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Published on: June 30, 2016
Discovery of PqsE Thioesterase Inhibitors for Pseudomonas aeruginosa Using DNA-Encoded Small Molecule Library
Julie S Valastyan1,2, Michael R Tota3, Isabelle R Taylor1
1Department of Molecular Biology , Princeton University , Princeton , New Jersey 08544 , United States.
Insights
Researchers identified novel inhibitors for PqsE, a key enzyme in Pseudomonas aeruginosa virulence. DNA-encoded library screening rapidly discovered potent compounds, offering a new strategy against hospital-acquired infections.
Area of Science:
- Microbiology
- Biochemistry
- Drug Discovery
Background:
- Pseudomonas aeruginosa is a major cause of hospital-acquired infections.
- The PqsE enzyme is crucial for P. aeruginosa virulence, making it a potential therapeutic target.
- The specific function and substrates of PqsE remain largely unknown.
Purpose of the Study:
- To identify small molecules that bind to and inhibit the PqsE enzyme.
- To validate DNA-encoded compound library screening as a method for discovering inhibitors of bacterial proteins with unknown functions.
Main Methods:
- Screening of a 550 million molecule DNA-encoded library against purified PqsE protein.
- High-throughput sequencing to identify DNA barcodes of bound molecules.
- In vitro assays to assess PqsE thioesterase inhibition and thermal stability of PqsE inhibitors.
Main Results:
- Identification of a series of 2-(phenylcarbamoyl)benzoic acids as noncompetitive PqsE inhibitors.
- The most potent analog exhibited an IC50 of 5 microM.
- Early structure-activity relationship studies showed increased potency with analog refinement.
Conclusions:
- DNA-encoded library screening is an effective method for identifying inhibitors of bacterial enzymes like PqsE.
- The identified compounds show potential but require further optimization for in vivo efficacy due to poor cytosolic accumulation in P. aeruginosa.
Abstract:
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 microM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.
