Chemical and pharmacological characterization of the TRPML calcium channel blockers ML-SI1 and ML-SI3

The members of the TRPML subfamily of non-selective cation channels (TRPML1-3) are involved in the regulation of important lysosomal and endosomal functions, and mutations in TRPML1 are associated with the neurodegenerative lysosomal storage disorder mucolipidosis type IV. For in-depth investigation of functions and (patho)physiological roles of TRPMLs, membrane-permeable chemical tools are urgently needed. But hitherto only two TRPML inhibitors, ML-SI1 and ML-SI3, have been published, albeit without clear information about stereochemical details. In this investigation we developed total syntheses of both inhibitors. ML-SI1 was only obtained as a racemic mixture of inseparable diastereomers and showed activator-dependent inhibitory activity. The more promising tool is ML-SI3, hence ML-SI1 was not further investigated. For ML-SI3 we confirmed by stereoselective synthesis that the trans-isomer is significantly more active than the cis-isomer. Separation of the enantiomers of trans-ML-SI3 further revealed that the (-)-isomer is a potent inhibitor of TRPML1 and TRPML2 (IC₅₀ values 1.6 and 2.3 μM) and a weak inhibitor (IC₅₀ 12.5 μM) of TRPML3, whereas the (+)-enantiomer is an inhibitor on TRPML1 (IC₅₀ 5.9 μM), but an activator on TRPML 2 and 3. This renders the pure (-)-trans-ML-SI3 more suitable as a chemical tool for the investigation of TRPML1 and 2 than the racemate. The analysis of 12 analogues of ML-SI3 gave first insights into structure-activity relationships in this chemotype, and showed that a broad variety of modifications in both the N-arylpiperazine and the sulfonamide moiety is tolerated. An aromatic analogue of ML-SI3 showed an interesting alternative selectivity profile (strong inhibitor of TRPML1 and strong activator of TRPML2).