Identification of Inhibitors of Bacterial Transcription/Translation Machinery Utilizing a Miniaturized 1536-Well Format Screen

Abstract

This report presents the miniaturization of a HTS screen to identify inhibitors of prokaryotic transcription-translation in a 1536-well format. The in vitro assay design utilized the bacterial expression machinery to drive expression of a firefly luciferase reporter gene, which was read as an endpoint luminesence measurement. This multicomponent system permits identification of inhibitors at different steps in this pathway. Successful miniaturization required integration of homogeneous assay formats, robust liquid-handling workstations, and second-generation imaging systems. Comparison of data from a triplicate 1536-well screen of a subset of a target library that had been previously validated and followed up for hit confirmation in a 384-well plate format confirmed that triplicate screening yields data of higher confidence and quality, eliminates the time-consuming and potentially error-prone step of cherry-picking, and reduces the number of false positives and negatives. The substantial savings of reagents and reduction of the numbers of plates to process obtained in a 1536-well format as compared to a 384-well format allowed a full triplicate evaluation of the entire library of 183,000 compounds at lower cost and in less time. The triplicate-screen statistics are consistent with a highly reliable data set with a coefficient of variation of 14.8% and Z' and Z values of 0.57 and 0.25, respectively. This screen resulted in the identification of 1,149 hits (0.63% hit rate), representing a compound population at 2.5 standard deviations from the mean cutoff. Furthermore, the data demonstrate good agreement between IC(50) values derived for this assay in a 1536-well format and 384-well format.

Novel Approach to Mapping of Resistance Mutations in Whole Genomes by Using Restriction Enzyme Modulation of Transformation Efficiency

“…Of the many possible applications for this technology, perhaps one of the most important is that it facilitates an ideal approach to the discovery of novel antibacterial agents. From a practical standpoint, researchers have had to choose between two extremes: cellular screening and molecular target screening, although pathway screens with cell extracts have also been pursued (12,24,35). Screening directly for inhibitors of bacterial proliferation results in compounds with antibacterial activity, but without a readily discovered molecular target to facilitate structure and/or mechanism-directed medicinal chemistry for lead optimization.…”

Free energy calculations for theophylline binding to an RNA aptamer: Comparison of MM‐PBSA and thermodynamic integration methods

“…Several studies have now focused on the first of these issues, using statistical approaches to setting hit cutoffs5, 6 or partitioning and clustering HTS hits according to similarity, based on pharmacophore information or quantitative structure‐activity‐relationship models such as comparative molecular field analysis (CoMFA) 7–14. In contrast, the second problem has received relatively scant attention, although it is well established that HTS hit lists contain numerous (in many cases a majority) false positives 15–18. The follow‐up hit confirmation assays required are laborious and costly, whether they involve simple retests in the same assay system (a weak, although common, form of verification that only corrects machine errors), control assays, or measurements in a completely independent system.…”

Microscale Adaptation of In Vitro Transcription/Translation for High‐Throughput Screening of Natural Product Extract Libraries

“…Previous studies using a coupled transcription/translation protocol focused only on discrete small-molecule libraries, leading to the identification of new antimicrobial leads at a 16 μ L scale in 384-well format (7,8), and demonstrating the feasibility of this method at a 4 μ L scale in 1536-well plates (12). We reduced the screening scale to a total volume of 2 μ L in low-volume 1536-well plates, a miniaturization that was further enhanced by the effective dilution of the working concentrations of the transcription/translation assay components.…”

“…Previous studies successfully optimized a coupled transcription/translation screen in 384-well plates at a total scale of 16 μ L using a luminescence-based reporter on a small-molecule library, which resulted in the discovery of a potential lead for a new, orthogonal class of ribosome inhibitor (7,8). This type of assay is amenable for use in 1536-well plates (12), and a number of secondary and counter screens are available to enable the decoupling of transcription from translation (7,8,13), remove intercalative inhibitors (14), discriminate compounds that interfere with luminescence (7,8), and identify general (eukaryotic) ribosome inhibitors (7,8,14). Our objectives were to create an improved method by significantly increasing efficiency through a reduction in reaction scale and to apply this screening technology directly to natural product extracts.…”