Protein arginine methyltransferase 5 (PRMT5) is known to symmetrically dimethylate numerous cytosolic and nuclear proteins that are involved in a variety of cellular processes. Recent findings have revealed its potential as a cancer therapeutic target. PRMT5 possesses a cysteine (C449) in the active site, unique to PRMT5. Therefore, covalent PRMT5 inhibition is an attractive chemical approach. Herein, we report an exciting discovery of a series of novel hemiaminals that under physiological conditions can be converted to aldehydes and react with C449 to form covalent adducts, which presumably undergo an unprecedented elimination to form the thiol-vinyl ethers, as indicated by electron density in the co-crystal structure of the PRMT5/MEP50 complex.

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The synthesis and optimization of a series of orally bioavailable 1-(1H-indol-4-yl)-3,5-disubstituted benzene analogues as antimitotic agents are described. A functionalized dibromobenzene intermediate was used as a key scaffold, which when modified by sequential Suzuki coupling and Buchwald-Hartwig amination provided a flexible entry to 1,3,5-trisubstituted phenyl compounds. A 1H-indol-4-yl moiety at the 1-position was determined to be a critical feature for optimal potency. The compounds have been shown to induce cell cycle arrest at the G2/M phase and demonstrate efficacy in both cell viability and cell proliferation assays. The primary site of action for these agents is revealed by their colchicine competitive inhibition of tubulin polymerization, and a computational model has been developed for the association of these compounds to tubulin. An optimized lead LP-261 significantly inhibits growth of a human non-small-cell lung tumor (NCI-H522) in a mouse xenograft model.

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Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMT that catalyzes the formation of symmetrical dimethyl arginine (SDMA) on protein substrates and plays important roles in various cellular pathways including tumorigenesis. PRMT5 can methylate histones H3 and H4, thereby leading to repression of tumor suppressor genes at specific loci. PRMT5 also regulates gene expression on a global level by methylating and altering the function of transcription factors such as p53, E2F-1, NF-κB and others. Additionally, PRMT5 has been shown to interact with members of the spliceosome complex and regulate pre-mRNA processing of key target genes. In this study, we describe the in vitro and in vivo activity of PRT543, a novel, potent and selective inhibitor of PRMT5, in hematological and solid tumor models. In a scintillation proximity based radiometric assay, PRT543 inhibited the methyltransferase activity of the PRMT5/MEP50 complex with an IC50 of 10.8 nM. It was also highly selective against a panel of 36 other methyltransferases. PRT543 robustly inhibited cell proliferation in a panel of >50 cancer cell lines, representing both solid tumors and cancers of hematological origin, in a concentration-dependent manner with IC50 values ranging from 10 - 1000 nM. This anti-proliferative activity correlated with a reduction of symmetrically dimethylated SmD3 protein, a known PRMT5 substrate, demonstrating effective inhibition of PRMT5 enzymatic activity in cells. PRT543 demonstrated good oral bioavailability and favorable pharmacokinetic properties. Oral administration of this compound led to significant tumor growth inhibition in several subcutaneous mouse xenograft models including mantle cell lymphoma (Granta-519, Z-138), acute myeloid leukemia (MV4-11, SET2, HEL), small cell lung cancer (NCI-H1048) and bladder cancer (5637) at well-tolerated doses. This in vivo activity was accompanied by a corresponding decrease in symmetrically dimethylated SmD3 in tumor tissue collected from the treated animals. PRT543 was also tested in combination with other approved targeted therapies both in vitro and in vivo. Combining PRT543 with the BCL2 inhibitor, venetoclax, revealed a potent synergistic interaction in models of leukemia and lymphoma. Further ex vivo testing in genetically-defined primary patient tumor samples is ongoing. PRT543 is currently under evaluation in a Phase I clinical trial in patients with advanced solid tumors and hematological malignancies (NCT03886831). Citation Format: Neha Bhagwat, Yang Zhang, Hong Lin, Min Wang, Dave Rominger, Tom Emm, Divya Chugani-Mahtani, Dimitrios Angelis, Rupa Shetty, Raul Leal, William Gowen-MacDonald, Alexander Grego, Juan Luengo, Taghi Manshouri, Friederike Pastore, Ross L. Levine, Srdan Verstovsek, Bruce Ruggeri, Peggy Scherle, Kris Vaddi. Preclinical characterization of PRT543, a potent and selective inhibitor of protein arginine methyltransferase 5 (PRMT5), with broad antitumor activity in in vitro and in vivo models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2915.