CLICK-enabled analogues reveal pregnenolone interactomes in cancer and immune cells

Abstract

Pregnenolone (P5) promotes prostate cancer cell growth, and <i>de novo</i> synthesis of intratumoural P5 is a potential cause of development of castration resistance. Immune cells can also synthesize P5 <i>de novo</i>. Despite its biological importance, little is known about P5's mode of actions, which appears to be context dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8⁺ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking and in P5's non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8⁺ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues, and organisms.