ICA-121431 targets voltage-gated sodium channels, with high selectivity for human Nav1.3 (IC50 = 18 ± 5 nM) and negligible inhibition of human Nav1.5 (IC50 >10 µM) and human Nav1.7 (IC50 >10 µM) [1]

Human Nav1.3 and the amino acid residues that might determine this channel's selectivity over other related Nav channels, such as Nav1.7 and Nav1.5, are interacting with ICA-121431. Data produced with a pulse protocol that involves an 8-s step to a voltage that inactivates half of the channels before a 20-ms test pulse using traditional patch clamp electrophysiological recording[1]. ICA-121431 exhibits IC50s of 0.013 µM, >30 µM, and 12 µM for Wild type hNav1.3, hNav1.5, and hNav1.7, respectively[1]. hNav1.3 M1 (S1510Y), hNav1.3 M2 (R1511W), hNav1.3 M3 (E1559D), hNav1.3 M1, 3 (S1510Y/E1559D), hNav1.3 M2, 3 (R1511W/E1559D), hNav1.3 M1, 2, 3 (S1510Y/R1511W/E1559D), and hNav1.7 M1, 2, 3 (Y1537S/W1538R/D1586E) have IC50 values of 0.1 µM, 0.37 µM, 1.1 µM, 1.3 µM, 11.6 µM, and 0.032 µM, respectively[1]. hNav1.3/hNav1.5 S1-S4, hNav1.3/hNav1.5 S3-S4, hNav1.3/hNav1.5 S5-S6, hNav1.3/hNav1.7 S1, hNav1.3/hNav1.7 S2, and hNav1.3/hNav1 are among the hNav channels that ICA-121431 is against. The IC50 values of hNav1.3/hNav1.7 S5-S6, 7 S3-S4, 1.2 µM, 11 µM, 2.0 µM, 0.045 µM, 0.030 µM, 0.30 µM, 1.0 µM, and 0.024 µM, respectively, were reported[1].

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1. ICA-121431 (1 µM) reduced the amplitude of human Nav1.3 current elicited by a 20-ms voltage step to 0 mV from −120 mV; after an 8-s conditioning voltage step to −60 mV (to inactivate ~50% of available channels), the inhibitor still exhibited inhibitory effects on Nav1.3 current. Current traces were normalized to ensure control traces had the same relative amplitude [1]
2. The voltage dependence of human Nav1.3 inactivation was altered by ICA-121431: the half-inactivation voltage (Vh) of Nav1.3 was −67 ± 2 mV under control conditions, while in the presence of 0.01 µM, 0.1 µM, and 1 µM ICA-121431, the Vh shifted to −78 ± 2 mV, −86 ± 2 mV, and −93 ± 2 mV, respectively. Peak current amplitudes were measured during test pulses to 0 mV after an 8-s conditioning depolarization to different potentials, and values were normalized to the largest amplitude current after a prepulse to −120 mV, with inactivation curves fitted by the Boltzmann equation [1]
3. ICA-121431 (1 µM) showed use-dependent inhibition of human Nav1.3: when a train of 20-ms voltage steps from −120 mV to 0 mV was applied at 10 Hz, the current amplitude (normalized to control) of Nav1.3 was progressively reduced, which was different from the inhibition pattern of 10 nM TTX [1]
4. ICA-121431 (1 µM) had no significant inhibitory effect on human Nav1.5 and Nav1.7 currents elicited by a 20-ms voltage step to 0 mV after an 8-s conditioning voltage step to inactivate ~50% of available channels [1]
5. Chimeric sodium channel experiments demonstrated that potent inhibition by ICA-121431 was only observed in chimeras containing Nav1.3 Domain 4; substitution of subregions of Nav1.3 Domain 4 with equivalent regions from Nav1.5 or Nav1.7 significantly reduced the inhibitory potency of ICA-121431 [1]
6. Mutagenesis studies identified three key residues (M1: S1510Y, M2: R1511W, M3: E1559D) in the S1-S4 voltage-sensor region of Nav1.3 Domain 4 that determine the selectivity of ICA-121431; single or combined mutations of these residues in Nav1.3 to the corresponding residues of Nav1.7 altered the IC50 of ICA-121431 inhibition, while amitriptyline (a local anesthetic) showed different sensitivity patterns to these mutations [1]
7. The triple mutant (S1510Y/R1511W/E1559D) of Nav1.3 and the reverse triple mutant (Y1537S/W1538R/D1586E) of Nav1.7 altered the inhibitory effects of ICA-121431 and tetracaine, and also affected biophysical properties (current-voltage relationships, voltage dependence of inactivation, time course of recovery from inactivation) of Nav1.3 and Nav1.7 channels [1]

1. PatchXpress automated electrophysiology assay: Human Nav1.3, Nav1.5, and Nav1.7 currents were recorded using PatchXpress. For Nav1.3, current traces were measured during a 20-ms voltage step to 0 mV from a holding potential (HP) of −120 mV, or after an 8-s conditioning voltage step to −60 mV (to inactivate ~50% of available channels). The effect of ICA-121431 (1 µM) on current amplitude was evaluated, and current traces were normalized to control traces [1]
2. IonWorks Quattro assay: Inhibition of sodium currents by ICA-121431 was assessed using a protocol consisting of a 500-ms step from −120 mV to 0 mV to inactivate channels, followed by a 100-ms recovery at −100 mV and a 20-ms test pulse to 0 mV to determine inhibition. This assay was used to measure the concentration dependence of ICA-121431 inhibition on Nav1.3 (IC50 = 18 ± 5 nM, n=6), Nav1.5 (IC50 >10 µM, n=4), and Nav1.7 (IC50 >10 µM, n=6) [1]
3. Conventional patch clamp assay: This assay was used to measure the concentration dependence of ICA-121431 inhibition on Nav1.3 chimeras (with subregions of Domain 4 replaced by Nav1.5/Nav1.7 sequences) and Nav1.3 mutants. The protocol included an 8-s conditioning voltage step from −120 mV to a potential producing half-maximal inactivation, followed by a 20-ms recovery at −100 mV and a 20-ms test pulse to 0 mV to measure inhibition. IC50 values for chimeras and mutants were calculated and compared with wild-type Nav1.3, Nav1.5, and Nav1.7 [1]
4. Biophysical property assessment using PatchXpress: Peak current amplitudes of wild-type and mutant Nav1.3/Nav1.7 were measured during 500-ms voltage steps to various membrane potentials from a holding potential of −120 mV (for current-voltage relationships); peak current amplitudes were also measured during test pulses to −20 mV after 500-ms conditioning depolarizations to different potentials (for voltage dependence of inactivation, fitted by Boltzmann equation: 1/1+exp[(Vh − V)/k]); recovery from inactivation was assessed by measuring peak current amplitudes during test pulses to −20 mV after a variable recovery time following a 500-ms conditioning depolarization to 0 mV (fitted by two-phase exponential association equation) [1]

[1]. Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels.Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):E2724-32.

1. ICA-121431 is a nanomolar potent voltage-gated sodium channel inhibitor, belonging to a unique class of inhibitors. It interacts with the S1-S4 voltage sensor fragment of the Nav channel homology domain 4, which differs from the pore regions and binding sites of other small molecule inhibitors (e.g., local anesthetics, TTX) [1]. 2. The interaction region of ICA-121431 with Nav1.3 domain 4 contains amino acid residue E1559, which is also crucial for site 3, which regulates Nav channel inactivation by α-scorpion venom and sea anemone polypeptide toxin [1]. 3. The amino acid residues in the extracellular regions of the S2 and S3 fragments of the Nav1.3 transmembrane region are the main factors determining the Nav channel subtype selectivity and species sensitivity of ICA-121431. [1]
4. ICA-121431 and PF-04856264 (a Nav1.7 selective inhibitor) are structurally related and both interact with the voltage sensor of the 4th domain of the Nav channel, providing a framework for developing subtype-selective Nav channel inhibitors targeting non-pore regions. [1]

C23H19N3O3S2

449.54

449.086

313254-51-2

998021

White to off-white solid powder

1.4±0.1 g/cm3

1.693

3.89

2

6

7

31

659

0

URSQNPPONHUJDL-UHFFFAOYSA-N

InChI=1S/C23H19N3O3S2/c27-22(21(17-7-3-1-4-8-17)18-9-5-2-6-10-18)25-19-11-13-20(14-12-19)31(28,29)26-23-24-15-16-30-23/h1-16,21H,(H,24,26)(H,25,27)

2,2-diphenyl-N-[4-(1,3-thiazol-2-ylsulfamoyl)phenyl]acetamide

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 (5.56 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 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 (5.56 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 (5.56 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 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.)