Design and characterisation of tool inhibitors of DNA damage response proteins

As a result of the high cost of drug discovery, it is imperative that promising targets with strong disease association are identified and validated before embarking on costly molecule discovery and development phases. Chemical inhibitors provide an excellent tool for target validation. This project was initiated with the aim of designing and characterising tool inhibitors of proteins involved in the DNA damage response, to enable detailed mechanistic biological investigation, disease validation and initiation of translational drug discovery projects. The SMC complexes are critically important in coordinating chromosome condensation, sister chromatid cohesion, DNA repair, homologous recombination and transcriptional regulation. They represent interesting oncology targets and are compelling targets for tool inhibitor development. The core SMC proteins and NSE subunits of SMC5/6 and cohesin SMC1 and SMC3 were modelled and their druggabilities assessed, with an aim to advance to inhibitor development for any of the proteins that proved druggable. Unfortunately, the SMC5/6 and cohesin complexes were established as not druggable. BAF180 represents a major clear cell renal cell carcinoma (ccRCC) cancer gene, exhibiting truncating mutations in 41% of samples in a series of primary ccRCCs.1 It has therefore been highlighted as a promising opportunity to target ccRCC using a synthetic lethal therapeutic approach.2 Hopkins et al. utilised a novel screening technique to identify several genes that are synthetic lethal with BAF180.3 In this work, the druggabilities of three of these genes were assessed, and KAT2A was identified as a promising target for tool inhibitor development. Druggable pockets were identified at the KAT2A histone acetyltransferase (HAT) domain and bromodomain. The catalytically active HAT domain was prioritised as the preferred target. Potential KAT2A HAT domain inhibitors were available from a high-throughput screen (HTS), which utilised a fluorescence based activity assay.4,5 Unfortunately, this assay format proved prohibitively unreliable, and moreover, after discounting the hits from the HTS as likely false positives, the inventory of KAT2A inhibitors was quickly exhausted and the target abandoned. Focus turned to the KAT2A bromodomain. Available X-ray crystal structures were utilised in a computational drug design effort, and Tm shift, TR-FRET, ITC and X-ray crystallography techniques were optimised and established in-house to enable characterisation of prospective KAT2A tool inhibitors. Over 3000 small molecules and fragments were screened, and an assortment of novel KAT2A bromodomain binders were identified. The optimised assays, novel chemical matter and ligand-bound crystal structures afford an exciting opportunity to develop potent and selective KAT2A bromodomain tool inhibitors.