A disk-diffusion-based target identification platform for antibacterials (TIPA): an inducible assay for profiling MOAs of antibacterial compounds

Abstract

One of the challenges in antibiotic lead discovery is the difficulty and time-consuming task of determining the mechanism of action (MOA) of antibacterial compounds. In this report, we describe the development and validation of a facile and inexpensive assay system utilizing disk diffusion of inhibitors on solid agar medium embedded with mixed pools of a comprehensive collection of Escherichia coli clones each containing a plasmid-borne inducible essential gene from E. coli. From individual clones, pilot small-scale (48 or 50 clones) assays, to full-scale target identification platform for antibacterials (TIPA) system, involving a variety of assay formats (liquid vs solid media, individual vs mix clones), we demonstrate that elevated resistance phenotypes of relevant cell clones were highly specific. In particular, the TIPA system was able to reveal cellular targets of several known antibacterial inhibitors: cerulenin, diazaborine, indolmycin, phosphomycin, and triclosan. Complementary to several existing MOA profiling schemes, the TIPA system offers a simple and low-cost method for elucidating the target proteins of antibacterial inhibitors, thus will facilitate discovery and development of novel antibacterial compounds to combat multidrug-resistant bacterial pathogens.

Identification of cellular targets of a series of boron heterocycles using TIPA II—A sensitive target identification platform

“…First, TIPA II was validated using several known antibiotics and antibacterial inhibitors, including triclosan 22 and phosphomycin 23 whose target protein over-expression conferred enhance cellular resistance to specific inhibitors. 14 Just like in TIPA system, TIPA II disk diffusion assays generated resistant colonies within the zones of inhibitions surrounding triclosan- or phosphomycin-soaked disks only on induced assay trays containing Pool #2 mixed clones (Fig. 1).…”

“…The AS19 clones were configured into six separate pools of mixed clones, with the number of clones between 39 and 48 per pool (Table 1). The compositions of the pools were rather random, primarily based on the numerical order of clone ID codes (JW numbers) described in the Pools #1 to #5 of TIPA, 14 with additions of newly acquired clones in various pools to account for the entire essential gene landscape of E. coli (Profiling of E.coli Chromosome, PEC). For uniformity, the previous Pool #6 in TIPA which consists of 50 cell clones constructed in DH5α host cells in our laboratory using pLEX5BA vector 14 was replaced with pCA24N-based clones obtained from Nara Institute of Science and Technology, 21 thus our entire collection of inducible essential gene cell clones can be maintained using chloramphenicol.…”

“…26 Some of these compounds were active against E. coli cells, while the majority were not, 26 which are perfect examples that can be used to validate our TIPA II system for identification of cellular targets of compounds, in particular of those with poor activity. A total of 28 boron heterocycles were tested in both TIPA 14 and TIPA II systems (Table 2). Due to a mutant outer membrane, AS19 cells are more permeable (thus more sensitive) to compounds as compared to AG1 host cells, as illustrated by disk diffusion assays conducted for both TIPA and TIPA II systems (Fig.…”

“…For example, over-producing an essential cofactor may confer resistance to the inhibitor but this cofactor may not be the primary target. Secondly, as discussed by us previously, 14 such approach of over-expressing single essential protein per clone has the limitation that target(s) of an inhibitor that interacts with multiple subunits of a protein or an enzyme complex would be difficult to identify.…”

“…Recently, we reported the development and validation of a facile and inexpensive t arget i dentification p latform for a ntibacterials (TIPA) which leverages inducible over-expression of essential genes in mixed pools of Escherichia coli clones harboring plasmid-borne essential genes. 14 This approach is based on the concept that elevated concentrations of a target protein confer resistance because higher levels of a specific antibacterial inhibitor are needed to bind the excess target in order to inhibit cell growth. 15 However, the host cells used in TIPA were “wild-type” laboratory strains of E. coli , which, like most Gram negative bacteria, possess an intact outer membrane that prevents penetration of many compounds, 16-20 making it difficult to identify novel chemical structures with limited potency.…”