Discovery Of Selective Inhibitors Of Carbamoyl Phosphate Synthetase I (Cps1) To Modulate Cancer Relevant Metabolic Pathways.

Abstract Carbamoyl Phosphate Synthetase 1 (CPS1) catalyzes the first and rate-limiting step in ammonia detoxification through the urea cycle, generating carbamoyl phosphate from ammonia, bicarbonate and ATP. CPS1 is overexpressed in several cancers, including LKB1-deficient non-small cell lung carcinoma (NSCLC), where its activity has been proposed to support tumor growth by generating carbamoyl phosphate for use in pyrimidine synthesis. In other cancers, CPS1 has been reported to remove toxic ammonia from growing tumors to allow for sustained growth. Currently, no small molecule inhibitors of CPS1 have been identified, limiting researcher's ability to dissect the involvement of CPS1 function in cancer biology. We describe here the discovery of the first known small molecule inhibitors of CPS1. From a high throughput screen, we identify two chemical series, which achieve inhibition of CPS1 by blocking bicarbonate phosphorylation in the first step of carbamoyl phosphate synthesis. Biochemical experiments reveal inhibition of bicarbonate phosphorylation occurs through competition between inhibitor and ATP. High-resolution co-crystal structures demonstrate that these novel inhibitors bind to a previously unidentified allosteric pocket located between the integrating domain and carbamate synthetase domain of CPS1. This allosteric mechanism of action allows for highly selective CPS1 inhibition, with no inhibition of CPS2 observed. Analogs with increased potency were developed, which are able to inhibit CPS1 cellular activity, blocking both urea production and the CPS1-mediated pyrimidine biosynthetic pathway in cultured cells. These novel CPS1 inhibitors are valuable, first-in-class tools for probing CPS1 biology. We will also discuss the opportunity to utilize these novel inhibitors of CPS1, along with genetic approaches, to probe the functional roles of CPS1 in LKB1-deficient NSCLC. Citation Format: Shihua Yao, Tuong-Vi Nguyen, Alan Rolfe, Anant A. Agrawal, Jiyuan Ke, Shouyong Peng, Federico Colombo, Sean Yu, Patricia Bouchard, Jiayi Wu, Kuan-Chun Huang, Xingfeng Bao, Kiyoyuki Omoto, Anand Selvaraj, Lihua Yu, Stephanos Ioannidis, Frédéric H. Vaillancourt, Ping Zhu, Nicholas A. Larsen, David M. Bolduc. Discovery of selective inhibitors of carbamoyl phosphate synthetase I (CPS1) to modulate cancer relevant metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2334.