Identification of new antibacterial agents in libraries of natural products

Author(s)

Fernanda Rodrigues Costa

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2020-07-30

Examining board members

Andréa Dessen; Ana Carolina de Mattos Zeri; Fabiana Fantinatti Garboggini; Andrea Balan; Rodrigo Cayô

• Advisor: Andréa Dessen

Abstract

Antibiotics have been used to treat infectious diseases for decades and are arguably the most successful drugs developed in history. However, the wide use and misuse of these drugs have selected multi-drug resistant organisms. Some pathogens have even become resistant to all classes of antibiotics, highlighting the urgent need for new antimicrobials in the clinical practice. Therefore, aiming at contributing to the global task force on antimicrobial resistance, the goal of this work was to screen natural product libraries, to identify new molecules that inhibited bacterial growth. The libraries used are composed of extracts and isolated compounds from the Brazilian biodiversity and are thus a yet untapped, totally novel potential source of new antibiotics. The libraries containing about 3510 compounds were screened through a high-throughput whole-cell screen. Hits were defined as compounds that inhibited bacterial growth in 50% or more of the level of the positive control (chloramphenicol 32 µg/mL). 339 hits that inhibited B. subtilis growth and 46 that inhibited E. coli growth were detected. The purification process of the hits lead to the isolation and identification of the merulinic acid C that presented synergism with gentamicin against E. faecium, exhibiting a minimum inhibitory concentration (MIC) of 4 µg/mL. Another six promising hits are in the isolation process, one of them was highly active with a MIC of 4 µg/mL against E. coli and 2 µg/mL against B. subtilis. A collection of synthetic compounds and the NIH clinical collection library were also screened, resulting in the detection of antibacterial activity in seven quinazolinone derivatives. Among them, the most active structure showed good activity against methicillin-resistant S. aureus (MIC 4 µg / mL) and vancomycin-resistant Enterococcus faecium (MIC 2 µg / mL). Regarding the NIH clinical collection, a candidate for the repurposing of a non-antimicrobial drug was detected, this drug exhibited excellent activity against P. aeruginosa clinical isolates (MIC 2 µg / mL). Another approach employed in this study was the characterization of the E. coli cell wall components that represents a promising new target for antibiotic development. This part of the project integrated the bolsa estágio pesquisa no exterior (BEPE) and was developed at the Institut of Biologie Structurale (Grenoble-FR). The elongation process in rod-shaped bacteria is dependent on the action of a highly organized machinery (elongasome), whose the components, such as PBP2 and MreC, interact forming complexes. Once the genes encoding such proteins are absents, or, when protein interactions are disrupted, the bacterial cell loses its rod shape and dies. Therefore, the objective of BEPE was the structural characterization of the E. coli PBP2: MreC complex. A great advance was achieved in this project, it was successfully established the purification protocol of the complex and the crystallization screenings were performed. The results obtained here will be essential to the structural elucidation of this potential new antibiotic target (AU)

• FAPESP's process: 15/19906-5 - Identification of new antibacterial agents in libraries of natural products
• Grantee: Fernanda Rodrigues da Costa
• Support Opportunities: Scholarships in Brazil - Doctorate