Diving into the Water: Inducible Binding Conformations for BRD4, TAF1(2), BRD9, and CECR2 Bromodomains

Abstract

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.

Large-scale analysis of water stability in bromodomain binding pockets with grand canonical Monte Carlo

“…Supplementary Figure 6 ranks bromodomains by the stability of the combinations of hydration sites that are expected to be most easily targetable with a single ligand (displacement of W1 + W2, W3 + W4, W1 + W2 + W3, and W2 + W3 + W4), and can be interpreted as it was done here for the whole network and the individual sites. It is interesting to compare the water stabilities calculated here with the work of Crawford et al 16 . The authors systematically modified the size of the aliphatic side chain in a 6-methyl pyrrolopyridone ligand (compound 2), targeting the conserved water network, and studied how this affected the affinity of the ligand for eight different bromodomains across five families.…”

“…The authors found that two different substituents could displace W3 and W4 from their canonical location in BRD4(1) and TAF1 (2). Specifically, replacing the methyl with a trans-crotyl (compound 4 in Crawford et al 16 ) or with 1-butene moiety (compound 5) resulted in the displacement of W3 and W4 from BRD4(1) and TAF1(2) (PDB-IDs 5I88 and 5I1Q). The authors studied the effect of these substitutions on ligand affinity also in BRD4(2), CREBBP, BRPF1B, BRD9, TAF1(1), and CECR(2).…”

“…The authors studied the effect of these substitutions on ligand affinity also in BRD4(2), CREBBP, BRPF1B, BRD9, TAF1(1), and CECR(2). However, it was shown how the longer aliphatic side chain is accommodated into the pocket without displacing any water in BRD9 and CECR2 16,32 . Extending the methyl group by three more carbon atoms generally resulted in different degrees of affinity loss for the six bromodomains in which displacement took place, which can be related to the free energy penalty associated with the displacement of W3 and W4 (-ΔG W3 -ΔG W4 ).…”

“…Extending the methyl group by three more carbon atoms generally resulted in different degrees of affinity loss for the six bromodomains in which displacement took place, which can be related to the free energy penalty associated with the displacement of W3 and W4 (-ΔG W3 -ΔG W4 ). To attempt a quantitative comparison to the data in Crawford et al 16 , we derive approximate binding free energies from the half-maximal inhibitory concentration values and assume an equal affinity (20 μM) for all measurements over the limit of detection of 20 μM. In such a way, we can directly compare the loss of affinity of ligands 4 and 5 over ligand 2 (ΔΔG binding ) to the free energy penalty for the displacement of W3 and W4 (-ΔG W3 -ΔG W4 ).…”

“…Cox et al 14 noticed the displacement of W1 from the binding pocket of PHIP(2) (in Family III) by a thiourea fragment, while Zhu and Caflisch 15 reported the displacement of W1 from BRPF1 (a member of Family IV) by an isoquinolinone fragment. Recently, Crawford et al 16 described the discovery of ligands causing a rearrangement of the water network in BRD4(1) and TAF1 (2) and the displacement of W3 and W4. The same behavior of TAF1 (2) was previously observed also by Flynn et al 17 while investigating bromodomain recognition of butyryllysine and crotonyllysine.…”