A nonhydrolyzable candesartan cilexetil analog reveals synergistic activation as a tractable mechanism for TMEM175 modulation.

TMEM175 is a lysosomal cation channel essential for maintaining lysosomal pH and function. Dysregulation of TMEM175 has been implicated in Parkinson's disease, highlighting the need for small-molecule modulators to probe its physiological and therapeutic roles. We previously screened an FDA-approved compound library for TMEM175 modulators using a plasma membrane overexpression system that enables functional analysis of channel activity. Here, we report the pharmacological characterization of the most potent hit, candesartan cilexetil. In fluorescence-based thallium flux, automated patch-clamp, and manual patch-clamp assays, candesartan cilexetil robustly activates TMEM175 with efficacy comparable with the reference activator DCPIB, whereas its hydrolyzed metabolite candesartan is inactive. Candesartan cilexetil is active only when applied to the extracellular (lysosome lumen-equivalent) side of the channel and is inactive from the cytosolic-facing side. To determine whether activation requires the intact prodrug, we generated analogs that modify or eliminate the cilexetil and ester functionalities. Structure-activity studies show that selected modifications of the cilexetil moiety reduce potency while preserving maximal efficacy, whereas more extensive modification markedly reduces intrinsic activity, indicating that it is an essential pharmacophoric element rather than a simple membrane-permeating handle. Manual patch-clamp wash-off experiments further demonstrate direct, reversible activation without requiring membrane permeation or hydrolysis. Unexpectedly, the nonhydrolyzable analog VU0982645 exhibits minimal intrinsic activity yet produces robust synergistic activation with DCPIB. Together, these findings establish the cilexetil handle as a key pharmacophoric element and support synergistic modulation as a tractable mechanism for activating TMEM175.