A Molecular Recognition Guidebook for Targeting Protein Loops with Covalent Ligands

Often considered simple connectors, protein loops are extraordinarily important structures, regulating essential biological functions by forming macromolecular interactions and initiating signaling cascades. When mutated, loops propagate cellular dysfunction and disease, transcending a multitude of infectious, hereditary, and physiological disorders. Despite their importance, we have a limited understanding of loop structures as well as how loops alter protein structure, activity, and allosteric communication. Historically, a powerful tool to elucidate such structure-function relationships has been small molecules. In the pursuit of drug discovery, several covalent ligands have been serendipitously discovered to target cysteine residues in loops, leading to potent and selective small molecule probes. However, there is little emphasis on understanding the molecular, biophysical, and/or structural basis of the observed selectivity; and thus, scientists are limited in extrapolating these discoveries to target novel loop structures. In our current work, we have utilized covalent ligands to build structure-affinity relationships for two unique protein loops, which have revealed the key role of i) ligand shape in loop recognition and differential regulation of protein activity; ii) small molecule feature diversity in predicting covalent bond formation to loops; and iii) loop motion in the rate of covalent bond formation. Building upon these observations, we identified an exceptional model system to systematically study small molecule:loop interactions. As one of the most abundant human protein domains, RNA Recognition Motifs (RRMs) have a well-defined globular fold, loops that orthosterically and allosterically regulate RNA binding, and > 125 cysteines in loop structures for covalent bond formation. Utilizing RRMs, this proposal will elucidate guiding principles that describe ligand and loop features critical for i) molecular recognition, ii) regulating protein activity and structure, and iii) altering orthosteric and allosteric communication. These first-in-kind principles can be harnessed to rationally design small molecule modulators of novel loop structures. As current state-of-the-art is limited to massive screening campaigns and/or serendipity, a rational approach to loop targeting will be paradigm-shifting for chemical probe and therapeutic discovery. Furthermore, ligands discovered for RRM domains will transform our fundamental understanding of disease; approximately 25% of disease-annotated mutations are in RNA-binding proteins, which are an intractable, underprivileged protein class ripe for innovative small molecule discovery strategies. In the future, we expect these principles to guide the targeting of other dynamic and/or disordered structures, such as interdomain-linkers and intrinsically disordered regions, that when selectively targeted, expands protein ligandability to encompass the entire human proteome. Project Number: 1R35GM160331-01 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Brittany Morgan | Institution: UNIVERSITY OF NOTRE DAME, NOTRE DAME, IN | Award Amount: $387,857 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - E Study Section[MRAE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11203007