Evidence for the in vitro bioactivation of aminopyrazole derivatives: trapping reactive aminopyrazole intermediates using glutathione ethyl ester in human liver microsomes.

Drug-induced toxicity is a leading cause of drug withdrawal from clinical development and clinical use and represents a major impediment to the development of new drugs. The mechanisms underlying drug-induced toxicities are varied; however, metabolic bioactivation to form reactive metabolites has been identified as a major contributor.1,2 These electrophilic species can covalently modify important biological macromolecules and thereby increase the risk of adverse drug reactions or idiosyncratic toxicity. Consequently, screening compounds for their propensity to form reactive metabolites has become an integral part of drug discovery programs. This screening process typically involves identification of structural alerts as well as the generation of reactive metabolites in vitro in subcellular hepatic fractions, followed by trapping the reactive species with nucleophiles and characterization via LC-MS. This article presents evidence for the bioactivation of a series of aminopyrazole derivatives via LC-MS detection of glutathione ethyl ester-trapped reactive intermediates formed in human liver microsomal incubations. These results indicate that the aminopyrazole motif, within specific contexts, may be considered a new structural alert for the potential formation of reactive metabolites.

ATP sensitive bi-quinoline activator of the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) regulates cellular and whole-body energy balance in response to changes in adenylate charge and hormonal signals. Activation of AMPK in tissues such as skeletal muscle and liver reverses many of the metabolic defects associated with obesity and Type 2 diabetes. Here we report a bi-quinoline (JJO-1) that allosterically activates all AMPK αßγ isoforms in vitro except complexes containing the γ3 subunit. JJO-1 does not directly activate the autoinhibited α subunit kinase domain and differs among other known direct activators of AMPK in that allosteric activation occurs only at low ATP concentrations, and is not influenced by either mutation of the γ subunit adenylate-nucleotide binding sites or deletion of the ß subunit carbohydrate-binding module. Our findings indicate that AMPK has multiple modes of allosteric activation that may be exploited to design isoform-specific activators as potential therapeutics for metabolic diseases.

Fragment screening for a protein-protein interaction inhibitor to WDR5.

The WD40-repeat protein WDR5 scaffolds various epigenetic writers and is a critical component of the mammalian SET/MLL histone methyltransferase complex. Dysregulation of the MLL1 catalytic function is associated with mixed-lineage leukemia, and antagonism of the WDR5-MLL1 interaction by small molecules has been proposed as a therapeutic strategy for MLL-rearranged cancers. Small molecule binders of the "WIN" site of WDR5 that cause displacement from chromatin have been additionally implicated to be of broader use in cancer treatment. In this study, a fragment screen with Surface Plasmon Resonance (SPR) was used to identify a highly ligand-efficient imidazole-containing compound that is bound in the WIN site. The subsequent medicinal chemistry campaign-guided by a suite of high-resolution cocrystal structures with WDR5-progressed the initial hit to a low micromolar binder. One outcome from this study is a moiety that substitutes well for the side chain of arginine; a tripeptide containing one such substitution was resolved in a high resolution structure (1.5 Å) with a binding mode analogous to the native tripeptide. SPR furthermore indicates a similar residence time (k d = ∼0.06 s-1) for these two analogs. This novel scaffold therefore represents a possible means to overcome the potential permeability issues of WDR5 ligands that possess highly basic groups like guanidine. The series reported here furthers the understanding of the WDR5 WIN site and functions as a starting point for the development of more potent WDR5 inhibitors that may serve as cancer therapeutics.

Structural Determinants for Small-Molecule Activation of Skeletal Muscle AMPK α2ß2γ1 by the Glucose Importagog SC4.

The AMP-activated protein kinase (AMPK) αßγ heterotrimer regulates cellular energy homeostasis with tissue-specific isoform distribution. Small-molecule activation of skeletal muscle α2ß2 AMPK complexes may prove a valuable treatment strategy for type 2 diabetes and insulin resistance. Herein, we report the small-molecule SC4 is a potent, direct AMPK activator that preferentially activates α2 complexes and stimulates skeletal muscle glucose uptake. In parallel with the term secretagog, we propose "importagog" to define a substance that induces or augments cellular uptake of another substance. Three-dimensional structures of the glucose importagog SC4 bound to activated α2ß2γ1 and α2ß1γ1 complexes reveal binding determinants, in particular a key interaction between the SC4 imidazopyridine 4'-nitrogen and ß2-Asp111, which provide a design paradigm for ß2-AMPK therapeutics. The α2ß2γ1/SC4 structure reveals an interaction between a ß2 N-terminal α helix and the α2 autoinhibitory domain. Our results provide a structure-function guide to accelerate development of potent, but importantly tissue-specific, ß2-AMPK therapeutics.