Structural and functional characterization of ABC transporters from ESKAPE microorganisms: tackling antimicrobial resistance

• Grant number: 24/10255-0
• Support Opportunities: Research Projects - SPEC Program
• Start date: March 01, 2025
• End date: February 28, 2029
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Marko Juhana Hyvönen
• Grantee: Marko Juhana Hyvönen
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• City of the host institution: São Paulo
• Principal investigators: Andrea Balan Fernandes
• Associated research grant(s): 25/07229-0 - Multiuser equipment granted in process 2024/10255-0: Nanotemper Prometheus Panta, AP.EMU
• Associated scholarship(s): 25/17834-9 - Evaluation of druggability of MsbA proteins, BP.PD; 25/11265-2 - Structural and functional characterization of ABC transporters from ESKAPE microorganisms: tackling antimicrobial resistance, BP.TT; 25/07714-6 - Protein production and analysis specialist, BP.TT; 25/06330-0 - Structural studies of MsbA efflux pumps from ESKAPE organisms, BP.PD

Abstract

Antimicrobial resistance (AMR) is a growing problem threatening public health worldwide. Failure to control microbial infections is already resulting increased deaths and last-resort antibiotics for the treatment of resistant microorganisms are gradually losing efficacy as well, as demonstrated by increase in multi-drug resistant (MDR) species. Among bacterial pathogens, the ESKAPE group bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are classified as the top priority organisms in terms of tackling AMR. Our aim in this project is to study the molecular mechanisms by which these bacteria generate resistance for antimicrobial agents. The mechanisms for bacteria to acquire AMR are many-fold, including active secretion of antibiotics from the cells. We propose to study key drug efflux transporters from ESKAPE pathogens, the MsbA-like proteins, which belong to the ATP-Binding Cassette class of membrane transporters (ABC transporters). These membrane proteins utilize energy from ATP hydrolysis to pump a wide range of substrates across cellular membranes. MsbA-like proteins are normally involved in lipid translocation, membrane recycling and biofilm formation, but have also a moonlighting function as exporters of foreign chemicals from cells. For this reason, they are important players in drug efflux and transport of antimicrobial outside the cell in many pathogenic bacteria. Stopping these transporters would impair the bacterial growth and make bacteria vulnerable to drugs again, re-enabling the use of existing, tried-and-tested antibiotics. We will use structural and biophysical methods to evaluate the function of MsbA proteins and explore possibilities for their inhibition by small molecules.To do this, we will take a three-pronged approach to studying ESKAPE MsbA proteins: (i) to characterize their structure and function; (ii) to develop methods and assays to evaluate the transport activities front to different substrates; and (iii) to establish a platform for structure-guided drug discovery on MsbAs and apply fragment-based methods to reveal potential sites for inhibition of these enzymes. With these approaches we will initiate a new, broader programme that will bring information and expertise to an important, but under-explored class of therapeutic targets. We will build new tools for heterologous protein expression, X-ray crystallography and cryo-electron microscopy for structural analyses. Biochemical and biophysical methods will be used to evaluate the transport activity and to screen small molecule libraries against the targets. The structures in complex with small molecules will be the trigger for optimisation of these molecules for subsequent structure-guided drug discovery campaign to turn the hits into specific, high-affinity inhibitors. (AU)