Discovery of novel small molecules targeting a circadian regulatory complex for cancer therapeutics

/Abstract Circadian rhythms are ~24-hour endogenous cycles observed across diverse biological processes. Altered circadian rhythms have been shown to be associated with various diseases, including cancer. Accumulating evidence has demonstrated that the core circadian components, particularly Brain and Muscle ARNT-Like Protein 1 (BMAL1) and Circadian Locomotor Output Cycles Kaput (CLOCK), play important roles in different types of cancer, such as glioblastoma (GBM), acute myeloid leukemia (AML), and hepatocellular carcinoma (HCC). These discoveries underline circadian clocks as a promising novel target for cancer treatment. Currently, however, effective BMAL1- and CLOCK-specific chemical probes are still lacking, which prevents the development of novel cancer therapies that directly target the central clock. To discover small-molecule compounds that can specifically modulate BMAL1:CLOCK and have anti-cancer efficacy, we introduced a new strategy by targeting BMAL1:CLOCK in the context of a holoprotein complex that reflects their native conformation. We developed and validated a new drug discovery pipeline that consists of an affinity selection mass spectrometry (AS-MS) based binding assay combined with a series of cell-based HiBiT reporter assays that can assess the endogenous protein properties and dynamics for hit validation. We have implemented this new pipeline in a small-scale pilot screen and identified two small molecules that selectively target BMAL1 and CLOCK, respectively. In this proposal, we aim to implement our new platform in a large-scale high-throughput screening (HTS) campaign, followed by rigorous hit validation. Meanwhile, we will integrate advanced cheminformatics analysis and medicinal chemistry inspection to prioritize the hit set, as a prelude to understanding structure-activity relationships, and optimization of chosen leads. The success of this project will discover more potent compounds for BMAL1 and CLOCK, which will open the door for novel cancer drug development by targeting the circadian system. Project Number: 1R01CA308442-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: STEVE KAY | Institution: UNIVERSITY OF SOUTHERN CALIFORNIA, Los Angeles, CA | Award Amount: $680,452 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 CTH-T (81)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11440396