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High throughput screening for small-molecule inhibitors of type III secretion in Yersinia pestis
Ning Pan1, Chrono Lee, Jon Goguen
1Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, USA. ning.pan@umassmed.edu
Abstract:
Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, utilize a plasmid encoded type III secretion system (T3SS) to promote infection by delivering Yersinia outer proteins (Yops) into the cytosol of mammalian cells. This T3SS is absolutely required for Yersinia virulence, which makes T3SS an attractive target in the development of novel therapeutics for treatment of plague and other Yersinia infections. In this study, a new method for high throughput screening (HTS) of small molecules for the ability to inhibit type III secretion (T3S) in Y. pestis has been developed. In comparison with screening assays employed by others, this method is very simple and rapid, and thus well suited for examining very large compound sets. Using this method, we screened a diverse collection of libraries at the US National Screening Laboratory. The initial examination of 70,966 compounds and mixtures from 13 libraries resulted in 431 primary hits. Strong positive indications of inhibition were observed at a rate of 0.01%, while moderate and weak but potentially meaningful signals were observed at rates of 0.056% and 0.54% respectively. Further characterizations were conducted on selected primary hits in Y. pestis. Of the eight compounds examined in secondary assays, four show good promise as leads for structure activity relationship studies. They are a diverse group, each having chemical scaffolds not only distinct from one another, but also distinct from previously described candidate T3S inhibitors.
Insights
Researchers developed a rapid, high-throughput screening method to find small molecules inhibiting the Yersinia type III secretion system (T3SS), crucial for bacterial virulence. Four promising compounds were identified as potential leads for new anti-plague therapeutics.
Area of Science:
- Microbiology and Infectious Diseases
- Drug Discovery and Development
Background:
- Yersinia species employ a plasmid-encoded type III secretion system (T3SS) to inject effector proteins (Yops) into host cells, a critical virulence factor.
- The T3SS is essential for Yersinia pathogenesis, making it a key target for developing novel anti-infective therapies against diseases like plague.
Purpose of the Study:
- To develop and implement a novel, rapid, and high-throughput screening (HTS) method for identifying small molecules that inhibit Yersinia type III secretion (T3S).
- To screen large compound libraries using the developed HTS assay and identify potential T3S inhibitors for Yersinia pestis.
Main Methods:
- Development of a simplified and rapid HTS assay to detect inhibition of type III secretion (T3S) in Yersinia pestis.
- Screening of 70,966 compounds from diverse libraries using the HTS assay.
- Secondary characterization of selected primary hits to evaluate their potential as T3S inhibitors.
Main Results:
- The HTS assay successfully screened a large compound set, identifying 431 primary hits out of 70,966 tested compounds.
- A hit rate of 0.01% for strong positive inhibition was observed, with additional moderate and weak signals identified.
- Secondary assays confirmed four out of eight examined compounds as promising lead candidates with novel chemical scaffolds.
Conclusions:
- A highly efficient HTS method for T3S inhibitors was established, suitable for screening extensive compound collections.
- The study identified novel chemical entities with potential for structure-activity relationship studies, offering new avenues for anti-Yersinia drug development.
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