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Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening - PubMed

  • ️Fri Jan 01 2016

Accelerating the discovery of antibacterial compounds using pathway-directed whole cell screening

Leigh M Matano et al. Bioorg Med Chem. 2016.

Abstract

Since the introduction of penicillin into the clinic in 1942, antibiotics have saved the lives of millions of people around the world. While penicillin and other traditional broad spectrum antibiotics were effective as monotherapies, the inexorable spread of antibiotic resistance has made alternative therapeutic approaches necessary. Compound combinations are increasingly seen as attractive options. Such combinations may include: lethal compounds; synthetically lethal compounds; or administering a lethal compound with a nonlethal compound that targets a virulence factor or a resistance factor. Regardless of the therapeutic strategy, high throughput screening is a key approach to discover potential leads. Unfortunately, the discovery of biologically active compounds that inhibit a desired pathway can be a very slow process, and an inordinate amount of time is often spent following up on compounds that do not have the desired biological activity. Here we describe a pathway-directed high throughput screening paradigm that combines the advantages of target-based and whole cell screens while minimizing the disadvantages. By exploiting this paradigm, it is possible to rapidly identify biologically active compounds that inhibit a pathway of interest. We describe some previous successful applications of this paradigm and report the discovery of a new class of d-alanylation inhibitors that may be useful as components of compound combinations to treat methicillin-resistant Staphylococcus aureus (MRSA).

Keywords: High throughput screen; Pathway-directed drug discovery; Teichoic acid; Transposon sequencing; d-Alanylation.

Copyright © 2016 Elsevier Ltd. All rights reserved.

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Figures

Figure 1
Figure 1

Schematic of important cell envelope biosynthetic pathways in Staphylococcus aureus. The S. aureus cell wall is composed of thick layers of peptidoglycan containing covalently bound wall teichoic acids (WTA). S. aureus also contains membrane-bound lipoteichoic acids (LTA). LTA and WTA are modified with D-alanine esters installed by DltABCD. In the schematic, selected WTA enzymes are shown in yellow, Dlt pathway enzymes are shown in orange, and LTA pathway enzymes are shown in pink. The targets of selected inhibitors mentioned in the text are indicated. Figure adapted from Rajagopal and Walker (2016)

Figure 2
Figure 2

Biologically active compounds with activity against preselected pathways can be identified by screening for compounds that differentially inhibit growth of different bacterial strains. (A) Schematic of a plot depicting growth of library compounds against wildtype S. aureus and ΔtarO. Hit compounds are depicted in green or red depending on whether they are lethal to the the ΔtarO or wildtype strain, respectfully. The former are possible late stage WTA inhibitors and the latter inhibit a pathway that becomes essential when WTA biosynthesis is prevented. (B) Structures of two compounds previously identified using the pathway-directed whole cell screening approach depicted in 2A. Amsacrine inhibits DltB, which is required to install D-alanine esters on LTA (see Figure 1). Targocil inhibits TarG, which transports WTA precursors to the cell surface for attachment to peptidoglycan.

Figure 3
Figure 3

Schematic showing selected synthetic lethal interactions between three cell envelope pathways in Staphylococcus aureus. Synthetic lethal interactions were identified by probing a high density transposon mutant library with tunicamycin, which inhibits TarO and prevents WTA synthesis, and amsacrine, which inhibits DltB and prevents D-alanylation of lipoteichoic acids. Inhibiting D-alanylation is lethal to mutants that make abnormal LTA due to deletion of ypfP or ltaA, but not to mutants that make no LTA (ΔltaS strains). Information on synthetic lethal interactions between the Dlt pathway and other pathways enabled design of a strain panel diagnostic for Dlt pathway inhibitors (see Figure 4).

Figure 4
Figure 4

A new DltB inhibitor was rapidly identified by testing synthetic lethal hits from a high throughput screen against a strain panel diagnostic for Dlt pathway inhibitors. (A) Schematic of a plate showing possible outcomes of treatment of five different strains against test compounds. Amsacrine, a validated DltB inhibitor, was used as a positive control. It inhibits growth of the ΔtarO, ΔypfP and tn::1050 strains, but not of the wildtype or ΔltaS strains. 200 synthetic lethal hits from a 230,000 compound screen were tested against these five strains and three possible Dlt pathway inhibitors were identified. (B) Plots showing growth as a function of inhibitor concentration for DBI-1, one of the three compounds identified as a Dlt pathway inhibitor, against the test strains. (C) Structure of DBI-1 and minimum inhibitory concentrations against a panel of mutants selected by plating amsacrine (gray boxes) or DBI-1 (red box) on a strain containing an inactivating transposon insertion in SAOUHSC_01050 (tn::1050). The A355E mutant was identified in independent selections on DBI-1. (D) PAGE autoradiograph showing 14C-D-Ala LTA after treatment of cells with increasing concentrations of DBI-1.

Figure 5
Figure 5

Summary of results from a pathway-directed screen of 230,000 small molecules. Approximately 2,000 compounds had some biological activity. The majority of these inhibited growth of both wildtype and ΔtarO S. aureus and were not considered further. We identified two possible WTA inhibitors, of which one has been confirmed as a new TarG inhibitor and will be reported elsewhere. We identified 200 synthetic lethal compounds and designed a cherry-pick screening panel to sort these compounds into Dlt pathway inhibitors and other types of inhibitors. Of three possible Dlt pathway inhibitors, one has been confirmed as a new DltB inhibitor.

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