Discovery and Development of Small Molecule Probes for the SUMO protease SENP1, a Novel Target for Advanced Prostate Cancer Therapy

Abstract

Androgen signaling through the androgen receptor (AR) is essential for normal growth and function of the prostate gland. In prostate cancer (PC), androgens provide the main proliferative drive for the disease, making androgen-deprivation one of the primary therapeutic strategies. Although initially effective, such treatments select for tumor cells that are able to sustain proliferation in a reduced androgen environment. This allows for the emergence of castration resistant PC, an incurable disease where both the AR transcriptional program is subverted and cellular senescence is evaded. SUMOylation is a post-translational modification that regulates both of these processes. SUMOylation of AR inhibits both basal and androgen-stimulated transcription in a promoter context manner and enhanced global SUMO modification induces prostate cell senescence. Advanced PC cells evade these mechanisms at least in part through the upregulation of SENP1, a SUMO-specific cysteine protease that reverses SUMOylation. In addition, AR is a direct activator of the SENP1 gene, creating a self-reinforcing loop that promotes and sustains its own activity and PC progression. This dissertation is aimed at the discovery and development of small molecule inhibitors of SENP1 as the basis for novel prostate cancer therapeutics. Using a robust FRET-based assay, we defined the kinetic properties of SENP1 and its closest paralog SENP2. This analysis revealed significant product inhibition and a differential sensitivity to ionic strength. Using this assay, an extensive high-throughput screening campaign led to the identification of two structurally distinct inhibitor classes. Characterization of these compounds indicates that they display significant selectivity towards SENP1 relative to SENP2 and that they act in both a reversible and competitive manner. Furthermore, these compounds inhibit native full-length SENP1 acting on endogenous SUMOylated substrates. Notably, both groups of compounds are known to display activity as purinergic receptor antagonists. The remarkable parallel pharmacology to P2X1 receptors led to our discovery that ATP, the endogenous P2X ligand, is a SENP1 selective inhibitor. We have thus revealed a novel nucleotide mediated regulation of SENP1. Using a combination of mutagenesis, biochemical assays, and fluorescence and NMR spectroscopy, we have characterized the binding of inhibitors and identified key enzyme residues involved in the interactions as well as residues responsible for SENP isoform selectivity. These findings reveal that SENP1 harbors a unique binding site for nucleotides that can be targeted by small molecules. This knowledge can guide novel strategies for further inhibitor development for evaluation of the therapeutic efficacy of SENP1 inhibitors in advanced prostate cancer models.