Optimization of a "bump-and-hole" approach to allele-selective BET bromodomain inhibition.

Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins - BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound - 9-ME-1 - shows ∼200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression, likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.

Concordant mRNA expression of UCP-3, but not UCP-2, with mitochondrial thioesterase-1 in brown adipose tissue and skeletal muscle in db/db diabetic mice.

A recent hypothesis concerning the function of uncoupling protein-3 (UCP-3) depends upon a positive relationship with mitochondrial thioesterase (MTE-1) in situations where fatty acid beta-oxidation is increased. MTE-1 mRNA levels are raised in transgenic mice overexpressing UCP-3 in skeletal muscle and we sought to extend these findings by quantifying in vivo expression of endogenous MTE-1, UCP-1, UCP-2, and UCP-3 mRNA levels in white adipose tissue, interscapular brown adipose tissue, and skeletal muscle in db/db mice. In this study we show that changes in MTE-1 mRNA levels as a result of differences between db/db vs db/+ mice or following long-term treatment of db/db mice with rosiglitazone or Wy-14,643 were more closely correlated with changes in UCP-3 than either UCP-1 or UCP-2 mRNA levels in the tissues examined. The present data contribute to the argument that UCP-3 and MTE-1 are linked within the same metabolic pathway either in response to, or as regulators of, fatty acid beta-oxidation.