TRPM4 (IC50 = 1.5 μM)

TRPM4-IN-1 (compound 5) is a potent selective for TRPM4 currents in HEK293 cells that overexpress TRPM4 [1]. In LNCaP (cancer carcinoma) cells, TRPM4-IN-1 reversibly increases endogenous TRPM4 current. At night, TRPM4-IN-1 (50 μM) restores the functional expression of transcribed TRPM4 variant A432T [1].

Na+‐influx screening assay[1]
TRPM4‐expressing cells were plated in 96‐well black‐walled clear bottomed poly‐D‐lysine coated plates at a density of 30 000 cells per well in 100 μL of induction medium (described above). After being plated, cells were incubated for 48 h at 37°C in a 5% CO2 incubator. On the day of the assay, the induction medium was replaced with 100 μL per well of the assay buffer [in mM: 140 NMDG‐Cl, 10 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.2) with NMDG‐OH and 290 to 300 mOsM] and incubated for 45 min at room temperature (RT). Later, the assay buffer was replaced with 95 μL per well of dye loading solution prepared in assay buffer supplemented with 0.1% pluronic acid F‐127 (Teflabs, TX, USA) and 5 μM of Na+ sensitive dye, Asante Natrium Green–II (ANGII). The cells were incubated in the dark at RT for 45 min. After incubation, the dye loading solution was completely drained and replaced with 72 μL assay buffer and incubated in the dark for 20 min, followed by fluorescence readout on FLIPR TETRA® (Molecular Devices, CA, USA). For evaluation of the effects of TRPM4 inhibitors using the Na+ influx assay, 10 compound plates were prepared in assay buffer in 96‐well polystyrene plates. All stocks such as TRPM4-IN-5 were dissolved in DMSO. To initiate Na+ influx through activation of TRPM4, a 5× stimulus buffer plate containing (in mM) 700 NaCl, 4 CaCl2, 4 MgCl2, 40 HEPES, (pH 7.2 with NaOH) along with 50 μM of ionomycin was prepared in a 96‐well polystyrene plate. The background control wells contained stimulus buffer without ionomycin (W/O). Fluorophores were excited by the 488 nm line of an argon laser. Emission was filtered with a 540 ± 30 nm bandpass filter. Initial baseline fluorescence was measured at 1 Hz from the plate loaded with ANGII dye for 1 min. Followed by addition of 8 μL solution from 10× compound plate, fluorescence was monitored at an interval of 1 Hz for 5 min. Finally, to activate TRPM4, 20 μL of solution from the stimulus buffer plate was added, and fluorescence was acquired at 0.5 Hz intervals during and after addition for 5 min. Data from individual assay wells were normalized to initial baseline fluorescence. The initial rise in fluorescence was measured as area under the curve (AUC), and the counts were normalized using the formula {[(Ionomycin − Compound)/(Ionomycin − W/O Ionomycin)] × 100} to plot concentration–response curves for inhibitor hits. The experimenter for the screening experiments (L.C.O.) was blinded and was not aware of any chemical properties of the compounds (e.g. TRPM4-IN-5) until the best hits were validated. The steps are summarized as workflow, presented in Table 1.

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Electrophysiology[1]
Cells were plated in 35 mm poly‐D‐lysine coated dishes in induction medium [phenol red free DMEM medium] supplemented with 10% FBS and 1 μg·mL−1 tetracycline (Invitrogen). After being plated, cells were incubated for 48 h at 37°C in a 5% CO2 incubator. Electrophysiological recordings were performed in the inside‐out patch clamp configuration with patch pipettes (1–2 μm tip opening) pulled from 1.5 mm borosilicate glass capillaries using DMZ Universal puller. Pipette tips were polished to have a pipette resistance of 2–4 M Ω in the bath solution. The pipette solution contained (in mM) 150 NaCl, 10 HEPES, 2 CaCl2 (pH 7.4 with NaOH). The bath solution contained (in mM) 150 NaCl, 10 HEPES, 2 HEDTA (pH 7.4 with NaOH) as Ca2+‐free solutions. Solutions containing 0.1 to 2 mM Ca2+ were prepared by adding the appropriate concentration of CaCl2 without a Ca2+ chelator to a solution containing (in mM) 150 NaCl, 10 HEPES (pH 7.4 with NaOH) as reported previously (Zhang et al., 2005). Bath solutions with different Ca2+ concentrations were applied to cells by a modified rapid solution exchanger. Inhibitors in DMSO stock were diluted to appropriate concentrations in the pipette solution and applied to the extracellular side of the cells. Membrane currents were recorded with a Multiclamp 700B amplifier controlled by Clampex 10 via a Digidata 1332A. Data were low‐pass filtered at 5 kHz and sampled at 10 kHz. Experiments were performed at RT (20–25°C). For I‐V relations, currents were recorded using a stimulation protocol consisting of voltage steps of 200 ms from a holding potential of 0 mV ranging from −80 to +100 mV, followed by a tail voltage at −100 mV. For constructing concentration–response curves, steady‐state currents at +100 mV recorded in 300 μM Ca2+ were normalized to current in absence of any inhibitor. For rescue experiments with TRPM4-IN-5, before performing patch clamp recordings the culture medium was replaced with bath solution without any added inhibitor.

Western Blot Analysis[1]
Cell Types: HEK293 Cell
Tested Concentrations: 50 μM
Incubation Duration:
Incubation Duration: Overnight
Experimental Results: Partial rescue of total and surface expression of A432T.

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Compound cytotoxicity assay[1]
The cytotoxicity of compounds 4, 5 (TRPM4-IN-5) and 6 was studied in RAW 264.7 and HeLa cells for anti‐proliferative effects. The cells were seeded at 2000 cells per well in 100 μL culture media in a 96‐well plate. Following overnight incubation, different concentrations of the compounds were added and incubated together with the solvent control for 72 h at 37°C. After that, the cells were incubated with MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide, final concentration 0.5 mg·mL−1) for 4 h at 37°C and solubilized with DMSO. The plates were measured at 550 nm (absorbance) and the IC50 value related to cell survival/proliferation was calculated and plotted. Paclitaxel was used as a positive control. Experiments were performed in at least three independent experiments, each in triplicate.

[1]. Identification of potent and selective small molecule inhibitors of the cation channel TRPM4. Br J Pharmacol. 2018 Jun; 175(12): 2504–2519.

[2]. Optimizing TRPM4 inhibitors in the MHFP6 chemical space. Eur J Med Chem. 2019 Mar 15;166:167-177.

Background and Objectives: TRPM4 is a calcium-activated, non-selective cation channel expressed in multiple tissues and associated with various diseases. Due to the lack of highly efficient and selective inhibitors, TRPM4 has not yet been identified as a therapeutic target. We aimed to discover a series of novel small-molecule inhibitors with sub-micromolar activity and higher selectivity than previously reported TRPM4 inhibitors by combining computer simulations and cell screening methods. Experimental Methods: We developed a high-throughput screening-compatible assay that uses sodium-sensitive dyes to record TRPM4-mediated sodium influx in cells. We used this method to screen a group of compounds obtained through ligand-based virtual screening, using known weakly active and non-selective TRPM4 inhibitors as seed molecules. We validated the activity and selectivity of these lead compounds in TRPM4-overexpressing HEK293 cells and the endogenously TRPM4-expressing prostate cancer cell line LNCaP using conventional electrophysiological methods. This study investigated the chemical chaperone properties of compound 5 using Western blotting and electrophysiological experiments. Main results: We identified a series of halo-o-aminobenzoamide compounds with sub-micromolar inhibitory activity and good selectivity that inhibit the TRPM4 protein. In addition, we demonstrated for the first time that the most promising compound 5 discovered in this study was able to rescue a naturally occurring TRPM4 variant that exhibited loss of expression and function. Conclusions and implications: The discovery of compound 5—a highly potent and selective TRPM4 inhibitor with additional chemical chaperone properties—provides new opportunities for studying the role of TRPM4 in human diseases and developing clinical drug candidates. [1] We recently reported that 4-chloro-2-(2-chlorophenoxy)acetamamido)benzoic acid (CBA) is the first highly potent TRPM4 inhibitor, TRPM4 being a cation channel associated with heart disease and prostate cancer. This paper reports the structure-activity relationship (SAR) study of CBA and the resulting two new highly potent analogues. To design and interpret our structure-activity relationship (SAR), we used interactive, color-coded 3D plots that demonstrate the similarity between compounds calculated using MHFP6 (a MinHash fingerprint of up to six bonds). MHFP6 is a novel molecular fingerprint that outperforms other fingerprints in benchmarks for virtual screening studies. We further illustrate the universality of our approach by visualizing the structural diversity of active compounds with decoy molecules and drugs in benchmark studies. MHFP6 chemical space 3D plots may be universally helpful for the design, interpretation, and communication of results in structure-activity relationship studies. The improved WebMolCS is available at http://gdb.unibe.ch, and its code is available at https://github.com/reymond-group/webMolCS for offline use. [2]

C15H11CL2NO4

340.16

339.006

C, 52.97; H, 3.26; Cl, 20.84; N, 4.12; O, 18.81

351424-20-9

2264067

White to yellow solid powder

1.5±0.1 g/cm3

576.4±50.0 °C at 760 mmHg

302.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.656

4.95

2

4

5

22

407

0

C1=CC=C(C(=C1)OCC(=O)NC2=C(C=CC(=C2)Cl)C(=O)O)Cl

CVQCJPCMPGKEDH-UHFFFAOYSA-N

InChI=1S/C15H11Cl2NO4/c16-9-5-6-10(15(20)21)12(7-9)18-14(19)8-22-13-4-2-1-3-11(13)17/h1-7H,8H2,(H,18,19)(H,20,21)

4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid

TRPM4IN-5; TRPM4 IN5; TRPM4-IN-1; 4-chloro-2-[[2-(2-chlorophenoxy)acetyl]amino]benzoic acid; CBA; TRPM4-IN-5; CHEMBL4525597; 4-Chloro-2-(2-(2-chlorophenoxy)acetamido)benzoic acid; 4-chloro-2-{[(2-chlorophenoxy)acetyl]amino}benzoic acid; TRPM4-IN-5

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~244.97 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.11 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)