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.

A High-Throughput Screen Identifies a New Natural Product with Broad-Spectrum Antibacterial Activity

“…First of all, the screen uses whole cells rather than purified targets [7]. Secondly, it is an assay for cell viability and, therefore, is biased toward bactericidal agents [22], [23]. Thirdly, because it does not require cell growth, it is rapid.…”

Perspective on Antibacterial Lead Identification Challenges and the Role of Hypothesis-Driven Strategies

“…However, lack of mechanistic bias can result in large numbers of nonspecific hits. Variations of the phenotypic screening formats that are biased toward inhibitors of specific antibacterial targets or pathways are typically accomplished with engineered bacterial strains (e.g., through sensitization by target underexpression or through use of a target or pathway-specific reporter genes 35,36 ). Although phenotypic screens may seem to bias toward a specific pathway or target, the complexity of systems biology means hits may be identified that are difficult to relate back to the primary target or pathway.…”

“…It is also worth noting that if a compound does bind the (presumed) target, then increasing levels of that protein's expression may reduce the amount of free compound available to interact with other targets, meaning that expression levels could alter the MIC of a compound, even if growth inhibition is not occurring via inhibition of that target. 35,36 Genomics. Expression profiling (transcriptome, proteome, metabolome) can help to establish the mechanism of action of an inhibitor.…”

Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii

“…Many MKs have been characterized and studied, as they have been implicated in human diseases such as mevalonic aciduria, hyperimmunoglobulinemia D and periodic fever syndrome (Favier & Schulert, 2016). MKs are also potentially interesting drug targets, as they play essential roles in the metabolism of pathogenic organisms such as Leishmania (Shafi et al, 2021;Prasad et al, 2022) and in some eubacteria, for example Streptococcus pneumoniae (Kudoh et al, 2010) and Staphylococcus aureus (Ferrand et al, 2011).…”