title: Screening for mevalonate biosynthetic pathway inhibitors using sensitized bacterial strains.
abstract: A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.
authors: Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.
authors: Sandrine
authors: S
authors: Ferrand
authors: Jianshi
authors: J
authors: Tao
authors: Xiaoyu
authors: X
authors: Shen
authors: Dorothy
authors: D
authors: McGuire
authors: Andres
authors: A
authors: Schmid
authors: J Fraser
authors: JF
authors: Glickman
authors: Ulrich
authors: U
authors: Schopfer