (classical inhibitors) are different to those produced when cells are knocked down with Hsp90a. Pull-down assays using classical inhibitors suggest that these molecules bind to multiple targets other than Hsp90. Classical inhibitors also induce similar protein markers as other anti-cancer therapies Cisplatin and bortezomib that do not target Hsp90. Together these data suggest that AUY922 and 17-AAG acts on multiple targets and likely kills cells through multiple mechanisms. Comparing these classical inhibitors to the effects seen when treating cells with C-terminal Hsp90 modulators reveals that C-terminal modulators effectively bind to Hsp90, and induce phenotypic markers consistent with the Hsp90a CRISPR knockdown data. Our findings challenge the current interpretation of Hsp90 inhibitors and suggest that a large body of literature that describes the Hsp90 phenotype and inhibitors is re-examined in this context.

How selective are Hsp90 inhibitors for cancer cells over normal cells?

Yao Wang, Yen Chin Koay, and Shelli R. McAlpine*ChemMedChem V12 p353-357 2017

Selectively inhibiting target proteins in cancer cells over normal cells is one of the most critical features of a successful protein inhibitor for clinical applications. By evaluating and comparing the impact of a clinical N-terminal heat shock protein 90 (Hsp90) inhibitor, AUY922 (luminespib), on Hsp90 inhibition-associated cellular events in cancer cells versus normal cells, we found that it produces similar phenotype characteristics in both cell types, indicating that AUY922 is not selective for targeting Hsp90 in tumor cells. By comparison, the C-terminal Hsp90 modulator SM258 suppresses cell proliferation, triggers apoptosis, regulates the expression of Hsp90-associated heat shock proteins, and enhances the degradation of Hsp90's client proteins preferentially in cancer cells over normal cells. Our findings support a new paradigm that AUY922 is not tumor selective, whereas SM258 is more selective and likely acts through an Hsp90-dependent mechanism.

Allosteric Modulators of Heat Shock Protein 90 (HSP90)

Yen Chin Koay and Shelli R. McAlpine * RSC Drug discovery series: “Allosterism in Drug Discovery” DOI:10.1039/9781782629276, p404-426

Three classes of allosteric inhibitors have been described in this chapter. The first were molecules targeting the ATP binding site at the N-terminus of HSP90 (classical inhibitors), while influencing clients that bind to the middle domain. These molecules are the only ones that have reached clinical trials, but they had significant problems including induction of pro-survival responses and dose-limiting toxicities. Thus, these classical inhibitors are being used as part of dual inhibition regiments in current clinical trials. Data was presented in this chapter to suggest that the poor results are also due to the classical inhibitor’s poor selectivity for HSP90 in a cellular environment. Indeed, knocking down HSP90 in cells produces a phenotype that is distinct from that reported with these classical inhibitors. The second class of molecules described are those that bind to the C-terminus (e.g., Novobiocin and KU-174), which have anti-cancer cellular activity without inducing high levels of HSF-1, HSP70, or HSP27. Although these compounds affected several clients that bound to the middle domain, efforts focused on improving the potency and pharmacokinetics of these molecules for in vivo efficacy.

The third class of compounds described are the SM series; these molecules bind between the N- and middle domains of HSP90 but affect the C-terminus. The SM molecules bind to the flexible charged linker region and stop the N-terminus from rotating, thereby blocking access to the C-terminal MEEVD region. By inhibiting access to the MEEVD region, the SM series blocks all co-chaperones with a TRP domain from binding to HSP90. Downstream effects attributed to blocking these co-chaperones produce a reduction of immunophilins FKBP52, and FKBP51, and hormone receptor levels. These SM molecules are also the first to be proven to target HSP90 in the cell. Specifically, as determined using pulldown assays with tagged variants of the molecules in multiple cell lysates, the compounds selectively pull out HSP90. Evaluating their efficacy in cells treated with siRNA for HSP90 we see

that the compounds are more effective when HSP90 is knocked down and less effective when HSP90 is over-expressed. Finally, cells treated with the SM compounds match the phenotype produced when the HSP90 alpha gene is knocked down. Thus, it is possible that there has not yet been a clinical evaluation of HSP90 inhibitors. However, as with all allosteric modulators, there are significant challenges associated with predicting active structures, and as such new direct C-terminal modulators are currently under evaluation.

Reinventing Hsp90 inhibitors: Blocking C-terminal binding events to Hsp90 using dimerized inhibitors

Yen Chin Koay, Hendra Wahyudi,and Shelli R. McAlpine*Chem. Eur. J. V22 p18572-18582 2016

Heat shock protein 90 (Hsp90) is a 90 kDa molecular chaperone that functions as a dimer. It facilitates the folding, assembly and stabilization of more than 400 proteins that are responsible for cancer development and progression. The reliance of the clients on Hsp90 has made it a promising anticancer target. Classical inhibitors that block the binding of ATP to the Hsp90’s N-terminus are highly toxic to cells and, trigger a resistance mechanism within cells. This resistance mechanism comprises of a large increase in the pro-survival heat shock proteins (HSF-1, Hsp70 and Hsp27). Molecules that modulate the C-terminus of Hsp90 are effective at inducing cancer cell death without activating the resistance mechanism. Herein we describe here the design, synthesis and biological binding affinity for a series of dimerized C-terminal Hsp90 modulators. We show that dimers of these C-terminal modulators synergistically inhibit Hsp90 compared to monomers.

A novel class of Hsp90 C-terminal modulators have preclinical efficacy in prostate tumor cells without induction of a heat shock response

Heather K. Armstrong, Yen Chin Koay, Swati Irani, Rajdeep Das, Zeyad D. Nassar, The Australian Prostate Cancer Bio resource, Luke A. Set, Margaret M. Centenera, Shelli R. McAlpine* and Lisa A. Butler* The Prostate V76, p1546-1559 2016

While there is compelling rationale to use heat shock protein 90 (Hsp90) inhibitors for treatment of advanced prostate cancer, agents that target the N-terminal ATP-binding site of Hsp90 have shown little clinical benefit. These N-terminal binding agents induce a heat shock response that activates compensatory HSP chaperones, which is believed to contribute in part to the agents’ lack of efficacy. Here, we describe the functional characterization of two novel agents, SM253 and SM258, that bind the N-middle linker region of Hsp90, resulting in reduced client protein activation and preventing C-terminal co-chaperones and client proteins from binding to Hsp90.SM253 and SM258 exhibit antiproliferative and pro-apoptotic activity in multiple prostate cancer cell lines (LNCaP, 22Rv1and PC-3) at low micromolar concentrations. Unlike the N-terminal inhibitors AUY922 and 17-AAG, these SM agents do not induce expression of Hsp27, Hsp40 or Hsp70, proteins that are characteristic of the heat shock response, in any of the prostate cell lines analyzed. Notably, SM258 significantly reduced proliferation within 2 days in human primary prostate tumors cultured ex vivo, without the significant induction of Hsp70 that was caused by AUY922 in the tissues. Our findings provide the first evidence of efficacy of this class of C-terminal modulators of Hsp90 in human prostate tumors, and indicate that further evaluation of these promising new agents is warranted.

The first report of direct inhibitors that target the C-terminus MEEVD region on heat shock protein 90

Laura K. Buckton, Hendra Wahyudi, and Shelli R. McAlpine* Chem. Commun. V52, p501-504 2016

Sixteen linear and cyclic peptides were designed de novo and synthesised. Protein binding data indicates that three compounds directly block acccess to heat shock protein 90 (hsp90)’s C-terminus. These molecules are valuable tools useful for investigating the impact of inhibiting hsp90 via a novel mechanism.

Hitting a moving target: How does an N-Methyl group impact biological activity?

Yen Chin Koay, Nicole L. Richardson, Samantha S. Zaiter, Jessica Kho, Sheena Y. Nguyen, Daniel H. Tran, Ka Wai Lee, Laura K. Buckton, and Shelli R. McAlpine* ChemMedChem. V11, p881-892 2016

Macrocycles have several advantages over small-moleculedrugs when it comes to addressing specific protein–protein interactionsas therapeutic targets. Herein we report the synthesisof seven new cyclic peptide molecules and their biologicalactivity. These macrocycles were designed to understand howmoving an N-methyl moiety around the peptide backbone impactsbiological activity. Because the lead non-methylated structure inhibits the oncogenic regulator heat-shock protein90 (Hsp90), two of the most potent analogues were evaluated for their Hsp90 inhibitory activity. We show that incorporating an N-methyl moiety controls the conformation of the macrocycle, which dramatically impacts cytotoxicity and binding affinity for Hsp90. Thus, the placement of an N-methylated amino acid within a macrocycle generates an unpredictable change to the compound’s conformation and hence biological activity.

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Hsp90 Inhibitors

Our Recent publications in the Hsp90 Field

Shelli R. McALpine, PhD., Visiting Professor at University of California, Irvine and Handling Editor Bioorganic Chemistry (Elsevier) mcalpine@uci.edu, www.coachingstemmacademics.com