A new cellular assay to identify novel ligands for the essential enzyme MurA of Neisseria gonorrhoeae

• Grant number: 21/08960-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2021
• End date: June 30, 2022
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Rafael Lemos Miguez Couñago
• Grantee: Sophia Pivatto Serra
• Host Institution: Centro de Biologia Molecular e Engenharia Genética (CBMEG). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

The growing number of bacteria resistant to current antibiotics poses a serious threat to public health services worldwide. At the same time, large pharmaceutical companies have exited this clinical area. A major obstacle in antibiotic discovery is that potent biochemical inhibitors to target essential enzymes often lack in-cell activity. This lack bactericidal activity can be linked to poor compound cell permeability or efflux by bacterial pumps. Thus, the development of new assays that can reconcile biochemical activity and on-target in-cell activity hold the promise to accelerate the discovery of novel antibiotics. In this project, we will develop an assay based on Bioluminescent Energy Transfer (BRET) to assess the engagement of ligands to a target protein in live bacterial cells. For its operation, this test depends on the development of two components. The first, called the BRET donor, will be obtained throughout this project by fusing the target protein MurA from Neisseria gonorrhoeae to a bright luciferase (NanoLuc). The MurA enzyme (UDP-N-acetylglucosamine enolpyruvyl transferase) catalyzes the first step in the biosynthesis of peptidoglycans, essential components of the bacterial cell wall. Genetic and pharmacological methods have already demonstrated that the loss of MurA function is bactericidal, which makes this enzyme an attractive target for the development of new antibiotics that can help fight multiresistant strains of N. gonorrhoeae. The second component of this new BRET-based assay is a MurA ligand modified by the addition of a fluorescent motif compatible with the luminescence generated by NanoLuc. The BRET acceptor will be developed by CQMED researchers in an open-science, collaborative project with Eurofarma Pharmaceutical Laboratories. We hope that the development of this new assay will accelerate the discovery of new MurA ligands capable of permeating the various bacterial envelope barriers. (AU)