Covalent Rpn13-Binding Inhibitors for the Treatment of Ovarian Cancer

Abstract

Substitution of the m,p-chloro groups of bis-benzylidinepiperidone RA190 for p-nitro, generating RA183, enhanced covalent drug binding to Cys88 of RPN13. Treatment of cancer cell lines with RA183 inhibited ubiquitin-mediated protein degradation, resulting in rapid accumulation of high-molecular-weight polyubiquitinated proteins, blockade of NFκB signaling, endoplasmic reticulum stress, an unfolded protein response, production of reactive oxygen species, and apoptotic cell death. High-grade ovarian cancer, triple-negative breast cancer, and multiple myeloma cell lines were particularly vulnerable to RA183. RA183 stabilized a tetraubiquitin-linked firefly luciferase reporter protein in cancer cell lines and mice, demonstrating in vitro and in vivo proteasomal inhibition, respectively. However, RA183 was rapidly cleared from plasma, likely reflecting its rapid degradation to the active compound RA9, as seen in human liver microsomes. Intraperitoneal administration of RA183 inhibited proteasome function and orthotopic tumor growth in mice bearing human ovarian cancer model ES2-luc ascites or syngeneic ID8-luc tumor.

Structure-function analyses of candidate small molecule RPN13 inhibitors with antitumor properties

“…The flexibility of the core unit allows numerous modifications around the pharmacophore without disturbing target specificity. Building upon our previous findings with our RA-chemical series [10,13,[43][44][45], and related inhibitors [19], herein we have identified important structure-activity relationships and analogs with a second warhead (RA371 and RA375) were identified as having increased potency. It is noteworthy that the related RPN13 inhibitors CLEFMA and EF24 have also demonstrated therapeutic activity against preclinical cancer models via related mechanisms [19].…”

“…This study provides insight into the structural requirements necessary for drug binding to RPN13, presumably by adducting Cys-88 [6,12,13], and informs design of molecules more potent than our prototype RA190 [10]. The flexibility of the core unit allows numerous modifications around the pharmacophore without disturbing target specificity.…”

“…Although binding to UCH37 can also occur in vitro [6], several lines of evidence including cell labeling [10], degrader [11] and knockout studies [12] suggest that RPN13 is RA190's principle cellular target. RA190 and the related RA183 [13] exhibit antineoplastic activity, including against bortezomib-resistant MM, and against solid tumors in pre-clinical models of ovarian cancer [10,13,14], human papillomavirus (HPV)-associated and several other solid cancers [6,12,[15][16][17][18]. Like RA190 and RA183 [13], the diphenyldihaloketones CLEFMA and EF-24 also adduct to RPN13 and inhibit proteasome function [19], and herein we seek to further understand the pharmacophore.…”

“…RA190 and the related RA183 [13] exhibit antineoplastic activity, including against bortezomib-resistant MM, and against solid tumors in pre-clinical models of ovarian cancer [10,13,14], human papillomavirus (HPV)-associated and several other solid cancers [6,12,[15][16][17][18]. Like RA190 and RA183 [13], the diphenyldihaloketones CLEFMA and EF-24 also adduct to RPN13 and inhibit proteasome function [19], and herein we seek to further understand the pharmacophore. Ovarian cancer is a promising target because RPN13, which is encoded by ADRM1, is consistently over-expressed in high grade serous carcinoma, and this occurs in the precursor lesion [17].…”

Small‐Molecule Inhibitors of the Proteasome's Regulatory Particle

“…Changes in RA190’s structure, such as the introduction of electron‐withdrawing p ‐nitro groups in compound RA183 (Table , below), have enhanced potency and binding to Rpn‐13’s Cys88 residue. Despite this enhancement, RA183 was still shown to be nonselective and also bound to Rpn‐13’s DEUBAD domain, as well as to DUBs ubiquitin‐specific protease 14 (Usp14) and Uch37 …”