TRPC4/TRPC5

ML204 has a 19-fold coupling to muscarinic receptor-coupled TRPC6 channel activation and inhibits TRPC4β-mediated intracellular Ca2+ rise with an IC50 value of 0.96 μM (HEK293 cells) [1]. The activation of Gq/11 by the M2 muscarinic receptor or endogenous M3-like muscarinic receptor, u-opioid, 5HT1A hematin, Gi/o, and TRPC4β is blocked by ML204[1]. ML204 inhibits TRPC5 channel activity that is triggered by LPS [3].

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ML204 was identified as a novel TRPC4 channel inhibitor following a high throughput fluorescent screen of the MLSMR library and SAR analysis of active compounds. ML204 inhibited calcium influx through TRPC4 channels activated by μ-opioid receptor stimulation with an IC50 value of 0.96 μM and exhibited 19-fold selectivity against TRPC6 channels in similar fluorescent assays. ML204 blocked TRPC4 channels in an electrophysiological assay with an IC value of 2.6 μM and was also active in fluorescent and electrophysiological assays in which TRPC4 channels were activated by different mechanisms, indicating direct block of TRPC4 channels. Selectivity for block of TRPC4 channels was examined in fluorescent and electrophysiological experiments against closely related TRPC channels and more distantly related TRPV, TRPA and TRPM channels, and against non-TRP ion channels. ML204 afforded good selectivity (19-fold) against TRPC6 channels and more modest selectivity against TRPC3 and TRPC5 (9-fold) channels. Little or no block of TRPV, TRPA, TRPM or voltage-gated ion channels was observed. ML204 exhibited properties useful for a variety of in vitro investigations[2].

In LPS-injected mice, ML204 (1 mg/kg; subcutaneous injection; twice daily; for 5 days) produced activity linked to increased hypothermia and decreased quantities of peritoneal leukocytes and cytokines [4]. Dual TRPC4/TRPC5 blockade by ML204 increased mortality and hypothermia in thioredoxin-treated LPS mice but preserved macrophage's ability to phagocytose. TRPC5 deletion did not alter body temperature but promoted additional accumulation of peritoneal leukocytes and inflammatory mediator release in thioredoxin-administered LPS mice. Thioredoxin diminished macrophage-mediated phagocytosis in wild-type but not TRPC5 knockout animals. TRPC5 ablation did not affect LPS-induced responses. However, ML204 caused mortality associated with exacerbated hypothermia and decreased peritoneal leukocyte numbers and cytokines in LPS-injected mice. These results suggest that bacterial thioredoxin effects under LPS stimuli are mediated by TRPC4 and TRPC5, shedding light on the additional mechanisms of bacterial virulence and on the pathophysiological roles of these receptors.

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Notably, ML204-perfused mice were protected from PS-induced FPE (Figure 7A). These results were quantified in a blinded fashion by counting the number of FPs over a measured length of GBM in TEM images (n = 90–105 images per group). By this analysis, ML204-perfused mice showed significant protection from the effects of PS (Figure 7B). These results are in line with our observations in Trpc5-KO mice.[3]
Next, we tested the effect of ML204 in the LPS model. ML204 was injected (20 mg/kg/d i.p.) twice at 12-hour intervals after injection of LPS. Control PBS-injected mice had no observable structural changes and no albuminuria (Figure 7, C–E). LPS injection resulted in FPE (Figure 7D), although this was milder than the PS-induced FPE. Importantly, the ML204-treated mice were protected from LPS-induced FPE and albuminuria[3].

Rac1 activation assay.[3]
Rac1 activation assays were done as previously described, with some modifications. Podocytes were treated with 300 μg/ml PS for 1 hour, followed by harvest and Rac1 pulldown experiments. In the ML204 experiments, cells were pretreated with 30 μM ML204 for 20 minutes before PS was applied. Activated Rac1 was analyzed with a commercial Rac1 activation assay kit using a GST-tagged fusion protein corresponding to the p21-binding domain (PBD; residues 67–150) of human PAK-1, according to the manufacturer’s instructions. After pulldown, the eluted active Rac1 was detected by immunoblotting using a mouse monoclonal Rac1 antibody. Total Rac1 and GADPH were measured in the cell lysates used for the pulldown studies and served as loading controls.

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Electrophysiology.[3]
Patch-clamp electrophysiology was performed in the whole-cell configuration or on outside-out patches. Patch pipettes with resistances of 3–4 MΩ were pulled from borosilicate glass with a P-97 puller and filled with a solution containing 135 mM CH3SO3Cs, 10 mM CsCl, 3 mM MgATP, 0.2 mM NaGTP, 0.2 mM EGTA, 0.13 mM CaCl2, and 10 mM HEPES (pH 7.3) with CsOH. The bath solution contained 135 mM CH3SO3Na, 5 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4) with NaOH. Angiotensin II (500 nM), LPS (100 μg/ml), and ML204 (10 μM) were applied to the bath solution. Whole-cell currents were recorded from –100 mV to +100 mV voltage ramps over 400 ms and a holding potential of –60 mV. For single-channel recordings in the outside-out configuration, we used a voltage step protocol from –100 mV to +100 mV delivered at 20-mV intervals and a holding potential of 0 mV. Average pipette resistance filled with pipette solution was 3–5 MΩ. Data were sampled at 10 kHz and filtered at 5 kHz. Single-channel data were further off-line filtered at 500 Hz before analysis. In single-channel traces, currents were idealized using a manually defined amplitude criterion to assign ion channel opening and closing transitions. Ensemble averages were expressed as Po (average current divided by unitary current amplitude and number of channels per patch) and plotted as histograms. All data were acquired at room temperature and analyzed using pClamp 10.

PS, LPS, and Cch treatment of cultured podocytes.[3]
Differentiated cultured podocytes grown at >90% confluence were incubated with 1–30 μM ML204 for 20 minutes, and then exposed to 300 μg/ml PS, 100 μg/ml LPS, or 100 μM Cch as appropriate. For PS experiments, as soon as changes in cell morphology could be seen by light microscopy (70–90 min), cells were fixed with 4% paraformaldehyde in PBS for 15 minutes before permeabilization with 0.1% Triton X-100 for 10 minutes. For LPS and Cch experiments, cells were fixed as above at 24 hours after treatment. For immunostaining, podocytes were incubated with synaptopodin “NT” antibody and detected with Alexa Fluor 488–conjugated secondary antibody. Actin structures were labeled with Alexa Fluor 594–conjugated phalloidin as described previously. 3 independent trials were analyzed, with 3 dishes per condition in each trial and 10 images per dish with comparable cell density. A total of 1,600–2,000 cells was analyzed for the PS/ML204 experiment and 1,400–1,600 cells for the PS/KD experiments. A total of 1,000 cells was analyzed for the LPS experiment and 1,400 cells for the Cch experiment. The number of cells was counted by DAPI staining and analysis with an automated script in ImageJ, which was subsequently corrected manually. Affected cells were defined as either collapsed with a very bright, condensed actin staining (for PS experiments), or cells without clearly visible stress fibers (for LPS and Cch experiments), as previously described. Images were acquired with a Zeiss LSM510 upright confocal microscope. Images from an optical slice of 3–4 μm were acquired using Zeiss Pascal software. Statistical significance was evaluated by ANOVA and Dunnett’s multiple-comparison test.

Animal/Disease Models: Non-fasted male C57BL/6 (2-3 months) [4]
Doses: 1 mg/kg
Route of Administration: subcutaneous injection twice (two times) daily for 5 days (before LPS injection)
Experimental Results: LPS-small Lower body temperature in mice induces systemic inflammatory responses associated with increased mortality.

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LPS-induced albuminuria.[3]
Induction of albuminuria in male WT and Trpc5-KO mice (20–25 g BW) by LPS injection was done as previously described, with some modifications. At 48 hours prior to injection, baseline urine was collected for 24 hours in metabolic cages. LPS (15 μg/g i.p., 1 mg/ml) was injected twice, at the 0- and 24-hour time points. PBS was injected i.p. twice, at 12 and 36 hours, to avoid dehydration. ML204 (20 mg/kg/d i.p.) was injected at 12 and 24 hours. Urine was collected for a 24-hour period beginning 24 hours after initial LPS injection using metabolic cages. To quantify the levels of albuminuria, 10 μl urine was analyzed by SDS-PAGE. Bovine serum albumin standards (0.25, 0.5, 1.0, 2.5, and 5.0 μg) were run on the same gel and used to identify and quantify urinary albumin bands. Coomassie signals were quantified using ImageJ. The resulting values — the product of area size and mean gray value of each albumin standard band — were used for construction of a standard curve and its associated mathematical function. Subsequently, the values of the sample bands were translated into albumin concentrations, which were extrapolated to the 24-hour total urine volume. Results were assessed by ANOVA and Bonferroni’s multiple-comparison test.
For preparation of glomerular lysates for Western blotting, mice were treated as above and killed 36 hours after initial LPS injection. Mouse kidneys were perfused through the renal artery with Dynabeads for magnetic isolation of highly purified glomeruli. Protein extraction from isolated glomeruli, SDS-PAGE, and Western blotting was done as described previously, and proteins were detected with appropriate primary and secondary antibodies.

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PS model.[3]
Adult WT (n = 15) and Trpc5-KO (n = 7) littermate mice were anesthetized with pentobarbital and placed on a heat pad set at 37°C, and their kidneys were perfused in situ through the renal artery at a pressure of approximately 240 mm Hg and an infusion rate of 9 ml/min as previously described (58), with some modifications. First, kidneys were flushed with HBSS or with HBSS plus 10 μM ML204 at 37°C for 2 minutes, followed by perfusion with 2 mg/ml PS in HBSS or with PS plus ML204 at 37°C for 15 minutes. All vascular perfusion solutions were kept at 37°C throughout the duration of the experiment.

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Pharmacological Treatments[4]
C57BL/6, TRPC5+/+, and TRPC5−/− mice received a subcutaneous (s.c.) injection of phosphate-buffered saline (PBS) containing bacterial Trx (20 μg/150 μl/animal, twice a day; from E. coli) for 3 days prior to the induction of SIRS. In order to assess the role of TRPC4 and TRPC5 complexes in LPS-induced responses, C57BL/6 mice received ML204 [16, 21] (1 mg/kg, 150 μl/animal, twice a day) for 5 days and then LPS. In a separate set of experiments, C57BL/6 animals received ML204 (1 mg/kg, twice a day; in 6% dimethyl sulfoxide (DMSO) in PBS) for 2 days alone, and then, this drug was coinjected with bacterial Trx (20 μg/animal, twice a day) for another 3 days prior to LPS challenge. Vehicle-treated mice were used as controls.

[1]. Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels. J Biol Chem. 2011 Sep 23;286(38):33436-46.

[2]. Novel Chemical Inhibitor of TRPC4 Channels. Probe Reports from the NIH Molecular Libraries Program [Internet].

[3]. Inhibition of the TRPC5 ion channel protects the kidney filter. J Clin Invest. 2013 Dec 2; 123(12): 5298–5309.

[4]. Transient Receptor Potential Canonical Channels 4 and 5 Mediate Escherichia coli-Derived Thioredoxin Effects in Lipopolysaccharide-Injected Mice. Oxid Med Cell Longev. 2018 Jun 10;2018:4904696.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4β by μ-opioid receptors. ML204 inhibited TRPC4β-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 μm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4β currents activated through either μ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTPγS), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 μm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTPγS, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.[1]

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ML204 was identified as a novel TRPC4 channel inhibitor following a high throughput fluorescent screen of the MLSMR library and SAR analysis of active compounds. ML204 inhibited calcium influx through TRPC4 channels activated by μ-opioid receptor stimulation with an IC50 value of 0.96 μM and exhibited 19-fold selectivity against TRPC6 channels in similar fluorescent assays. ML204 blocked TRPC4 channels in an electrophysiological assay with an IC value of 2.6 μM and was also active in fluorescent and electrophysiological assays in which TRPC4 channels were activated by different mechanisms, indicating direct block of TRPC4 channels. Selectivity for block of TRPC4 channels was examined in fluorescent and electrophysiological experiments against closely related TRPC channels and more distantly related TRPV, TRPA and TRPM channels, and against non-TRP ion channels. ML204 afforded good selectivity (19-fold) against TRPC6 channels and more modest selectivity against TRPC3 and TRPC5 (9-fold) channels. Little or no block of TRPV, TRPA, TRPM or voltage-gated ion channels was observed. ML204 exhibited properties useful for a variety of in vitro investigations.[2]

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An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.[3]

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Thioredoxin plays an essential role in bacterial antioxidant machinery and virulence; however, its regulatory actions in the host are less well understood. Reduced human Trx activates transient receptor potential canonical 5 (TRPC5) in inflammation, but there is no evidence of whether these receptors mediate bacterial thioredoxin effects in the host. Importantly, TRPC5 can form functional complexes with other subunits such as TRPC4. Herein, E. coli-derived thioredoxin induced mortality in lipopolysaccharide- (LPS-) injected mice, accompanied by reduction of leukocyte accumulation, regulation of cytokine release into the peritoneum, and impairment of peritoneal macrophage-mediated phagocytosis.[4]

C15H18N2

226.32

226.147

5465-86-1

ML204 hydrochloride;2070015-10-8

230710

Light yellow to yellow ointment

1.1±0.1 g/cm3

404.4±33.0 °C at 760 mmHg

198.4±25.4 °C

0.0±0.9 mmHg at 25°C

1.617

3.92

0

2

1

17

247

0

N1(C2C([H])=C(C([H])([H])[H])C3=C([H])C([H])=C([H])C([H])=C3N=2)C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

USYRQXDHKXGTCK-UHFFFAOYSA-N

InChI=1S/C15H18N2/c1-12-11-15(17-9-5-2-6-10-17)16-14-8-4-3-7-13(12)14/h3-4,7-8,11H,2,5-6,9-10H2,1H3

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (11.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (11.05 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (11.05 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 25.0 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.)