UC Irvine

DEL technology has gained popularity as an adjunct to high-throughput screening techniques, offering expanded capabilities for investigating novel chemical entities. Typically, DELs comprise mixtures of DNA-small molecule conjugates, in which the DNA tag reflects the synthesis history of the combinatorial library member. These DELs, termed "on-DNA" DELs, undergo screening using affinity selection, and the DNA is sequenced to decode compounds which bind to specific protein targets. With modern DNA sequencing technologies, DEL can explore unique binding interactions in a single experiment, which has led to the discovery of ligands for various targets and the identification of numerous clinical candidates. More recently, the use of DELs in drug discovery has also spurred computational intervention, as the inherent properties of combinatorial chemistry yield highly structured and modular datasets ripe for informatics and machine learning-based analyses.

We have previously pioneered solid-phase one-bead-one-compound (OBOC) DEL technology, introducing an innovative approach to DEL synthesis that unlocks new avenues for screening. In this method, a DEL library member is polyvalently coupled to a bead via a photolabile bond, alongside covalently bound DNA-encoding tags. Upon UV-exposure, the library member is released into the surrounding solution where it may engage with and modulate the activity of a target of interest. We have previously demonstrated the efficacy of activity-based OBOC-DEL "off-DNA" screening for novel enzyme inhibitors, in competition-based fluorescence polarization screening, and for the prediction of compound potency.

In this dissertation, I build on this prior work and present advances in DEL library design and synthesis principles alongside the development of assays that enable the use of DEL in in vitro systems. Chapter 1 contains a detailed protocol summarizing improved synthesis and quality control procedures for OBOC-DELs, accessible by both academic and industry groups. Chapter 2 presents design considerations for a diversity-oriented DEL, proposing principles that increase the compatibility of DEL members for lead optimization processes. Chapter 3 details the development of a miniaturized in vitro transcription and translation assay and the design of a bespoke scaffold-oriented DEL, culminating in the discovery of novel translation inhibitors. Lastly, Chapter 4 discloses the development of a cell-based phenotypic screening platform for OBOC-DELs, and reports preliminary screening results from a library inspired by agonists of the immunomodulatory cGAS-STING pathway.