Structural characterization and inhibition of efflux pumps from the ESKAPE group using sybodies

• Grant number: 24/02417-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2024
• End date: February 29, 2028
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Vinícius Carneiro Barbosa
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Membrane proteins account for between 25% and 30% of all sequences transcribed in the genomes of organisms, performing a wide variety of vital functions for cellular metabolism, one of the reasons that make them relevant to the pharmaceutical industry, accounting for a total of 50% of all targets for drug production. Antibodies, nanobodies and sybodies (synthetic antibodies) have been an interesting approach for understanding the function, modulation, inhibition and increased stability for structural studies of these proteins, but their production still requires purified proteins. Studies show that hydrophilic regions exposed to the outside of the plasma membrane are the main targets of antibodies and derivatives. In this proposal, we bring a different approach to the production of sybodies against a class of membrane proteins found in all organisms, of great relevance in the phenomenon of multiple drug resistance, and which are structurally conserved. The proteins are ABC transporters (ATP-Binding Cassette) of the exporter type (class IV) that constitute lipid recycling pumps in the membrane and drug and antibiotic efflux pumps in multidrug-resistant bacteria of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). We will use the Sav1866 transporter from Staphylococcus aureus, extensively characterized from a functional and structural point of view, as an anchor for the presentation of the hydrophilic regions of orthologs present in multidrug-resistant bacteria. Furthermore, we intend to characterize the inhibition potential of the sybodies in relation to drug transport carried out by these proteins and as the stabilizing potential of the proteins for structural studies. To produce transporters, we will use protocols established in the literature and in our laboratory and for the construction of sybodies, phage display, ribosome display and ELISA (Enzyme Linked Immuno Sorbent Assay) methodologies will be used. The structures of transporters that do not yet have characterization will be resolved by cryo-electron microscopy. The group has the collaboration of Prof. Markus Seeger from the University of Zurich who developed the methodology and a platform for producing sybodies and Dr. Daniela Luz from the Butantan Institute to carry out the phage-display and ribosome display assays under GLP conditions. Additionally, interaction tests of the transporters with the sybodies using surface plasmon resonance and biophysical tests will be carried out in collaboration with Prof. Marko Hyvonen from the University of Cambridge and structural studies of transporters in collaboration with Prof. Albert Guskov from the University of Groningen, an expert in the structural characterization of ABC transporters by cryo-electron microscopy. The proposal also has a high potential for innovation and can lead to other projects for modulation and inhibition studies of multidrug-resistant bacteria.