Novel strategies to expand the chemical diversity of phage display libraries for inhibitor discovery

/Abstract Peptides are an attractive scaffold for therapeutic development due to their unique ability to combine desirable features of both small molecules and larger, protein-based biologics. Like small molecules, peptides are synthetically accessible and can penetrate tissues effectively, yet they offer the superior binding affinity and specificity typical of biologics. Another key advantage of peptides lies in the availability of powerful screening technologies, such as phage display, which enables the rapid screening of vast peptide libraries to identify novel ligands for a chosen target. However, phage display libraries are traditionally limited to the 20 natural amino acids, which lack useful chemical functionalities found in non-ribosomal peptides and synthetic pharmaceuticals. As a result, the full potential of phage display in therapeutic peptide discovery remains underutilized. The overarching goals of my research are: (1) to develop synthetic and chemical biology approaches to expand the chemical diversity of phage display libraries, thereby improving their utility in early-stage drug discovery, and (2) to apply these expanded libraries to identify novel antimicrobial peptides. With this MIRA application, we seek to accomplish these goals by pursuing two interrelated research directions. Direction 1 focuses on inhibitor discovery using a new chemically and genetically augmented phage display platform. In preliminary studies, we developed a novel approach to introduce diverse chemical moieties into phage-displayed peptides by combining unnatural amino acid (uAA) mutagenesis with chemical post-translational modification. Over the next five years, we will use this strategy to produce peptide libraries containing, for example, reactive chemical warheads and metal-chelating groups, granting access to highly diverse libraries with unexplored chemical space. We will evaluate these expanded libraries for their ability to produce potent and selective inhibitors, with an initial focus on enzymes from multidrug-resistant Acinetobacter baumannii. This work will establish key principles for implementing this nascent technology for de novo inhibitor discovery and provide first-in-class inhibitors for bacterial enzymes of significant medical concern. Direction 2 focuses on expanding our repertoire of cysteine- reactive uAAs to produce cyclic peptides with unique molecular architectures. Cyclic peptides offer several advantages over their linear counterparts, but current methods for producing phage-displayed cyclic peptide libraries are technically challenging and/or limited in scope. Encouraged by strong preliminary data, we will develop two entirely new classes of uAAs, to produce ribosomally synthesized bicyclic and backbone-to-side- chain cyclic peptides. This work will provide access to unique cyclic peptide libraries that are not accessible with existing phage display technology. Moreover, as we push the limits of in vivo protein synthesis, incidental discoveries will likely offer new insights into the fundamental mechanisms of gene translation. Collectively, the proposed work will greatly expand the capabilities of phage display technology and accelerate the discovery of new peptides with broad research, diagnostic, and therapeutic applications. Project Number: 1R35GM162266-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Jeffery Tharp | Institution: INDIANA UNIVERSITY INDIANAPOLIS, INDIANAPOLIS, IN | Award Amount: $383,880 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MBBC-A (57)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11271667