Background and PurposeTRPM4 is a calcium‐activated non‐selective cation channel expressed in many tissues and implicated in several diseases, and has not yet been validated as a therapeutic target due to the lack of potent and selective inhibitors. We sought to discover a novel series of small‐molecule inhibitors by combining in silico methods and cell‐based screening assay, with sub‐micromolar potency and improved selectivity from previously reported TRPM4 inhibitors.Experimental ApproachHere, we developed a high throughput screening compatible assay to record TRPM4‐mediated Na+ influx in cells using a Na+‐sensitive dye and used this assay to screen a small set of compounds selected by ligand‐based virtual screening using previously known weakly active and non‐selective TRPM4 inhibitors as seed molecules. Conventional electrophysiological methods were used to validate the potency and selectivity of the hit compounds in HEK293 cells overexpressing TRPM4 and in endogenously expressing prostate cancer cell line LNCaP. Chemical chaperone property of compound 5 was studied using Western blots and electrophysiology experiments.Key ResultsA series of halogenated anthranilic amides were identified with TRPM4 inhibitory properties with sub‐micromolar potency and adequate selectivity. We also showed for the first time that a naturally occurring variant of TRPM4, which displays loss‐of‐expression and function, is rescued by the most promising compound 5 identified in this study.Conclusions and ImplicationsThe discovery of compound 5, a potent and selective inhibitor of TRPM4 with an additional chemical chaperone feature, revealed new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates.

Abbreviations CD, current density; CRC, colorectal cancer; TBC, tumor border configuration; TRPM4, transient receptor potential melastatin-4 channel.

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4−/− mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.

The cardiac voltage-gated sodium channel Na v 1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Na v 1.5 N-terminal domain (NTD) exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Na v 1.5 NTD: Y87C-here newly identified-, R104W, and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Na v 1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Na v 1.5 at the cell surface and in whole-cell lysates. Localization of Na v 1.5 WT channel with the ER did not change in the presence of variants, as shown by transfected and stained rat neonatal cardiomyocytes. We demonstrated that calmodulin binds the Na v 1.5 NTD using in silico modeling, SPOTS, pull-down, and proximity ligation assays. Calmodulin binding to the R121W variant and to a Na v 1.5 construct missing residues 80-105, a predicted calmodulin-binding site, is impaired. In conclusion, we describe the new natural BrS Na v 1.5 variant Y87C and present first evidence that calmodulin binds to the Na v 1.5 NTD, which seems to be a determinant for the DNE.

Yeast p20 is a small, acidic protein that binds eIF4E, the cap-binding protein. It has been proposed to affect mRNA translation and degradation, however p20′s function as an eIF4E-binding protein (4E-BP) and its physiological significance has not been clearly established. In this paper we present data demonstrating that p20 is capable of binding directly to mRNA due to electrostatic interaction of a stretch of arginine and histidine residues in the protein with negatively charged phosphates in the mRNA backbone. This interaction contributes to formation of a ternary eIF4E/p20/capped mRNA complex that is more stable than complexes composed of capped mRNA bound to eIF4E in the absence of p20. eIF4E/p20 complex was found to have a more pronounced stimulatory effect on capped mRNA translation than purified eIF4E alone. Addition of peptides containing the eIF4E-binding domains present in p20 (motif  YTIDELF), in eIF4G (motif  YGPTFLL) or Eap1 (motif  YSMNELY) completely inhibited eIF4E-dependent capped mRNA translation (in vitro), but had a greatly reduced inhibitory effect when eIF4E/p20 complex was present. We propose that the eIF4E/p20/mRNA complex serves as a stable depository of mRNAs existing in a dynamic equilibrium with other complexes such as eIF4E/eIF4G (required for translation) and eIF4E/Eap1 (required for mRNA degradation).