Thermal proteome profiling and proteome analysis using high‐definition mass spectrometry demonstrate modulation of cholesterol biosynthesis by next‐generation galeterone analog <scp>VNPP</scp> 433‐3β in castration‐resistant prostate cancer

Cholesterol (CHOL) homeostasis is significantly modulated in prostate cancer (PCa) suggesting an active role in PCa development and progression. Several studies indicate a strong correlation between elevated CHOL levels and increased PCa risk and severity. Inhibition of CHOL biosynthesis at different steps, including lanosterol synthase (LSS), has shown significant efficacy against both hormone-dependent and castration-resistant PCa. Earlier, we reported proteasomal degradation of androgen receptor (AR)/AR-Vs and Mnk1/2 as the primary mechanisms of action of VNPP433-3β in inhibiting PCa cell proliferation and tumor growth. Through thermal proteome profiling, comparative proteomics and cellular thermal shift assay, we identified VNPP433-3β's ancillary effect of lowering CHOL by binding to LSS and lanosterol 14-alpha demethylase, potentially inhibiting CHOL biosynthesis in PCa cells and tumors. Additionally, in conjunction with our previously reported transcriptome analysis, proteomics reveals that VNPP433-3β modulated upstream regulators and pathways critical for PCa stem cell maintenance and recurrence. The inhibition of CHOL biosynthesis by VNPP433-3β reinforces its multifaceted effects in PCa across all stages, highlighting its potential as a single-agent therapy. Achieving reduced CHOL levels aligns with better treatment outcomes, further substantiating VNPP433-3β's therapeutic potential.