High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases Bacteria that colonize the human mucosa can become virulent and invasive, causing chronic and acute diseases including drug-resistant gonorrhea, pneumonia, and meningitis. New drugs targeting colonization and invasion would be welcome additions to the antibacterial arsenal, but interactions among bacterial factors, host tissues, and host immune defenses are poorly understood. For instance, immunoglobulin A1 (IgA1) represents an important barrier to colonization of mucosal surfaces, and pathogenic strains of several Gram- positive and Gram-negative bacteria produce proteases that cleave IgA1. These include Neisserial, Haemophilus, and Streptococcus strains that cause chronic ear infections (otitis media), bronchitis, drug- resistant gonorrhea, bacterial pneumonia and bacterial meningitis. Despite a large body of evidence that IgAPs are virulence factors with diverse functions, they have been vastly understudied because there were few assays for measuring their activity and no selective inhibitors. Importantly, the potential for targeting IgAPs as an ani-virulence therapy remains untested, and will remain so until selective inhibitors are identified. The Kritzer lab uses synthesis, biophysical chemistry, and cell biology to develop inhibitors of difficult-to-target proteins. Recently, we designed fluorescence probes to quantitate IgAP activity from diverse human pathogens. Despite forty years of research into IgAP biology, these probes are the first and only of their kind and enable the first-ever high-throughput screens for IgAP inhibitors. The goal of this R01 proposal is to leverage these probes into high-throughput screens that will deliver selective inhibitors of IgAPs. We will accomplish this goal by: testing additional substrate sequences, developing the substrates into fluorescent probes, incorporating the probes into HTS assays for IgAP inhibitors, performing pilot screens, and testing hits in follow-up assays that measure their effects in vitro and on live bacteria. We have partnered with the Broad Institute to ensure smooth transitions from probe development to pilot assays to a full screening campaign. The assays developed in this project will be of general use to microbiologists and immunologists for quantifying IgAP activity. They could also find clinical applications in the diagnosis of bacterial meningitis and other infections. Most importantly, the inhibitors developed from high-throughput screening will allow us and others to investigate host-pathogen interactions involving IgAPs. The inhibitors will also represent starting points for evaluating IgAPs as drug targets for treating urgent infectious disease threats, including drug- resistant gonorrhea, bacterial pneumonia, and bacterial meningitis.

Public Health Relevance

High-Throughput Assays for Inhibitors of Understudied Bacterial Proteases Drug-resistant forms of the bacteria that cause gonorrhea, pneumonia, and meningitis are emerging rapidly. Targeting virulence factors of these bacteria, including the proteins known as Immunoglobulin A1 proteases (IgAPs), represents a promising anti-bacterial strategy. Until our recent work, there were few tools, inhibitors, or screening assays that could be used to study IgAPs. This project describes the first-ever high-throughput screening assays for IgAP inhibitors, which will be valuable tools for understanding host-pathogen interactions as well as potential lead compounds for treating several devastating infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM111042-03
Application #
9321116
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Barski, Oleg
Project Start
2015-08-01
Project End
2018-12-31
Budget Start
2017-08-01
Budget End
2018-12-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Tufts University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
073134835
City
Boston
State
MA
Country
United States
Zip Code
02111