Project Summary/Abstract Natural products (NP) are a mainstay of drug discovery, accounting for up to 50% of approved drugs either as direct natural molecules or as inspiration for synthetic molecules. High-throughput screening of compound libraries is a common starting point for drug development campaigns. The quality of these libraries is therefore critical to high-throughput screening campaign success. NP compound library design is particularly challenging given redundancies in NP production between isolates and greater costs of compound production and isolation. Evidence-based and scientifically rigorous methods to optimize NP library design are therefore urgently required. MPIs previously demonstrated using the fungus Alternaria that liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analysis of fungal extracts can reveal the minimal number of extracts to include in a chemical library, to achieve saturation of chemical diversity. Strikingly, this could be as few as 39 isolate extracts, depending on Alternaria clades. The overall objective of this proposal is to demonstrate the broader utility of this bioanalytical approach, to the significant biological problem of high-throughput screening NP chemical library design. The central hypothesis of this proposal is that untargeted metabolomics will enable rational NP library generation and provide quantitative evidence as to whether current fungal culture and library generation approaches improve library diversity or lead to unnecessary redundancy. This proposal builds on MPI’s extensive expertise in metabolomics, NP analysis and drug development. In addition, it is enabled by the MPI’s access to the large collection of fungal isolates from the University of Oklahoma Citizen Science Soil Collection Program. This collection currently totals >88,000 isolates from 893 fungal genera. We will focus on three common library design approaches, in three independent aims. Aim 1 will assess whether comparable chemical diversity and improved bioassay hit rates can be obtained from focused, rationally-designed NP libraries, compared to current large-scale library build approaches (historical sampling, cosmopolitan collections, morphological diversity, deep local collections, etc.). Aim 2 will systematically assess the impact of co-culture on elicited chemical diversity and hit rates, comparing sympatric vs allopatric co-culture systems. Aim 3 will systematically quantify the impact of environment- mimicking culture conditions such as soil or bacterial-derived signals, on elicited chemical diversity and hit rates. The proposed research is innovative because it applies a bioanalytical approach to NP library design and because it challenges existing dogmas in this field. The proposed research is significant because it will lead to an improved understanding of the determinants of NP chemical diversity and enable improved NP library design. Overall, our results will lead to validation of a new approac...