ABSTRACT
IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,ß-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.
Subject(s)
Drug Discovery , Enzyme Inhibitors/pharmacology , Histidine , Isocitrate Dehydrogenase/antagonists & inhibitors , Isocitrate Dehydrogenase/chemistry , Cell Line, Tumor , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Isocitrate Dehydrogenase/genetics , Isocitrate Dehydrogenase/metabolism , Ligands , Molecular Docking Simulation , Mutation , Protein Conformation , Structure-Activity RelationshipABSTRACT
Drug-induced liver injury (DILI) has been the most frequent cause of post-marketing drug withdrawals in the last 50years. The multifactorial nature of events that precede severe liver injury in human patients is difficult to model in rodents due to a variety of confounding or contributing factors that include disease state, concurrent medications, and translational species differences. In retrospective analyses, a consistent risk factor for DILI has been the inhibition of the Bile Salt Export Pump (BSEP). One compound known for potent BSEP inhibition and severe DILI is troglitazone. The purpose of the current study is to determine if serum profiling of 19 individual bile acids by liquid chromatography-mass spectrometry (LC/MS) can detect perturbations in bile acid homeostasis in rats after acute intravenous (IV) administration of vehicle or 5, 25, or 50mg/kg troglitazone. Minimal serum transaminase elevations (approximately two-fold) were observed with no evidence of microscopic liver injury. However, marked changes in individual serum bile acids occurred, with dose-dependent increases in the majority of the bile acids profiled. When compared to predose baseline values, tauromuricholic acid and taurocholic acid had the most robust increase in serum levels and dynamic range, with a maximum fold increase from baseline of 34-fold and 29-fold, respectively. Peak bile acid increases occurred within 2hours (h) after dosing and returned to baseline values before 24h. In conclusion, serum bile acid profiling can potentially identify a mechanistic risk of clinical DILI that could be poorly detected by traditional toxicity endpoints.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/antagonists & inhibitors , Bile Acids and Salts/blood , Chemical and Drug Induced Liver Injury/etiology , Risk Assessment , Animals , Chromans/toxicity , Female , Male , Rats , Rats, Sprague-Dawley , Thiazolidinediones/toxicity , TroglitazoneABSTRACT
Cancer cells are highly reliant on NAD+-dependent processes, including glucose metabolism, calcium signaling, DNA repair, and regulation of gene expression. Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ salvage from nicotinamide, has been investigated as a target for anticancer therapy. Known NAMPT inhibitors with potent cell activity are composed of a nitrogen-containing aromatic group, which is phosphoribosylated by the enzyme. Here, we identified two novel types of NAM-competitive NAMPT inhibitors, only one of which contains a modifiable, aromatic nitrogen that could be a phosphoribosyl acceptor. Both types of compound effectively deplete cellular NAD+, and subsequently ATP, and produce cell death when NAMPT is inhibited in cultured cells for more than 48 hours. Careful characterization of the kinetics of NAMPT inhibition in vivo allowed us to optimize dosing to produce sufficient NAD+ depletion over time that resulted in efficacy in an HCT116 xenograft model. Our data demonstrate that direct phosphoribosylation of competitive inhibitors by the NAMPT enzyme is not required for potent in vitro cellular activity or in vivo antitumor efficacy. Mol Cancer Ther; 16(7); 1236-45. ©2017 AACR.
Subject(s)
Colorectal Neoplasms/drug therapy , Cytokines/antagonists & inhibitors , Enzyme Inhibitors/administration & dosage , Nicotinamide Phosphoribosyltransferase/antagonists & inhibitors , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Calcium Signaling/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Cytokines/genetics , DNA Repair/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , Humans , Mice , NAD/metabolism , Nicotinamide Phosphoribosyltransferase/genetics , Xenograft Model Antitumor AssaysABSTRACT
Stearoyl-CoA desaturase 1 (SCD1) catalyzes the committed step in the biosynthesis of monounsaturated fatty acids from saturated, long-chain fatty acids. Studies with SCD1 knockout mice have established that these animals are lean and protected from leptin deficiency-induced and diet-induced obesity, with greater whole body insulin sensitivity than wild-type animals. In this work, we have discovered a series of potent, selective, orally bioavailable SCD1 inhibitors based on a known pyridazine carboxamide template. The representative lead inhibitor 28c also demonstrates excellent cellular activity in blocking the conversion of saturated long-chain fatty acid-CoAs (LCFA-CoAs) to monounsaturated LCFA-CoAs in HepG2 cells.
