Development of Glucose Regulated Protein 94-Selective Inhibitors Based on the BnIm and Radamide Scaffold

Abstract

Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum resident of the heat shock protein 90 kDa (Hsp90) family of molecular chaperones. Grp94 associates with many proteins involved in cell adhesion and signaling, including integrins, Toll-like receptors, immunoglobulins, and mutant myocilin. Grp94 has been implicated as a target for several therapeutic areas including glaucoma, cancer metastasis, and multiple myeloma. While 85% identical to other Hsp90 isoforms, the N-terminal ATP-binding site of Grp94 possesses a unique hydrophobic pocket that was used to design isoform-selective inhibitors. Incorporation of a cis-amide bioisostere into the radamide scaffold led to development of the original Grp94-selective inhibitor, BnIm. Structure–activity relationship studies have now been performed on the aryl side chain of BnIm, which resulted in improved analogues that exhibit better potency and selectivity for Grp94. These analogues also manifest superior antimigratory activity in a metastasis model as well as enhanced mutant myocilin degradation in a glaucoma model compared to BnIm.

Second Generation Grp94‐Selective Inhibitors Provide Opportunities for the Inhibition of Metastatic Cancer

“…The resorcinol ring anchored within the N-terminal ATP-binding site through direct interactions with Asp149 and Thr245 and a hydrogen bonding network with water molecules (Figrue 3c and 3g). [11–12] Interestingly, both inhibitors were best modeled via two partially occupied binding modes. The first, termed “benzyl in”, aligns with the previously observed binding mode of the first generation Grp94-selective inhibitor, KUNG38, where the benzylic side chain is oriented towards the Grp94-exclusive binding pocket.…”

“…The first, termed “benzyl in”, aligns with the previously observed binding mode of the first generation Grp94-selective inhibitor, KUNG38, where the benzylic side chain is oriented towards the Grp94-exclusive binding pocket. [11] When crystallized with 6 , Grp94 loop residues 167–170, which make up a portion of the aforementioned unique binding region, appeared disordered, suggesting that this interaction was not stabilizing this loop (Figure 3a). Alternatively, when crystallized with 30 , the main chain of these loop residues could be built into visible electron density, suggesting that the hydrophobic interactions mediated by the fluoride substituent stabilized these residues, leading to slightly improved Grp94 binding affinity (Figure 3e).…”

“…[11] Purified NΔ41 protein was exchanged into 100 mM bicine buffer pH 7.8 and concentrated to 30 mg/mL; protein concentration was determined by Bradford assay (Amresco), with BSA used as a calibration standard. Apo NΔ41 crystals were grown using the sitting drop vapor diffusion method.…”

“…[10] Subsequent structure-activity relationship studies on the benzyl side chain of 2 resulted in the development of KUNG29 ( 3 ), which exhibited improved affinity and selectivity for Grp94 as compared to 2 (Figure 1). [11] Interestingly, substitutions at the 2-position of the benzyl side chain ( 3 – 5 ) manifested the greatest Grp94-selectivity (Figure 1), and suggested that projection of the side chain into this region could improve selectivity. Analysis of the co-crystal structure of KUNG38 bound to Grp94 suggested that modification of the cis -amide bioisostere (imidazole) could orient the side chain to mimic substitutions present in 3–5 and increase both selectivity and affinity.…”

HSP90 inhibitors in the context of heat shock and the unfolded protein response: effects on a primary canine pulmonary adenocarcinoma cell line*

“…Notably, heat shock protein 90 (HSP90, HSP90A family) and its ER paralog glucose regulated protein 94 (GRP94, HSP90B1) are considered high-value targets for pharmacologic development [2, 12–18]. Both of these proteins are important players in the heat shock and unfolded protein responses; their upregulated expression in tumors begs the question, are these proteins viable targets in stressed tumors?…”