Validation of fragment-like molecules inhibiting DNA Gyrase from pathogenic bacteria

• Grant number: 24/19106-8
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: February 28, 2025
• End date: June 27, 2025
• Field of knowledge: Biological Sciences - Microbiology
• Principal Investigator: Marcio Vinicius Bertacine Dias
• Grantee: Sabrina Tami Shiroma
• Supervisor: Neil Osheroff
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Vanderbilt University (VU), United States
• Associated to the scholarship: 23/06554-0 - Identification of compounds that interact with DNA gyrase from Mycobacterium tuberculosis through fragment screening, BP.IC

Abstract

As highlighted by recent global health reports, millions of deaths annually occur from bacterial infections. Although there is a broad availability of antimicrobial agents, the growing resistance of bacteria to these treatments is becoming an increasingly public health problem. Therefore, the development of new antimicrobial agents is critical. Type II topoisomerases have proven to be effective targets for therapeutic agents, as they play crucial roles in bacteria. These enzymes maintain DNA topology through the introduction of negative supercoils or the decatenation of interlinked DNA. In Mycobacterium tuberculosis (Mtb), DNA gyrase is the only type II topoisomerase that is expressed and is, therefore, the sole target for fluoroquinolone antibacterials in tuberculosis treatment. Fluoroquinolones effectively target this bacterial enzyme by stabilizing gyrase-DNA cleavage complexes, ultimately causing cell death. However, increasing resistance to these drugs, often driven by mutations in the quinolone resistance-determining region (QRDR) of gyrase, renders them ineffective against some infections. Therefore, altering existing fluoroquinolones or discovering new molecules with different mechanisms of action targeting DNA gyrase is a potential strategy to manage this obstacle. To this end, fragment-based drug design (FBDD) allows the optimization of lead compounds with therapeutic potential by screening small organic fragments that bind to target proteins. Recently, we screened an in-house fragment library, identifying 28 fragments that significantly altered the melting temperature of M. tuberculosis DNA gyrase. Fourteen fragments were further validated for binding through Saturation Transfer Difference (STD) NMR. We also attempted to perform a DNA cleavage assay to verify whether the selected compounds interact with the enzyme at the appropriate site (e.g., the fluoroquinolone binding site) and exert their desired inhibitory effect, but it proved quite challenging. Therefore, this project aims to further understand the mechanism of action of these compounds against M. tuberculosis and other orthologous bacterial DNA gyrases. We expect to determine whether compounds inhibit DNA gyrase by interfering with DNA binding, preventing its supercoiling function, or stabilizing the cleavage complex, offering insights into the potential of these compounds as antibacterial drug candidates.