HDX reveals unique fragment ligands for the vitamin D receptor.

Modulation of the vitamin D receptor (VDR) with a ligand has the potential to be useful for the oral treatment of osteoporosis. One component of our lead generation strategy to identify synthetic ligands for VDR included a fragment based drug design approach. Screening of ligands in a VDR fluorescence polarization assay and a RXR/VDR conformation sensing assay resulted in the identification of multiple fragment hits (lean >0.30). These fragment scaffolds were subsequently evaluated for interaction with the VDR ligand binding domain using hydrogen-deuterium exchange (HDX) mass spectrometry. Significant protection of H/D exchange was observed for some fragments in helixes 3, 7, and 8 of the ligand binding domain, regions which are similar to those seen for the natural hormone VD3. The fragments appear to mimic the A-ring of VD3 thereby providing viable starting points for synthetic expansion.

Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.

Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion.

Glycine levels are inversely associated with branched-chain amino acids (BCAAs) and cardiometabolic disease phenotypes, but biochemical mechanisms that explain these relationships remain uncharted. Metabolites and genes related to BCAA metabolism and nitrogen handling were strongly associated with glycine in correlation analyses. Stable isotope labeling in Zucker fatty rats (ZFRs) shows that glycine acts as a carbon donor for the pyruvate-alanine cycle in a BCAA-regulated manner. Inhibition of the BCAA transaminase (BCAT) enzymes depletes plasma pools of alanine and raises glycine levels. In high-fat-fed ZFRs, dietary glycine supplementation raises urinary acyl-glycine content and lowers circulating triglycerides but also results in accumulation of long-chain acyl-coenzyme As (acyl-CoAs), lower 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in muscle, and no improvement in glucose tolerance. Collectively, these studies frame a mechanism for explaining obesity-related glycine depletion and also provide insight into the impact of glycine supplementation on systemic glucose, lipid, and amino acid metabolism.

An intra/intermolecular suzuki sequence to benzopyridyloxepines containing geometrically pure exocyclic tetrasubstituted alkenes.

A route to enable the preparation of 5-benzylidenyl-benzopyridyloxepine analogues was developed to continue our research in the field of nuclear hormone receptor modulators. The key steps are 1) a syn-stereoselective diboration of a tethered aryl alkyne; 2) an intramolecular Suzuki cross-coupling reaction, which forms in a stereo- and regiocontrolled fashion, the 5-exoalkylidenyl 7-membered ring imbedded within the core of the scaffold and; 3) an intermolecular Suzuki to furnish the final tetra-substituted olefinic benzopyridyloxepines.

Cyclocarbopalladation of alkynes: a stereoselective method for preparing dibenzoxapine containing tetrasubstituted exocyclic alkenes.

[reaction: see text] A palladium-catalyzed cascade carbometalation-cross coupling of alkyne route was developed for the preparation of tetrasubstituted exocyclic alkenes with high stereo- and regiocontrol. The effectiveness of this novel methodology was demonstrated by the synthesis of a number of dibenzoxapines in sufficient quantities to support their further development.

Indole Glucocorticoid Receptor Antagonists Active in a Model of Dyslipidemia Act via a Unique Association with an Agonist Binding Site.

To further elucidate the structural activity correlation of glucocorticoid receptor (GR) antagonism, the crystal structure of the GR ligand-binding domain (GR LBD) complex with a nonsteroidal antagonist, compound 8, was determined. This novel indole sulfonamide shows in vitro activity comparable to known GR antagonists such as mifepristone, and notably, this molecule lowers LDL (-74%) and raises HDL (+73%) in a hamster model of dyslipidemia. This is the first reported crystal structure of the GR LBD bound to a nonsteroidal antagonist, and this article provides additional elements for the design and pharmacology of clinically relevant nonsteroidal GR antagonists that may have greater selectivity and fewer side effects than their steroidal counterparts.

Glucocorticoid receptor modulators informed by crystallography lead to a new rationale for receptor selectivity, function, and implications for structure-based design.

The structural basis of the pharmacology enabling the use of glucocorticoids as reliable treatments for inflammation and autoimmune diseases has been augmented with a new group of glucocorticoid receptor (GR) ligands. Compound 10, the archetype of a new family of dibenzoxepane and dibenzosuberane sulfonamides, is a potent anti-inflammatory agent with selectivity for the GR versus other steroid receptors and a differentiated gene expression profile versus clinical glucocorticoids (lower GR transactivation with comparable transrepression). A stereospecific synthesis of this chiral molecule provides the unique topology needed for biological activity and structural biology. In vivo activity of 10 in acute and chronic models of inflammation is equivalent to prednisolone. The crystal structure of compound 10 within the GR ligand binding domain (LBD) unveils a novel binding conformation distinct from the classic model adopted by cognate ligands. The overall conformation of the GR LBD/10 complex provides a new basis for binding, selectivity, and anti-inflammatory activity and a path for further insights into structure-based ligand design.