A ‘bump-&-hole’ approach for engineering allele-selective inhibition of the BET bromodomains
Chemical tools can be used to investigate the function of proteins and their importance to biological processes and disease. This approach has many advantages over classical genetic tools, but requires compounds with high selectivity for their targets. When single-target selectivity cannot be achieved through normal means it can be engineered through the development of orthogonal protein:ligand pairs, wherein target proteins are mutated and an existing ligand is modified to become mutant-selective. An ideal model for this engineered, allele-specific chemical genetic work is the BET family of bromodomains – 8 closely related, and structurally conserved, epigenetic acetyl-lysine reader domains that so far has defied single-bromodomain inhibition. The Alessio Ciulli lab has previously designed a proof-of-concept ‘bump-&-hole’ system to selectively target these bromodomains by mutating a conserved leucine to an alanine (L/A mutation) and adding an ethyl bump to an existing inhibitor scaffold (ET compound). This system was promising, but required optimisation of the mutation and modified compound before it could be used to answer biological questions.Here the L/A mutation is replaced with a more conservative leucine/valine (L/V) mutation, which is shown to be significantly more functional with regards to acetylated chromatin binding and downstream gene expression. Simultaneously, a large number of new modifications were incorporated into a library of bumped compounds, which were then screened for their selectivity for L/V BET bromodomains (over WT). This lead to the selection of ‘9-ME-1’ as the preferred L/V-selective compound. This optimised BET bump-&-hole system was validated through a number of cellular assays. By repurposing some of these cellular assays the bump-&-hole system was then used to answer biological questions – specifically the relative importance of different BET bromodomains to BET protein function. These results, in connection to recent advances in the literature, provides evidence for a new model of how the BET bromodomains work together to drive downstream gene expression.Finally, the need to use CRISPR/Cas9 gene-editing technology to generate L/V-mutant cell-lines was identified, and an attempt was made to knock-in the L/V mutation to the first bromodomain of BRD4 in a cancer cell-line.
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