The application's broad objective is to provide new molecular insights into the biosynthetic assembly of medicinally relevant fungal polycyclic compounds by nonribosomal peptide synthetases (NRPSs). Two major classes of amino acid-derived multicyclic fungal metabolites are the benzodiazepinones and quinazolinones. Core structures are derived from combination of the beta amino acid anthranilate (ortho-aminobenzoate) with proteinogenic amino acids, resulting in the formation of scaffolds with potent bioactivities, both as therapeutics (such as the asperlicins) and toxins (such as acetylaszonalenin). The anthranilate-containing fungal metabolite asperlicin is a cholecystokinin (CCK) receptor antagonist of nanomolar potency. CCK receptor isoforms are found in the brain, central nervous system and alimentary canal;CCK receptor antagonists provide a route to treat both gastrointestinal and neurological disorders. The NRPS-based morphing of linear peptide chains into architecturally constrained fused-ring systems with diverse substituents underlies the high affinities and biological selectivities observed for these classes of compounds. Previous studies have provided structures and synthetic routes for many compounds of these classes, but little is known regarding their biosynthesis beyond building block composition through precursor feeding studies. The proposed research aims to provide a fundamental understanding of: 1) the selection, activation, and loading of anthranilate by fungal NRPSs, and 2) the chemical processes mediated by the biosynthetic enzymes to cyclize and tailor the linear peptide into the final bioactive product. We predict that the number and order or anthranilate building blocks incorporated into the peptide backbone are key determinants to multicylic scaffold construction. Completion of the proposed research will provide knowledge that may be applied to the directed incorporation of anthranilate into peptide backbones to engineer non-natural cyclization patterns and substituents. Through genome mining, biochemical experimentation, and structural analysis we expect to characterize the molecular basis of anthranilate selection and loading by NRPSs. Through deconstruction and reconstitution of the biosynthetic machinery activity, we will explore a predicted new mode of NRPS-based cyclization of the peptide backbone for multicylic scaffold construction.
Understanding the biological assembly of anthranilate-containing fungal natural products will provide a foundation for producing molecules to screen as therapeutic agents, and will identify biological targets from toxin producing pathways for drug design.
