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A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein-Protein Interactions in an Academic Screening Facility.

ABSTRACT: Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein-protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration-response curves with IC50 values of <40 ?M, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

SUBMITTER: Voter AF

PROVIDER: S-EPMC5667550 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein-Protein Interactions in an Academic Screening Facility.

Voter Andrew F AF Killoran Michael P MP Ananiev Gene E GE Wildman Scott A SA Hoffmann F Michael FM Keck James L JL

SLAS discovery : advancing life sciences R & D 20170601 1

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein-protein interactions (PPIs) to recruit more than a dozen important ...[more]

PMID: 28570838

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Project description:Protein-protein interactions play important roles in virtually all cellular processes, making them enticing targets for modulation by small-molecule therapeutics: specific examples have been well validated in diseases ranging from cancer and autoimmune disorders, to bacterial and viral infections. Despite several notable successes, however, overall these remain a very challenging target class. Protein interaction sites are especially challenging for computational approaches, because the target protein surface often undergoes a conformational change to enable ligand binding: this confounds traditional approaches for virtual screening. Through previous studies, we demonstrated that biased "pocket optimization" simulations could be used to build collections of low-energy pocket-containing conformations, starting from an unbound protein structure. Here, we demonstrate that these pockets can further be used to identify ligands that complement the protein surface. To do so, we first build from a given pocket its "exemplar": a perfect, but nonphysical, pseudoligand that would optimally match the shape and chemical features of the pocket. In our previous studies, we used these exemplars to quantitatively compare protein surface pockets to one another. Here, we now introduce this exemplar as a template for pharmacophore-based screening of chemical libraries. Through a series of benchmark experiments, we demonstrate that this approach exhibits comparable performance as traditional docking methods for identifying known inhibitors acting at protein interaction sites. However, because this approach is predicated on ligand/exemplar overlays, and thus does not require explicit calculation of protein-ligand interactions, exemplar screening provides a tremendous speed advantage over docking: 6 million compounds can be screened in about 15 min on a single 16-core, dual-GPU computer. The extreme speed at which large compound libraries can be traversed easily enables screening against a "pocket-optimized" ensemble of protein conformations, which in turn facilitates identification of more diverse classes of active compounds for a given protein target.

Dataset Information

A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein-Protein Interactions in an Academic Screening Facility.

Publications

A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein-Protein Interactions in an Academic Screening Facility.

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