Development Of A Screening Cascade To Identify Selective Small Molecule Inhibitors Of Dnmt1

DNA Methyltransferases (or DNMTs) play an important role in genomic integrity. The DNMT family of enzymes catalyse the transfer of methyl groups to specific CpG structures on DNA, with DNMT1 acting as a maintenance methyltransferase and DNMT3a and 3b acting as de novo methyltransferases. Upon cell division, epigenetic profiles within DNA strands are copied, with DNMT1 ensuring that methyl marks are transferred correctly to the newly synthesised DNA strand, thereby conferring heritability. Aberrant DNA hypermethylation within DNA promoter regions can lead to gene silencing, which is a hallmark of human cancer. Treatment with hypomethylating agents such as azacytidine and decitabine reverse methyl marks and show clinical benefit for the treatment of Acute Myeloid Leukemia (AML) and Chronic Myelomonocytic Leukemia (CMML) and are also the standard of care for Myelodysplastic Syndrome (MDS). However, these agents also show dose limiting toxicity due to their irreversible and non-selective mechanism of action and generally poor pharmacokinetic properties. Over the past decades both the Pharma and Biotech industries have deployed significant resource in a bid to identify and develop potent, selective DNMT1 inhibitors. These attempts have delivered little, if any, success until now. Collaboration between the CRUK Manchester Institute and GSK has resulted in the successful development of a screening cascade to identify compounds that selectively inhibit DNMT1. A collection of over 1 million compounds was screened at GSK using a radioactive scintillation proximity assay (SPA). Preliminary hits were identified and triaged through our screening cascade to determine genuine hits from false positive results. Firstly, ‘hits9 from the SPA screen were tested in rapid-fire mass spectrometry and also in fluorescence intensity coupled ‘break-light9 assays. Though these orthogonal screens apparently confirmed activity, IC50 screening of many of these compounds revealed high Hill slopes and high maximal inhibition profiles which are indicative of non-specific inhibition mechanisms. Detailed mechanism of inhibition studies alongside differential scanning fluorimetry was employed to rule out those compounds that were binding non-specifically to the oligonucleotide substrate. One series of compounds displayed IC50 profiles consistent with specific inhibition of DNMT1, with a 1:1 stoichiometry and no evidence for non-specific binding to the oligonucleotide substrate. Selectivity screening against DNMT3a and 3b also showed that within the DNMT family this series of compounds was selective for DNMT1 only. All compounds that displayed reasonable biochemical potency, (sub 1µΜ), were tested further in cellular assays to confirm target engagement. Through our extensive range of assays and thorough profiling of hit compounds, we were able to quickly de-validate the significant number of false positive hits that have plagued previous drug discovery screens in this area and identify a series of genuine, selective, small molecule inhibitors of DNMT1. Citation Format: Alexandra Stowell, Graeme Thomson, Mark Cockerill, Charlotte Burt, Emma Fairweather, Ian Waddell, Ali Raoof, Allan Jordan, Donald 0gilvie, Melissa Pappalardi, Juan Luengo, Mehul Patel, Ryan Kruger, Chris Carpenter. Development of a screening cascade to identify selective small molecule inhibitors of DNMT1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-068.