Targeting Ion Channel TRPM4.

The transient receptor potential melastatin 4 (TRPM4) ion channel is ubiquitously expressed. Dysregulation and/or functional mutations of TRPM4 lead to several diseases. Within our studies, we screened for TRPM4 inhibitors and identified small molecules that block TRPM4 in the low µM range. Furthermore, we investigated the pathophysiology of TRPM4 in cardiac conditions, immune diseases and cancer using these novel inhibitors, molecular biology techniques and functional assays.

CBA (4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) inhibits TMEM206 mediated currents and TMEM206 does not contribute to acid-induced cell death in colorectal cancer cells.

Introduction: Upon activation at low pH, TMEM206 conducts Cl- ions across plasma and vesicular membranes. In a (patho)physiological context, TMEM206 was reported to contribute to acid-induced cell death in neurons, kidney and cervical epithelial cells. We investigated the role of TMEM206 in acid-induced cell death in colorectal cancer cells. In addition, we studied CBA as a new small molecule inhibitor for TMEM206. Methods: The role of TMEM206 in acid-induced cell death was studied with CRISPR/Cas9-mediated knockout and FACS analysis. The pharmacology of TMEM206 was determined with the patch clamp technique. Results: In colorectal cancer cells, TMEM206 is not a critical mediator of acid-induced cell death. CBA is a small molecule inhibitor of TMEM206 (IC50 = 9.55 µM) at low pH, at pH 6.0 inhibition is limited. Conclusion: CBA demonstrates effective and specific inhibition of TMEM206; however, its inhibitory efficacy is limited at pH 6.0. Despite this limitation, CBA is a potent inhibitor for functional studies at pH 4.5 and may be a promising scaffold for the development of future TMEM206 inhibitors.

Hypersensitive quantification of major astringency markers in food and wine by substoichiometric quenching of silicon-rhodamine conjugates.

Tannins are chemically diverse polyphenols in plant-derived products that not only show diverse biological activities but also play a crucial role in determining the sensory attributes of food and beverages. Therefore, their accurate and cost-effective quantification is essential. Here, we identified a novel fluorescence quenching mechanism of different synthetic rhodamine fluorophores, with a high selectivity towards tannic acid (TA) and catechin-3-gallate (C3G) compared to a structurally diverse panel of tannins and polyphenols. Specific chemical conjugates of silicon-rhodamine with alkyl linkers attached to bulky apolar moieties had a limit of detection near 500 pM and a linear range spanning 5-100 nM for TA. We validated the assay on 18 distinct red wine samples, which showed high linearity (R2 = 0.92) with methylcellulose precipitation with no interference from anthocyanins. In conclusion, a novel assay was developed and validated that allows the sensitive and selective quantification of major astringency markers abundant in food and beverages.