BACR Roger Griffin Prize for Cancer Drug Discovery

Engineering potency and selectivity of chemical probes for functional elucidation and target validation

Matthias Baud, University of Southampton, UK

Designing selective chemical probes is crucial for accurate functional elucidation and target validation in disease. However, this can become a daunting task when the target is part of a family of structurally related proteins. One example of such a family of proteins includes Bromo and Extra-Terminal (BET) proteins brd2, brd3, brd4 and brdt, which are important transcriptional co-regulators modulating gene expression in the nucleus. Key to their activity are small modular domains called bromodomains that recognise and interact with acetylated lysine residues from chromatin. The eight BET bromodomains (two per BET protein) have attracted particular attention lately for drug development. Disregulation of their transcriptional activity has been linked to a number of aggressive cancers that are usually associated with relatively poor prognosis, such as NUT midline carcinoma, multiple myeloma, mixed-lineage leukemia and myeloid leukaemia. These findings have fuelled the interest of medicinal chemists for developing new routes towards synthetic and potent small molecules modulating the activity of BET bromodomains by disrupting their interaction with their chromatin substrates. This eventually resulted in the development of a range of small molecules showing efficacy against several cancers. A number of such compounds are currently evaluated in the clinic as anticancer agents. Despite their promising
pharmacologies, these molecules bind to all BET bromodomains rather unspecifically in cells and all past attempts at modulating an individual BET bromodomain with a small molecule proved largely unsuccessful. This in turn hampered accurate functional elucidation of individual BET bromodomains, making it unclear which of these domains should be the ideal target of further medicinal chemistry efforts.

We recently developed a chemical genetics approach called “bump-and-hole”, aimed at delivering a new tool that would allow us to selectively modulate an individual BET bromodomains with a small molecule. This approach is based on the generation of orthogonal and high-affinity protein/ligand pairs and involves introducing a single point mutation (the “hole”) onto the BET bromodomain of interest together with making a synthetic modification (the “bump”) onto the parent BET bromodomain binder to complement a newly created protein subpocket. We discovered that compound “ET”, a derivative of I-BET762, was able to target engineered bromodomains bearing a leucine to alanine (L/A) mutation in their Z/A loop with very high potency (< 100nM) and selectivity (up to 500 fold) both in vitro and in cells. This orthogonal protein/ligand pair provided the first chemical tool that allows controlling a single BET bromodomain while leaving the remaining seven domains unaffected.

Following-up on these results, we are currently using our approach to gain deeper understanding of the biological function of BET proteins, which we expect will further shed light on their potential as targets in oncology. We also anticipate that this approach will
be of benefit to address a wide range of other protein targets where chemical probes development has been hampered by selectivity and potency issues.