Pharmacokinetics-Based DNA-Encoded Library Screening - PROJECT ABSTRACT This R00 proposal seeks to expand the space of druggable protein targets through the development of novel analytical technology that will guide the design of next-generation, non-Lipinski molecular therapeutics and diagnostics. Cyclic peptides, which are not drug-like (i.e., Lipinski-like), hold great promise for addressing undruggable targets, especially protein-protein interactions (PPIs). However, the larger molecular mass and more complex secondary structures result in unpredictable and almost always low cell permeation, significantly blunting their utility as drug candidates and even as preclinical tool compounds in cells. Rarely, a cyclic peptide will exhibit anomalously high permeation. However, insufficient data exist to uncover the rules dictating permeation because high-throughput measurements of cell permeation simply do not exist. In K99 phase, a high-throughput in vitro permeation assay compatible with DNA-encoded combinatorial library (DEL) screening was developed and miniaturized to the microfluidic droplet scale. In this proposal, the permeation assay will be applied to screen DELs to identify the species that efficiently permeate membrane bilayers. The permeable macrocycle hits will be further validated in a cell-based permeation assay that was also developed in K99 phase and will be further investigated in the project. The large screening data sets will reveal relationships between permeability and structure, especially for molecules of beyond the Rule of 5 (bRo5), which will be further analyzed using machine learning. With these and other empirically derived models of macrocyclic peptide pharmacokinetic properties in hand, we may finally be able to move such nonLipinski molecules from the lab to the clinic at scale. This proposal will significantly enhance the PI's career development and advance her goal of becoming a tenured professor and a leader in analytical assay and tool development to understand cell membrane permeability. San Diego State University offers an ideal research environment, boasting renowned experts in chemistry and chemical biology and fostering a collaborative intellectual community. Through this plan, the PI will engage in activities essential for achieving independence, including establishing a robust research program, honing grantsmanship skills, networking, and preparing for tenure evaluation. The proposed plan will facilitate the PI's scientific and professional growth, positioning her for future R01 funding success.
