TRPC3/transient receptor potential canonical channel 3 (IC50 = 700 nM, for TRPC3-mediated Ca2+ influx)

In TRPC family members, Pyr3 directly and selectively inhibits TRPC3 channels. With an IC50 value of 700 nM, Pyr3 application suppressed TRPC3-mediated Ca2+ influx in a dose-dependent manner. At 0.3 μM, Pyr3 becomes noticeable, and at 3 μM, it is nearly finished. It's interesting to note that cells coexpressing TRPC3 and TRPC6 were able to suppress Ca2+ influx using Pyr3, but not cells coexpressing TRPC1 and TRPC5. Pyr3 inhibits Ang II-induced NFAT translocation, but Pyr2's inhibition is mild and concentration-dependent (Pyr3's IC50 value is 0.05 μM, while Pyr2's IC50 value is 2 μM) [1].

In this study, researchers examined Pyr3 in pressure overload-induced cardiac hypertrophy in vivo. Importantly, systolic and diastolic blood pressure, heart rate, mortality, body weight, and weight for liver, lung, and heart were unaffected by chronic treatment with Pyr3 (0.1 mg·kg−1·day−1) in sham operated mice (Fig. S16A and Tables S1 and S2 in SI Appendix). In addition, transverse aortic constriction (TAC) operation significantly increased left ventricular end-systolic pressure (ESP) in mice treated with vehicle or Pyr3 (Tables S1 and S2 in SI Appendix), suggesting that pressure overload was equally induced in these mice. Strikingly, increased size of the heart by 1-week TAC operation was significantly attenuated by Pyr3 (Fig. 6 A and B and Fig. S16B in SI Appendix). The Pyr3 effect refers to concentric hypertrophy, because the ratio of internal ventricular radius at end diastole (r) to ventricular wall thickness (h) was significantly decreased in echocardiography in mid transverse heart sections (Fig. 6A and Fig. S16C in SI Appendix), in contrast to fractional shortening (FS) and the right ventricle unaffected by TAC (Fig. S16 D and E in SI Appendix). The TAC-induced increase in expression of atrial natriuretic peptide (ANP) mRNA, a reliable marker for cardiac hypertrophy, was also suppressed by Pyr3 (Fig. 6C). Six-weeks TAC operation induced an r/h ratio increase characteristic of dilated hypertrophy (Fig. S16F in SI Appendix), and deterioration of FS and elevation of weight-to-tibia length ratio of the left ventricle (LVW/TL) in good correlation with systolic pressure gradient. These symptoms were suppressed by Pyr3 (Fig. 6 D and E). Thus, Pyr3 is potent against concentric and dilated cardiac hypertrophy[1].

Electrophysiology. [1]
Whole-cell mode of the patch-clamp technique was performed on HEK293 or HEK293T cells at room temperature (22–25°C) with an EPC-9 or Axopatch 200B patch-clamp amplifier as previously described. Voltage-clamp experiments were performed at a holding potential of –50 mV or –60 mV, and recordings were sampled at 2.0 kHz and filtered at 2.9 kHz. The I-V relationships were determined using a 50-, 200-, or 400-ms voltage ramp from –100 mV to +100 mV or from –120 mV to +80 mV. 6 Pipette resistance ranged from 2 to 6 megohm when filled with the pipette solution described below. The series resistance was electronically compensated to > 50%. An external solution contained (in mM): For TRPC3 and C6, 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, 10 HEPES (pH 7.4 adjusted with NaOH). For TRPM4b, 110 NaCl, 5 CaCl2, 10 glucose, 10 HEPES (pH 7.4 adjusted with NaOH). The pipette solution contained (in mM): For mAChR-activated TRPC3 current, 95 CsOH, 95 aspartate, 40 CsCl, 4 MgCl2, 5 EGTA, 2 ATPNa2, 5 HEPES, 8 creatine phosphate (pH 7.2 adjusted with CsOH). For OAG-induced TRPC3 current, 130 CsOH, 130 glutamate, 3.1 MgCl2, 2.8 CaCl2, 10 EGTA, 2 ATPNa2, 0.3 GTPNa2, 10 HEPES (pH 7.2 adjusted with CsOH). For TRPC6 current, 145 CsOH, 145 aspartate, 2 MgCl2, 0.3 CaCl2, 10 EGTA, 10 HEPES (pH 7.2 adjusted with CsOH). For TRPM4b current, 110 CsOH, 110 aspartate, 4 MgCl2, 8.4 CaCl2, 10 EGTA, 4 ATPNa2, 10 HEPES (pH 7.4 adjusted with CsOH). The osmolarity of the external solutions was adjusted to about 300 mOSM. No significant difference (P = 0.74) was observed in membrane capacitance between the control (22.7 ± 2.4 pF) and Pyr3-treated (21.2 ± 2.5 pF) HEK293 cells expressing TRPC3.

Measurement of NFAT activity and hypertrophic growth of cardiomyocytes was performed as described. Briefly, neonatal rat cardiomyocytes were isolated from 1−2 day-old Sprague-Dawley rats, and cultured on the gelatin-coated dishes or laminin-coated silicon rubber dishes. The cDNAs (pNFAT-Luc, pBNP-Luc and pRL-SV40) were transfected by Fugene 6 and a recombinant adenovirus encoding GFP-fused amino terminal region of NFAT4 isoform was infected at 100 MOI in serum-free medium. Forty-eight h after transfection, cells were stimulated with Ang II (100 nM) or mechanical stretch by 20%. Cells were treated with Pyr2 or Pyr3 for 20 min prior to the stimulation with Ang II or mechanical stretch. The cells were washed, fixed, and then stained with Alexa Fluor 594-phalloidin to visualize actin filaments. Protein synthesis was measured by [3 H]leucine incorporation. Cells were treated with Pyr2 or Pyr3 for 20 min prior to the stimulation with Ang II (100 nM), and [3 H]leucine (1 10 μCi/ml) was add to the culture medium and further incubated for 6 h. The incorporated [3 H]leucine was measured using liquid-scintillation counter. IC50 values were determined by using GraphPad Prism.

TAC surgery, hemodynamic measurements, and histological analyses were performed as described. Briefly, pressure overload was evoked by the surgical TAC. TAC was performed on 6-week-old male C57BL6j mice. A mini-osmotic pump filled with polyethylene glycol or Pyr3 (0.1 mg/kg/day) was implanted intraperitoneally in mice 3 d before 1 week of TAC. In the case of 6 weeks of TAC, Pyr3 was treated 3 d after TAC. Six weeks after TAC operation, transthoracic echocardiography was performed using ALOKA ultrasonic image analyzing system (SSD-5500) equipped with 7.5 MHz imaging transducer. Hemodynamic measurements were taken by inserting a micronanometer catheter from the right common carotid artery into the aorta and then the left ventricle. To calculate pressure gradient, distal arterial pressure was measured using tail-cuff detection system. After heart weight was measured, total RNA was isolated. The quantitative measurement of ANP mRNA expression was performed by real time RT-PCR. Primers and probe for mouse ANP mRNA are as follow: forward primer, 5’-CATCACCCTGGGCTTCTTCCT-3’; reverse primer, 5’-TGGGCTCCAATCCTGTCAATC-3’; TaqMan® probe, 5’-ATTTCAAGAACCTGCTAGACCACCTGGA-3’.

[1]. Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound. Proc Natl Acad Sci U S A. 2009 Mar 31;106(13):5400-5.

Canonical transient receptor potential (TRPC) channels control influxes of Ca(2+) and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca(2+) influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca(2+) response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca(2+)-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.[1]

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Background and purpose: Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation. Experimental approach: Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca(2+) entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca(2+) entry mediated by Orai channels. The consequences of inhibitory effects on Ca(2+) signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation. Key results: Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca(2+) entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca(2+) entry as compared with TRPC3-mediated Ca(2+) entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation. Conclusions and implications: The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca(2+) entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca(2+) channels.https://pubmed.ncbi.nlm.nih.gov/22862290/

C16H11CL3F3N3O3

456.62

454.982

C, 42.09; H, 2.43; Cl, 23.29; F, 12.48; N, 9.20; O, 10.51

1160514-60-2

56964346

White to off-white solid powder

4.964

1

7

6

28

621

0

RZHGONNSASQOAY-UHFFFAOYSA-N

InChI=1S/C16H11Cl3F3N3O3/c1-2-28-15(27)10-7-23-25(12(10)16(20,21)22)9-5-3-8(4-6-9)24-14(26)11(17)13(18)19/h3-7H,2H2,1H3,(H,24,26)

ethyl 1-[4-(2,3,3-trichloroprop-2-enoylamino)phenyl]-5-(trifluoromethyl)pyrazole-4-carboxylate

Pyr 3; Pyr-3; 1160514-60-2; 1H-Pyrazole-4-carboxylic acid, 1-[4-[(2,3,3-trichloro-1-oxo-2-propen-1-yl)amino]phenyl]-5-(trifluoromethyl)-, ethyl ester; CHEMBL4177187; ethyl 1-(4-(2,3,3-trichloroacrylamido)phenyl)-5-(trifluoromethyl)-1h-pyrazole-4-carboxylate; RZHGONNSASQOAY-UHFFFAOYSA-N; GTPL4293; SCHEMBL12274132; Pyr3

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 : ≥ 125 mg/mL (~273.74 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.56 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 20.8 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: ≥ 2.08 mg/mL (4.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (4.56 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.)