Thermal proteome profilng of BMAL1 ligand CCM in U2OS cell lysate

The master transcription factor BMAL1-CLOCK is crucial for orchestrating rhythmic gene expression and governing circadian rhythms. Pharmacological manipulation of this central circadian regulator requires accessible protein cavities and selective compounds. By targeting BMAL1 through a resident cavity in its PAS-B domain, we demonstrate that the small-molecule Core Circadian Modulator (CCM) penetrates and substantially expands this pocket. Biochemical and cellular analyses validate CCM's target engagement selectivity, enabling direct access to BMAL1's transcriptional activities. Thermal proteome profiling of CCM in U2OS cell lysate was performed at two concentrations (50 µM and 200 µM) and compared to DMSO. The proteome integral stability assay (PISA) setup was used.