Design, Synthesis, and Biological Evaluation of Conformationally Constrained cis-Amide Hsp90 Inhibitors

Abstract

Conformationally constrained CIS-amide chimeric inhibitors of Hsp90 have been synthesized and evaluated for their Hsp90 inhibitory activity. These new compounds exhibited Hsp90 ATPase inhibition and induced Hsp90-dependent client protein degradation in a dose-dependent manner. Biological data reported herein suggests that amide bond isomerization of geldanamycin derivatives plays an important role in affinity for the heteroprotein complex present in cancer cells.The 90 kDa heat shock proteins (Hsp90) are ATP-dependent molecular chaperones overexpressed in response to cellular stress and necessary for the folding and rematuration of nascent and denatured polypeptides, respectively. 1,2,3,4 Two natural products, geldanamycin 5 (GDA, Figure 1) and radicicol 6 (RDC), bind the Hsp90 N-terminal binding pocket and competitively inhibit ATP binding resulting in degradation of Hsp90-dependent client proteins via the ubiquitin-proteasome pathway. 7 Hsp90 clientele play key roles in all six hallmarks of cancer 8 , therefore inhibition of the Hsp90 protein folding machinery results in simultaneous disruption of all six mechanisms of oncogenesis. 9,10 Consequently, not only has Hsp90 emerged as a promising anti-cancer target 11 , but GDA and RDC have proven to represent excellent models for which the development of new Hsp90 inhibitors can be pursued for drug development and mechanistic studies. 12Although RDC and GDA bind Hsp90 with high affinity, their modes of binding and inhibitory activities are different. Radicicol exists in the same bent conformation when bound and unbound to Hsp90 and produces a favorable entropy of 8.3 cal/mol upon binding. Not surprisingly, the predisposition of RDC to the bent conformation is believed to be responsible for its similar activity in both cellular and recombinant assays. 13 E-mail: bblagg@ku.edu. Supporting Information Available Experimental procedures and characterization for all compounds. This material is avalible free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptOrg Lett. Author manuscript; available in PMC 2010 June 4. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptIn contrast to the bent, cis-amide conformation of GDA when bound to Hsp90, both solution and crystal structures have demonstrated that this natural product exists in an extended, trans-amide conformation when unbound to Hsp90 (Figure 2). 14 Multiple studies have demonstrated that prior to binding Hsp90, GDA must undergo two conformational changes; the ansa ring must rotate over the benzoquinone moiety and the amide bond must isomerize from trans to cis by rotation about C 1 -N 22 and C 20 -N 22 (Figure 2). The first event is reported to occur spontaneously; however isomerization of the amide bond is estimated to exceed 20 kcal/mol. 15 Accordingly, isothermal titration calorimetry (ITC) experiments have shown that GDA exhibits an entropic penalty of −6.4 cal/mol upon binding Hsp90. 16,17As a consequence of these thermodynamic data, Santi and co...

A Mechanistic and Structural Analysis of the Inhibition of the 90-kDa Heat Shock Protein by the Benzoquinone and Hydroquinone Ansamycins

“…However, a recent study concluded that no isomerization step occurs in the binding of the benzoquinone ansamycins to Hsp90 (Onuoha et al, 2007) and proposed that small quantities of the cis-amide isomer in solution, identified in an NMR study (Thepchatri et al, 2007), may bind directly to Hsp90, and in vivo potency could be due to the direct binding of the hydroquinone of the cis-amide isomer. This counters the results of a study examining the effects of conformationally constrained cis-amide chimeric inhibitors of Hsp90 (Duerfeldt et al, 2009) and our molecular docking studies in which the cis-amide isomer would not dock into NQO1. However, the results of a binding affinity study using BODIPY-geldanamycin in the absence and presence of reducing agents reported by Onuoha et al may, in fact, support Hsp90-mediated isomerization.…”

“…The intrinsic structural properties of the ansamycin ring, the anchoring of the carbamate through interaction with Asp90 at the base of the nucleotide-binding domain, and the interaction of the ansa ring with amino acid residues via through-bond interactions with the specific solvent network in the nucleotide-binding domain may also aid to drive trans-cis isomerization (Pappenberger et al, 2001). The confirmation of Hsp90-mediated trans-cis isomerization of the benzoquinone and hydroquinone ansamycins is important because it could be exploited in the development of new Hsp90 inhibitors, and the effect of conformationally restrained cis-amide isomers of benzoquinone ansamycin analogs on Hsp90 inhibition has been reported (Duerfeldt et al, 2009).…”

Targeting the unfolded protein response for disease intervention

“…Geldanamycin may have adverse effects due to the reactive epoxide moiety and other adverse structural features [72]. A chimeric structure containing quinone moiety from geldanamycin and resorcinol from radicicol gave rise to radamide, a molecule with higher affinity for GRP94 and antiproliferative activities on various cancer cell lines [73,74]. The flavonol apigenin also led to a reduction in GRP94 expression in ER stressed cells [58].…”

Structure‐Based and in silico Design of Hsp90 Inhibitors

“…These molecules show low-micromolar inhibitory activity against purified Hsp90 (1 .5 mm, ATPase assay) and a~10fold increase in cellular activities against MCF7 and SKBr3 cancer cells with respect to the lead compound, radamide. [39] In a subsequent study, Lee and collaborators used quantum chemical calculations and mutagenesis experiments to propose that the trans/cis isomerization of GA was induced by Hsp90, and identified Ser 113 as a key residue involved in the catalysis of this conversion. [40] Conversely, using ITC binding assays Onuoha et al did not observe a dramatic reduction in binding affinity for the S113A mutant with respect to the wild-type protein, suggesting that Hsp90 does not catalyze the ligand isomerization.…”