The goal of the research in this Maximizing Investigators' Research Award (MIRA) is to develop transformative capabilities for discovering novel small molecules used to explore biology and advance medicine. The project aims to build on research enabled by 32 years of funding by NIGMS 038627 and 23 years of support by HHMI (ending Nov 2018). Although this research contributed to several important advances in biomedicine, it also revealed major gaps in our ability to translate insights from human biology into new medicines for public health. Overcoming these limitations is a primary aim of the proposed research. During the past five years of our GM038627-funded research, we used strategic planning principles of diversity-oriented synthesis (DOS) to synthesize a collection of novel, arrayed small molecules. Over 100 cell- based phenotypic screens were performed that yielded many novel small-molecule probes and candidates for novel therapeutics. We now propose to extend this use of modern, asymmetric synthesis to the discovery of compounds that bind target proteins. Discovering such `binders' remains a challenging first step towards developing compounds that confer specific activities on the target following binding – a key shortcoming in precision medicine guided by human genetics and functional genomics. To advance the discovery of small- molecule binders, we propose to pioneer diversity-oriented synthesis encoded by DNA oligonucleotides (DOSEDO) by synthesizing DNA bar-coded compounds resulting from DOS pathways. The resulting DNA- encoded libraries (DELs) will be incubated with tagged target proteins to affinity purify binders, which can be `decoded' using DNA sequencing. This project aims to meld advances in DOS with those of DELs, but benefitting simultaneously from the impressive advance of contemporary synthetic chemistry. A second key limitation has been the long time required to uncover the activities of newly synthesized small molecules. During the past five years, we introduced the concept of real-time annotation of small molecules, using cell painting to make > 1,000 cellular measurements of novel synthetic compounds within days of their synthesis. Our proposed research aims to develop an equally inexpensive and complementary method of real- time annotation – measuring compound-induced changes in the relative levels of thousands of cellular mRNA transcripts by bar-coding and pooling transcripts prior to a single RNA-Seq experiment (`HiTSeq'). Lastly, we will test the methods of DOSEDO, HiTSeq and others developed in the course of this MIRA by focusing on a cell state we recently showed to be adopted by cancer cells to confer resistance to chemothera- py, targeted therapeutics and immunotherapy. This lipophagy-induced, myofibroblastic cell state and its druggable vulnerability was discovered during the past five years of our GM038627-funded research. We will use this lipid-based pathway as a testing ground for new methods to discover smal...