Fragment-based drug discovery against oncogenic microRNA precursors using high-throughput crystallography.

Targeting structured RNAs with small molecules dramatically increases the number of potentially “druggable” therapeutic targets, representing a major opportunity for anti-cancer drug discovery. The precursors (pre- miRNAs) of oncogenic microRNAs (oncomiRs) fold into hairpin structures that are recognized and cleaved by microRNA (miRNA) processing enzymes. Thus, the pre-miRNA structures provide binding sites for small molecules that can inhibit the production of specific oncomiRs. Using this strategy, eleven previous studies have identified eighteen diverse small molecules that bind to prominent oncomiR precursors, pre-miR-21 and pre- miR155. However, most of these compounds lack drug-like properties and further improvement has been hampered by lack of underlying structural details. We propose to perform fragment-based drug discovery (FBDD) via high-throughput crystallographic screening, enabled by a newly established facility at the National Synchrotron Light Source II. FBDD focuses the initial screening on compounds with molecular masses less than 300 Da to reduce the size of the screens while sufficiently covering the chemical space. In Aim 1, we will execute fragment-based crystallographic screening campaigns against three prominent oncomiR precursors, pre-miR- 21, pre-miR-155, and pre-miR-10b. In preliminary studies, we have obtained diffraction-quality crystals of these pre-miRNAs and determined several structures of pre-miR-21, which revealed previously unknown rare structural features as potential drug-binding sites. Through a preliminary screen, we have identified fragment hits for a pre-miR-21 construct. Similar screens will be performed for other pre-miR-21 constructs, as well as pre-miR-155 and pre-miR-10b (Aim 1a). Since the fragment libraries currently used were tailored for targeting proteins, we will acquire and test an RNA-specific fragment library (Aim 1b) and make it available to the research community. We will also determine the structures of pre-miRNAs bound with previously reported ligands (Aim 1c), which will facilitate potential merging or linking of these ligands to fragment hits. In Aim 2, we will first confirm and optimize the fragment hits, then merge and link the fragment hits to drug-sized compounds that bind the pre-miRNAs tightly, and finally validate the drug-like hits using a comprehensive set of biochemical, structural, and cellular assays. The development of fragment hits into drug-like leads should be greatly facilitated by the structural information offered by our crystallographic approach. If successful, our study will identify novel small molecule inhibitors against oncomiRs that will be ready for lead optimization and pre-clinical studies. To our best knowledge, fragment-based crystallographic screening has not been performed for RNA-only targets. The proposed study will pave the way for similar studies in the future. Project Number: 1R01CA299144-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Feng Guo (+1 co-PI) | Institution: UNIVERSITY OF CALIFORNIA LOS ANGELES, LOS ANGELES, CA | Award Amount: $528,265 | Activity Code: R01 | Study Section: Drug Discovery and Molecular Pharmacology C Study Section[DMPC] View on NIH RePORTER: https://reporter.nih.gov/project-details/11368868