Preclinical studies of RA475, a guanidine-substituted spirocyclic candidate RPN13/ADRM1 inhibitor for treatment of ovarian cancer.

There is an urgent unmet need for more targeted and effective treatments for advanced epithelial ovarian cancer (EOC). The emergence of drug resistance is a particular challenge, but small molecule covalent inhibitors have promise for difficult targets and appear less prone to resistance. Michael acceptors are covalent inhibitors that form bonds with cysteines or other nucleophilic residues in the target protein. However, many are categorized as pan-assay interference compounds (PAINS) and considered unsuitable as drugs due to their tendency to react non-specifically. Targeting RPN13/ADRM1-mediated substrate recognition and deubiquitination by the proteasome 19S Regulatory Particle (RP) is a promising treatment strategy. Early candidate RPN13 inhibitors (iRPN13) produced a toxic accumulation of very high molecular weight polyubiquitinated substrates, resulting in therapeutic activity in mice bearing liquid or solid tumor models, including ovarian cancer; however, they were not drug-like (PAINS) because of their central piperidone core. Up284 instead has a central spiro-carbon ring. We hypothesized that adding a guanidine moiety to the central ring nitrogen of Up284 would produce a compound, RA475, with improved drug-like properties and therapeutic activity in murine models of ovarian cancer. RA475 produced a rapid accumulation of high molecular polyubiquitinated proteins in cancer cell lines associated with apoptosis, similar to Up284 although it was 3-fold less cytotoxic. RA475 competed binding of biotinylated Up284 to RPN13. RA475 shows improved solubility and distinct pharmacodynamic properties compared to Up284. Specifically, tetraubiquitin firefly luciferase expressed in leg muscle was stabilized in mice more effectively upon IP treatment with RA475 than with Up284. However, pharmacologic analysis showed that RA475 was more rapidly cleared from the circulation, and less orally available than Up284. RA475 shows reduced ability to cross the blood-brain barrier and in vitro inhibition of HERG. Treatment of mice with RA475 profoundly inhibited the intraperitoneal growth of the ID8-luciferase ovarian tumor model. Likewise, RA475 treatment of immunocompetent mice inhibited the growth of spontaneous genetically-engineered peritoneal tumor, as did weekly cisplatin dosing. The combination of RA475 and cisplatin significantly extended survival compared to individual treatments, consistent with synergistic cytotoxicity in vitro. In sum, RA475 is a promising candidate covalent RPN13i with potential utility for treatment of patients with advanced EOC in combination with cisplatin.
Competing Interests: RA475 is described in the Johns Hopkins University technology C16365 - ANTI-CANCER “SPIROCYCLIC-GUANIDINE COMPOUNDS” AND USES THERE OF, provisional application filed on May 18, 2020 as U.S. Patent Application No. 63/026,268, inventors Roden RB and Anchoori RK. The study described in this publication utilized compounds manufactured by Up Therapeutics LLC (MTA A36130) and covered by US patent US20190365726 and application 17/154,306. Dr. Roden is a consultant to Up Therapeutics LLC and owns stock in the company. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. Dr. Anchoori is the founder of Up Therapeutics LLC and owns stock in the company. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. Michelle Rudek reports a relationship with Geminus Therapeutics, LLC, that includes co-founder, board membership, and equity or stocks. Michelle Rudek reports a relationship with GlaxoSmithKline that includes employment (spouse). This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict of interest policies. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
(Copyright: © 2024 Anchoori et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)