High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes

ABSTRACT Penicillin binding protein (PBP) 1b of Escherichia coli has both transglycosylase and transpeptidase activities, which are attractive targets for the discovery of new antibacterial agents. A high-throughput assay that detects inhibitors of the PBPs was described previously, but it cannot distinguish them from inhibitors of the MraY, MurG, and lipid pyrophosphorylase. We report on a method that distinguishes inhibitors of both activities of the PBPs from those of the other three enzymes. Radioactive peptidoglycan was synthesized by using E. coli membranes. Following termination of the reaction the products were analyzed in three ways. Wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads were added to one set, and the same beads together with a detergent were added to a second set. Type A polyethylenimine-coated WGA-coated SPA beads were added to a third set. By comparison of the results of assays run in parallel under the first two conditions, inhibitors of the transpeptidase and transglycosylase could be distinguished from inhibitors of the other enzymes, as the inhibitors of the other enzymes showed similar inhibitory concentrations (IC50s) under both conditions but the inhibitors of the PBPs showed insignificant inhibition in the absence of detergent. Furthermore, comparison of the results of assays run under conditions two and three enabled the distinction of transpeptidase inhibitors. Penicillin and other β-lactams showed insignificant inhibition with type A beads compared with that shown with WGA-coated SPA beads plus detergent. However, inhibitors of the other four enzymes (tunicamycin, nisin, bacitracin, and moenomycin) showed similar IC50s under both conditions. We show that the main PBP being measured under these conditions is PBP 1b. This screen can be used to find novel transglycosylase or transpeptidase inhibitors.

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High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

PLoS ONE ◽

10.1371/journal.pone.0044918 ◽

2012 ◽

Vol 7 (9) ◽

pp. e44918 ◽

Cited By ~ 16

Author(s):

Alena Fedarovich ◽

Kevin A. Djordjevic ◽

Shauna M. Swanson ◽

Yuri K. Peterson ◽

Robert A. Nicholas ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

High Throughput ◽

High Throughput Screening ◽

Binding Protein ◽

Penicillin Binding Protein

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Turning high-throughput structural biology into predictive inhibitor design

10.1101/2021.10.15.464568 ◽

2021 ◽

Author(s):

Kadi L Saar ◽

Daren Fearon ◽

Frank von Delft ◽

John D Chodera ◽

Alpha Albert Lee ◽

...

Keyword(s):

Structural Biology ◽

High Throughput ◽

Chemical Space ◽

Learning To Rank ◽

Binding Pocket ◽

Binding Activity ◽

Binding Modes ◽

Main Protease ◽

One Step ◽

High Throughput Crystallography

A common challenge in drug design pertains to finding chemical modifications to a ligand that increases its affinity to the target protein. An underutilised advance is the increase in structural biology throughput, which has progressed from an artisanal endeavour to a monthly throughput of up to 100 different ligands against a protein in modern synchrotrons. However, the missing piece is a framework that turns high throughput crystallography data into predictive models for ligand design. Here we designed a simple machine learning approach that predicts protein-ligand affinity from experimental structures of diverse ligands against a single protein paired with biochemical measurements. Our key insight is using physics-based energy descriptors to represent protein-ligand complexes, and a learning-to-rank approach that infers the relevant differences between binding modes. We ran a high throughput crystallography campaign against the SARS-CoV-2 Main Protease (MPro), obtaining parallel measurements of over 200 protein-ligand complexes and the binding activity. This allows us to design a one-step library syntheses which improved the potency of two distinct micromolar hits by over 10-fold, arriving at a non-covalent and non-peptidomimetic inhibitor with 120 nM antiviral efficacy. Crucially, our approach successfully extends ligands to unexplored regions of the binding pocket, executing large and fruitful moves in chemical space with simple chemistry.

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Faculty Opinions recommendation of Deletion of penicillin-binding protein 5 (PBP5) sensitises Escherichia coli cells to beta-lactam agents.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1649962.1152060 ◽

2010 ◽

Author(s):

Samuel Kariuki

Keyword(s):

Escherichia Coli ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Beta Lactam ◽

Escherichia Coli Cells

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PenA, a penicillin-binding protein-type thioesterase specialized for small peptide cyclization

Journal of Industrial Microbiology & Biotechnology ◽

10.1093/jimb/kuab023 ◽

2021 ◽

Author(s):

Kenichi Matsuda ◽

Kei Fujita ◽

Toshiyuki Wakimoto

Keyword(s):

Enzymatic Activity ◽

Computational Model ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Small Peptide ◽

Peptide Cyclization ◽

Chain Lengths ◽

Non Ribosomal Peptides

Abstract Penicillin binding protein-type thioesterases (PBP-type TEs) are a recently identified group of peptide cyclases that catalyze head-to-tail macrolactamization of non-ribosomal peptides. PenA, a new member of this group, is involved in the biosyntheses of cyclic pentapeptides. In this study, we demonstrated the enzymatic activity of PenA in vitro, and analyzed its substrate scope with a series of synthetic substrates. A comparison of the reaction profiles between PenA and SurE, a representative PBP-type TE, showed that PenA is more specialized for small peptide cyclization. A computational model provided a possible structural rationale for the altered specificity for substrate chain lengths.

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