Project Grant F32GM167170

IDENTIFICATION OF METABOLIC MODULATORS VIA BIOCATALYSIS AND PROFILING OF SYNTHETIC LASSO PEPTIDE LIBRARIES - PROJECT SUMMARY/ABSTRACT CYCLIC PEPTIDE LIBRARIES CURRENTLY FAIL TO CAPTURE THE STRUCTURAL DIVERSITY FOUND IN NATURAL PRODUCT-DERIVED SCAFFOLDS, LIMITING THERAPEUTIC DISCOVERY TO A NARROW SUBSET OF SYNTHETICALLY ACCESSIBLE MACROCYCLES. RIBOSOMALLY SYNTHESIZED AND POST-TRANSLATIONALLY MODIFIED PEPTIDES EXPAND THIS SPACE WITH POST-TRANSLATIONAL TAILORING ENZYMES THAT GENERATE TOPOLOGIES THAT ARE INACCESSIBLE BY CONVENTIONAL PEPTIDE SYNTHESIS. AMONG THESE, LASSO PEPTIDES HAVE EMERGED AS ATTRACTIVE SCAFFOLDS DUE TO THEIR THREADED LARIAT STRUCTURE, REMARKABLE STABILITY, AND GROWING POTENTIAL AS THERAPEUTIC MODALITIES. NOTABLY, SEVERAL LASSO PEPTIDES APPEAR BROADLY REPRESENTED IN THE HUMAN GUT MICROBIOTA, SUGGESTING THEY MAY PARTICIPATE IN PREVIOUSLY UNRECOGNIZED HOST-MICROBE INTERACTIONS THAT INFLUENCE METABOLIC HOMEOSTASIS AND DISEASE. DESPITE THIS PROMISE, MAJOR GAPS REMAIN IN OUR ABILITY TO IDENTIFY NEW BIOACTIVE LASSO PEPTIDES, RELIABLY ACCESS THEM FOR FUNCTIONAL STUDIES, AND DIVERSIFY THEIR SCAFFOLDS. THESE LIMITATIONS HINDER BOTH THERAPEUTIC DEVELOPMENT AND EFFORTS TO UNDERSTAND THE BIOLOGICAL ROLES OF THESE METABOLITES. TO ADDRESS THIS, I WILL USE BI-32169, A CLASS III LASSO PEPTIDE FROM STREPTOMYCES WITH POTENT HUMAN GLUCAGON RECEPTOR ANTAGONISM, AS A MODEL SCAFFOLD FOR DEVELOPING A CHEMOENZYMATIC PLATFORM CAPABLE OF PRODUCING LIBRARIES OF BI-32169 ANALOGS. STRUCTURAL STUDIES INTO THE LASSO CYCLASE ENZYME RESPONSIBLE FOR BI-32169 WILL ALLOW FOR GUIDED MUTATIONS TO EXPAND THE SUBSTRATE SCOPE OF THE TAILORING ENZYMES. IN PARALLEL, BIOINFORMATIC MINING OF GUT MICROBES ASSOCIATED WITH PROTECTION AGAINST METABOLIC DISEASE HAS IDENTIFIED BI-32169 HOMOLOGS; THESE WILL SERVE AS ADDITIONAL SCAFFOLDS FOR DIVERSIFICATION AND PROFILING. TOGETHER, THESE EFFORTS WILL PRODUCE THE FIRST SYNTHETIC LIBRARIES OF LASSO PEPTIDES DESIGNED FOR HIGH-THROUGHPUT SCREENING AGAINST TARGETS INVOLVED IN METABOLIC SIGNALING PATHWAYS, SUCH AS GLUCAGON RECEPTOR MODULATION. THIS WORK WILL UNCOVER NEW LASSO PEPTIDES WITH ENHANCED THERAPEUTIC POTENTIAL AND ABILITY TO MODULATE HUMAN METABOLIC RECEPTORS, WHILE ALSO CLARIFYING THE ECOLOGICAL ROLE THESE PEPTIDES MAY PLAY WITHIN THE HUMAN MICROBIOME. MORE BROADLY, THE PROPOSED RESEARCH WILL ESTABLISH A GENERALIZABLE METHOD FOR LASSO PEPTIDE PRODUCTION AND LIBRARY DESIGN, EXPANDING THE NATURAL PRODUCT-DERIVED SCAFFOLDS AVAILABLE FOR THERAPEUTIC DISCOVERY AND ENABLING SYSTEMATIC EXPLORATION OF MICROBIOME-DERIVED RIPPS.