Discovery of Novel BKCa Channel Activators as Smooth Muscle Relaxants and TPST2 Inhibitors as Immuno-Oncology Therapeutics

Abstract

To address unmet medical needs in both smooth muscle disorders and cancer immunotherapy, this dissertation describes the identification and development of small-molecule modulators against two mechanistically distinct targets, the BKCa (large-conductance calcium-activated potassium) channel and the tyrosylprotein sulfotransferase 2 (TPST2) enzyme. As Part 1, we report the identification and optimization of novel BKCa channel activators based on a diphenyl ether scaffold. Starting from the screening hit compound 4d, structure–activity relationship (SAR) studies led to the discovery of compound 10b, which exhibited potent activity in cell-based assays (EC50 = 0.12 μM) and demonstrated in vivo efficacy in a spontaneous hypertensive rat (SHR) model of urinary incontinence. Furthermore, an orally bioavailable analog, compound 51b, showed dose-dependent efficacy in a citric acid-induced guinea pig cough model (ED50 = 11.8 mg/kg). The cryo-electron microscopy (cryo-EM) structures of the BKCa channel in complex with 10b and 51b were resolved at 2.8 Å and 3.4 Å, respectively, providing mechanistic insights into ligand-induced channel activation. As Part 2, we describe the discovery and biological evaluation of a novel TPST2 inhibitors. Optimization of the initial hit compound 44a yielded 77c, with an IC50 value of 946 nM. Through in silico docking analyses and molecular dynamic (MD) simulations, we proposed a putative binding mode of 77c for the TPST2 enzyme. In vivo, 77c suppressed tumor growth in the MC38 syngeneic mouse model and enhanced the efficacy of anti-PD-1 immune checkpoint therapy. These effects were accompanied by increased infiltration of effector CD8+ T cells and systemic immune activation, supporting its potential as a combination immunotherapy agent.