The bromodomain and extra-terminal domain (BET) family of proteins, bromodomain-containing (BRD)2, 3, 4 and T, are a class of epigenetic regulators that have been implicated in a number of diseases but most significantly cancer. Despite concentrated efforts in the development of BET inhibitors with a significant number currently in clinical trials,1 the understanding of how the BET-family of proteins regulate gene expression is not well established. In order to delineate some of the molecular mechanisms by which these proteins function we have been studying the ET domain of BRD3.2
Recently we have applied a fragment-based approach to develop inhibitors of BRD3-ET, utilising our in house REFiL (Rapid Elaboration of Fragments into Leads) technique. This strategy involves a computational analysis of validated fragment hits to select commercially available analogues for analysis. These enable suitable positions (vectors) on a hit fragment to be identified for chemical elaboration. Each vector is investigated by generating diverse libraries of elaborated compounds using parallel microscale synthesis. These are evaluated following minimal chemical work-up using Off-Rate Screening by SPR to identify higher affinity binders.3 Examination of the kinetics in the dissociation phase of the SPR data enabled the rapid identification of potential leads, from an initial hit. Reagent sets were designed that enabled the most efficient coverage of chemical space for each chemical transformation, and solution phase parallel synthesis in 96 well plates was developed to generate the library. This approach enabled us to improve binding affinity by > 30-fold in 2 rounds of screening from a single vector.
The work presented will demonstrate REFiL as applied to BRD3-ET and how this strategy could be applied to a number of proteins for an efficient method of fragment evolution to drug leads.