The novel quinoline derivative SKA-346 as a K_Ca3.1 channel selective activator

Abstract

The calcium-activated K_Ca3.1 channel plays a crucial role in T-cell immune response. Genetic manipulation of T-cells to upregulate the expression of K⁺ channels has been shown to boost T-cell cytotoxicity in cancer. Here, we aimed to identify and characterize an activator that would augment K_Ca3.1 currents without affecting other channels. We synthesized five quinoline derivatives and used electrophysiology to screen them on K_Ca3.1 and a panel of 14 other ion channels. One quinoline derivative, SKA-346, activated K_Ca3.1 with an EC₅₀ of 1.9 μM and showed selectivity against the other channels. In silico analysis using RosettaLigand and GLIDE demonstrated a well-converged pose of SKA-346 in a binding pocket at the interface between the calmodulin N-lobe and the S₄₅A helix in the S4–S5 linker of the K_Ca3.1 channel. SKA-346 (30 mg kg⁻¹), tolerated by mice after intra-peritoneal administration, exhibited a peak plasma concentration of 6.29 μg mL⁻¹ (29.2 μM) at 15 min and a circulating half-life (t_1/2) of 2.8 h. SKA-346 could serve as a template for the development of more potent K_Ca3.1 activators to enhance T-cell cytotoxicity in cancer.

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1 Introduction

Potassium (K⁺) channels play a key role in the immune function. Human T-cells use the voltage-dependent K_V1.3 channel and the intermediate-conductance Ca²⁺-activated K_Ca3.1 channel to regulate membrane potential and K⁺ efflux.¹ Following activation, both naïve and central memory T-cells transcriptionally up-regulate K_Ca3.1 channels (<20 channels per cell to >500 channels per cell) without a change in K_V1.3 expression, while effector memory T-cells upregulate K_V1.3 channels (200 channels per cell to 1500 channels per cell) without altering K_Ca3.1 expression. Consequently, K_Ca3.1 plays an important functional role in activated naïve and central memory T-cells, while K_V1.3 modulates the function of effector memory T-cells.² K_Ca3.1 channels are also expressed in other cell types, including macrophages,³ mast cells,⁴ microglia,⁵ fibroblasts,⁶ dedifferentiated proliferating smooth muscles,^7,8 airway epithelium⁹ and vascular endothelium.¹⁰

Unlike large-conductance Ca²⁺-activated K⁺ channels which directly bind Ca²⁺, intracellular Ca²⁺ interacts indirectly with K_Ca3.1,¹¹ through calmodulin, constitutively bound to the C-terminal region of K_Ca3.1. Binding of Ca²⁺ to channel-bound calmodulin induces a conformational change that opens the channel pore and increases K⁺ efflux.¹² K⁺ efflux results in hyperpolarization of the membrane potential, which provides the electrochemical gradient needed to drive Ca²⁺ influx into the cell. Ca²⁺-dependent cellular processes,¹³ important for T-cell activation and effector functions involving proliferation, differentiation, and migration, are thereby regulated by the dynamic and precise control of the membrane potential.^14–16

The death of cancer cells in tumours is associated with poor patient prognosis.¹⁷ Dying tumour cells release K⁺, the main intracellular cation, which accumulates in the tumour microenvironment (TME), reaching 5–10 times the levels encountered by T-cells in the bloodstream. T-cells in the TME exposed to the K⁺-rich fluid accumulate K⁺ intracellularly due to greater entry through ‘pump’ or ‘leak’ channels than exit through K_Ca3.1 and K_V1.3 K⁺ channels.^18,19 The increased intracellular K⁺ suppresses genes involved in a myriad of pathways in T-cells, nutrient consumption and metabolic changes are altered, the transition of T-cells from the resting to the tumour-killing effector stage is inhibited, and the transition to regulatory T-cells that dampen immune responses is promoted. These alterations occur without affecting the viability of T-cells.^18,19 Overexpression of K_V1.3 or K_Ca3.1 K⁺ channels in T-cells rescues T-cells from the immunosuppressive effects of the K⁺-rich TME.¹⁸ Moreover, defects in K_Ca3.1 activity hinder T-cell recruitment in the adenosine-rich TME, thereby promoting tumour immune escape.^14,20 Pharmacological activation of the K_Ca3.1 channels in T-cells thus represents a promising approach to boost anti-tumour T-cell immunity within the TME.²¹

While K_Ca3.1 inhibitors are in pre-clinical development to dampen T-cell hyperactivation in autoimmune diseases, there is limited information on K_Ca3.1 channel activators. A previously developed K_Ca3.1 activator, SKA-31, potently augmented K_Ca3.1 activity and lowered blood pressure in experimental mice,²² but it lacked selectivity over closely related small-conductance Ca²⁺-activated K_Ca2 channels.²³ Here, we synthesized a panel of five compounds structurally related to SKA-31 and demonstrate that one analogue, SKA-346, effectively activates K_Ca3.1 and exhibits selectivity for K_Ca3.1 over related channels. We further show in vivo tolerability, pharmacokinetic profiles, and tissue distribution of SKA-346 in mice. These findings will be helpful for evaluating SKA-346 as a novel therapy for conditions requiring specific activation of the K_Ca3.1 channel such as alleviating the immunosuppressive effects on tumour-infiltrating lymphocytes in the necrotic microenvironment of solid tumours.

2 Results and discussion

2.1. Chemistry of quinoline derivatives

The synthesis steps of the five quinoline derivative compounds (i) 5-methyl-[1,3]thiazolo[4,5-f]quinolin-2-amine (SKA-346), (ii) 8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-101), (iii) 5-methyl-8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-102), (iv) 5-fluoro-[1,3]thiazolo[4,5-f]quinolin-2-amine (NKV-103), and (v) 5-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-104) are provided in Scheme 1. Final products of all the five quinoline derivatives were confirmed by proton nuclear magnetic resonance spectroscopy (¹H NMR) and liquid chromatography-mass spectrometry (LC-MS) (ESI Fig. 1–5†).

Scheme 1

2.1.1. Synthesis of SKA-346. The synthesis procedure of SKA-346 is outlined in Scheme 1A. Briefly, 5-amino-8-methylquinoline (500 mg) (1) and potassium thiocyanate (KSCN, 3.5 mmol) were dissolved in glacial acetic acid at room temperature. In a dropwise manner, liquid bromine (Br₂, 1.2–1.5 equivalent) in glacial acetic acid was added and the reaction mixture was stirred for 2 h. Subsequently, solids were filtered and washed with glacial acetic acid followed by H₂O. The filtrate was diluted with H₂O (100 mL) and the pH was adjusted in the range of 7.0–7.5 prior to cooling in the refrigerator overnight for product precipitation. Finally, the product was filtered and washed with cold H₂O before drying under vacuum. The product was isolated as brownish powder and confirmed by ¹H NMR and LC-MS (110 mg, 16%); m.p. = 259 °C (CAS 1082847-64-0). ¹H NMR (800 MHz, DMSO-d₆): δ = 8.88 (dd, J = 4.1, 1.9 Hz, 1H), 8.66 (dd, J = 8.3, 1.9 Hz, 1H), 7.90 (t, J = 1.2 Hz, 1H), 7.63 (s, 2H), 7.54 (dd, J = 8.2, 4.1 Hz, 1H), 2.69 (d, J = 1.1 Hz, 3H). ¹³C NMR (201 MHz, DMSO-d₆): δ = 168.19, 148.72, 146.89, 146.02, 132.22, 129.13, 125.57, 122.45, 121.20, 121.14, 39.93, 18.38 (ESI Fig. 1†).

2.1.2. Synthesis of NKV-101. The 5-steps synthesis process (Scheme 1B) was initiated using 5-bromo-3-(trifluoromethyl)quinoline (0.30 g, 1.086 mmol) (2). In a stirred solution with toluene (10 mL), diphenylmethanimine (Ph₂C=NH) (1 equivalent, 0.19 g, 1.086 mmol) and cesium carbonate (Cs₂CO₃) (2 equivalent, 0.70 g, 2.173 mmol) were added. The reaction mixture was degassed with argon for 10 min prior to the addition of palladium acetate (Pd(OAc)₂) (0.1 equivalent, 0.04 g, 0.217 mmol) and Xantphos (0.2 equivalent, 0.0031 g, 0.005 mmol) and subsequently stirred at 120 °C for 16 h. After completion (monitored by thin layer chromatography, TLC), the mixture was quenched with ice-cold H₂O (10 mL), extracted with ethyl acetate (EtOAc) (2 × 50 mL) and washed with brine (10 mL). The organic layer was then dried over anhydrous sodium sulfate (Na₂SO₄) and concentrated under reduced pressure. The crude was then purified by combi-flash column chromatography (70% EtOAc/heptane) to afford 1,1-diphenyl-N-(3-(trifluoromethyl)quinolin-5-yl)methanimine (3; 0.20 g, 0.531 mmol, 48.89%). Compound 3 (0.03 g, 0.079 mmol), in a stirred solution of HCl (1 mL) at 0 °C, was stirred at room temperature for 2 h. Upon completion (monitored by TLC), the reaction mixture was quenched with ice-cold H₂O (5 mL) and extracted with EtOAc (2 × 10 mL). The aqueous layer was basified with saturated sodium bicarbonate (NaHCO₃; pH 8.0), washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford 3-(trifluoromethyl)quinolin-5-amine hydrochloride (4; 0.015 g, 0.070 mmol, 88.70%). Benzoyl isothiocyanate (PhCONCS) (2 equivalent, 0.19 g, 1.206 mmol) was then added to 4 (0.15 g, 0.603 mmol) in a stirred solution of acetone (CH₃COCH₃; 12 mL). Sequentially, the reaction mixture was stirred at room temperature for 1 h. After completion (monitored by TLC), the mixture was diluted with EtOAc and H₂O. The organic layer was separated, washed with brine, and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (40–60% EtOAc/hexane) to afford N-((3-(trifluoromethyl)quinolin-5-yl)carbamothioyl)benzamide (5; 0.18 g, 0.479 mmol, 79.50%). Sodium methoxide (NaOMe) (2 equivalent, 0.08 g, 1.598 mmol) was then added to the stirred solution N-((3-(trifluoromethyl)quinolin-5-yl)carbamothioyl) benzamide (0.30 g, 0.799 mmol) in methanol (MeOH, 10 mL) and stirred at room temperature for 1 h. Upon completion (monitored by TLC), the mixture was concentrated under reduced pressure and diluted with EtOAc and H₂O. The organic layer was separated, washed with brine, and concentrated under reduced pressure. The crude was then purified by combi-flash column chromatography (30–50% EtOAc/hexane) to afford 1-(3-(trifluoromethyl)quinolin-5-yl)thiourea (6; 0.20 g, 0.737 mmol, 92.29%). Br₂ (0.10 mL) was then added to a stirred solution of 6 (0.15 g, 0.552 mmol) in dichloromethane (CH₂Cl₂, 10 mL) and stirred at room temperature for 16 h. After completion (monitored by TLC), the mixture was quenched in ice-cold H₂O. The organic layer was separated, washed with brine, and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (50–80% EtOAc/hexane) to afford 8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-101; 0.06 g, 0.222 mmol, 40.32%) as a brown solid. LC-MS calculated for C₁₁H₆F₃N₃S: 269.02; found: 270 (M⁺¹); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.13 (d, J = 2.25 Hz, 1H), 8.93–8.95 (m, 1H), 8.27 (d, J = 8.88 Hz, 1H), 7.96 (s, 2H), 7.78 (d, J = 8.88 Hz, 1H) (ESI Fig. 2†).

2.1.3. Synthesis of NKV-102. NKV-102 was synthesized in an 8-steps reaction process (Scheme 1C). Briefly, 3-(trifluoromethyl)quinoline (7; 1.80 g, 9.129 mmol), in a stirred solution with sulfuric acid (H₂SO₄, 7 mL) was mixed with N-bromosuccinimide (NBS, 1.5 equivalent, 2.43 g, 13.694 mmol) and stirred at 50 °C for 6 h. After completion (monitored by TLC), the mixture was quenched with ice-cold H₂O (50 mL) and extracted with EtOAc (2 × 100 mL). The organic layer was washed with brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (30% EtOAc/hexane) to afford 5,8-dibromo-3-(trifluoromethyl)quinoline (8; 0.70 g, 1.972 mmol, 21.60%). Ph₂C=NH (0.8 equivalent, 0.28 g, 1.577 mmol) and Cs₂CO₃ (2 equivalent, 1.28 g, 3.944 mmol) was then added to a stirred solution of 8 (0.70 g, 1.972 mmol) in toluene (8 mL). The reaction mixture was degassed with argon for 10 min prior to the addition of Pd(OAc)₂ (0.2 equivalent, 0.08 g, 0.394 mmol) and Xantphos (0.4 equivalent, 0.45 g, 0.788 mmol). The reaction mixture was then stirred at 120 °C for 16 h. After completion (monitored by TLC), the mixture was quenched with ice-cold H₂O (10 mL) and extracted with EtOAc (2 × 50 mL) and washed with brine (10 mL). The organic layer was dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (70% EtOAc/Heptane) to afford mixture of N-(8-bromo-3-(trifluoromethyl)quinolin-5-yl)-1,1-diphenylmethanimine (9) and N-(5-bromo-3-(trifluoromethyl)quinolin-8-yl)-1,1-diphenylmethanimine (9a; 0.70 g, 1.537 mmol, 77.98%). In a mixture of 9 and 9a (0.70 g, 1.537 mmol) stirred in concentrated HCl (6 mL) at 0 °C, the reaction mixture was left to be stirred at room temperature for 2 h. Upon completion (monitored by TLC), the mixture was quenched with ice-cold H₂O (5 mL) and extracted with EtOAc (2 × 10 mL). The aqueous layer was basified with saturated NaHCO₃ (pH 8), washed with brine, dried over anhydrous Na₂SO₄ and concentrated under reduced pressure to afford mixture of 8-bromo-3-(trifluoromethyl)quinolin-5-amine hydrochloride (10) and 5-bromo-3-(trifluoromethyl)quinolin-8-amine hydrochloride (10a; 0.30 g, 0.915 mmol, 59.58%, HCl salt). PhCONCS (1 equivalent, 0.14 g, 0.915 mmol) was then added to the stirred solution of 10 and 10a (0.30 g, 0.915 mmol) in CH₃COCH₃ (10 mL). Following which, the reaction mixture was stirred at room temperature for 1 h. After completion (monitored by TLC), the mixture was diluted with EtOAc and H₂O. The organic layer was separated, washed with brine and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (30–50% EtOAc/hexane) to afford mixture of N-((8-bromo-3-(trifluoromethyl)quinolin-5-yl)carbamothioyl)benzamide (11) and N-((5-bromo-3-(trifluoromethyl)quinolin-8-yl)carbamothioyl)benzamide (11a; 0.40 g, 0.880 mmol, 96.15%). In a stirred solution of mixture of 11 and 11a; (0.40 g, 0.880 mmol) in MeOH (10 mL), NaOMe (2 equivalent, 0.09 g, 1.761 mmol) was added to it and stirred at room temperature for 1 h. Upon completion (monitored by TLC), the mixture was concentrated under reduced pressure and diluted with EtOAc and H₂O. The organic layer was separated, washed with brine and concentrated under reduced pressure to afford the crude. The crude was then purified by combi-flash column chromatography (30–50% EtOAc/hexane) to yield 1-(8-bromo-3-(trifluoromethyl)quinolin-5-yl)thiourea (12; 0.30 g, 0.856 mmol, 97.30%) which was directly used in the next step without purification. Br₂ (0.20 mL) was added to the stirred solution of 12 (0.30 g, 0.856 mmol) in CH₂Cl₂ (10 mL) and stirred at room temperature for 16 h. After completion (monitored by TLC), the mixture was quenched with ice-cold H₂O and diluted with CH₂Cl₂. The organic layer was separated, washed with brine and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (50–80% EtOAc/hexane) to afford 5-bromo-8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (13; 0.12 g, 0.344 mmol, 40.24%). Triethylamine (Et₃N) (5 equivalent, 0.30 mL, 2.154 mmol) and acetic anhydride (Ac₂O) (3 equivalent, 0.12 mL, 1.292 mmol) were then added to the stirred solution of 13 (0.15 g, 0.430 mmol) in CH₂Cl₂ (10 mL) at room temperature and stirred at 40 °C for 2 h. After completion (monitored by TLC), the mixture was concentrated under reduced pressure and diluted with CH₂Cl₂ and H₂O. The organic layer was separated and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (70–80% EtOAc/hexane) to afford N-(5-bromo-8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-yl)acetamide (14; 0.11 g, 0.281 mmol, 65.43%). Trimethylboroxine (4 equivalent, 0.14 g, 1.127 mmol) and Cs₂CO₃ (2 equivalent, 2.93 g, 9.021 mmol) were then added to the stirred solution of 14 (0.11 g, 0.281 mmol) in 1,4-dioxane (5 mL) and degassed with argon for 5 min prior to the addition of PdCl₂ (dppf) (0.1 equivalent, 0.02 g, 0.028 mmol). The reaction mixture was then stirred at 100 °C for 16 h before it was diluted with EtOAc and H₂O. The organic layer was separated and concentrated under reduced pressure. The crude was dissolved in MeOH (2 mL) followed by the addition of NaOMe (1.5 equivalent, 0.02 g, 0.422 mmol) and stirred at 100 °C for 16 h. After completion (monitored by TLC), the mixture was concentrated under reduced pressure, quenched with ice-cold H₂O, and extracted with EtOAc (2 × 10 mL). The organic layer was separated and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (70% EtOAc/hexane) to afford 5-methyl-8-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-102; 0.013 g, 0.045 mmol, 16.28%) as a solid. LC-MS calculated for C₁₂H₈F₃N₃S: 283.04; found: 284.10 (M¹⁺); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 9.18 (d, J = 2.32 Hz, 1H), 8.93 (d, J = 1.34 Hz, 1H), 8.15 (s, 1H), 7.84 (s, 2H), 2.73 (s, 3H) (ESI Fig. 3†).

2.1.4. Synthesis of NKV-103. 5-Fluoro-[1,3]thiazolo[4,5-f]quinolin-2-amine, CAS No. 1082821-76-8, NKV-103 (Scheme 1D) was purchased from ChemSpace (USA) (ESI Fig. 4†).

2.1.5. Synthesis of NKV-104. The synthesis of NKV-104 was achieved in a 3-steps reaction process (Scheme 1E). With 8-(trifluoromethyl)quinolin-5-amine (15; 0.25 g, 1.178 mmol) as the base substrate in CH₃COCH₃ (10 mL), PhCONCS (2 equivalent, 0.38 g, 2.356 mmol) was added to the mixture and stirred at room temperature for 1 h. Upon completion (monitored by TLC), the mixture was diluted with EtOAc and H₂O. The organic layer was separated and concentrated under reduced pressure. The crude was purified by combi-flash column chromatography (40–60% EtOAc/hexane) to afford N-((8-(trifluoromethyl)quinolin-5-yl)carbamothioyl)benzamide (16; 0.32 g, 0.852 mmol, 72.36%). Compound 16 (0.32 g, 0.852 mmol) was then stirred in 10% NaOH (20 mL) and stirred at 100 °C for 10 min. After completion (monitored by TLC), the mixture was neutralized with NaHCO₃ and diluted with EtOAc and H₂O. The organic layer was separated and concentrated under reduced pressure to afford 1-(8-(trifluoromethyl)quinolin-5-yl)thiourea (17; 0.18 g, 0.663 mmol, 77.85%) which was directly used in the next step without purification. Br₂ (0.20 mL) was then added to the stirred solution of stirred solution of 17 (0.18 g, 0.663 mmol) in CH₂Cl₂ (10 mL) and stirred at 0 °C for 1 h. Upon completion (monitored by TLC), the mixture was quenched with ice-cold H₂O and diluted with CH₂Cl₂ and H₂O. The organic layer was separated, washed with brine, and concentrated under reduced pressure to afford the crude. The crude was purified by combi-flash column chromatography (50–80% EtOAc/hexane) to afford 5-(trifluoromethyl)thiazolo[4,5-f]quinolin-2-amine (NKV-104; 0.06 g, 0.222 mmol, 33.59%) as an off-white solid. LC-MS calculated for C₁₁H₆F₃N₃S: 269.02; found: 269.70 (M⁺¹); ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.98 (dd, J = 4.22, 1.77 Hz, 1H), 8.76 (dd, J = 8.44, 1.83 Hz, 1H), 8.58 (s, 1H), 8.14 (s, 2H), 7.66 (dd, J = 8.38, 4.22 Hz, 1H) (ESI Fig. 5†).

2.2. Electrophysiology analysis identifies SKA-346 and NKV-104 as potential K_Ca3.1 activators

Using an automated QPatch-HTX electrophysiology platform (Sophion Biosciences), we performed initial screening of all five quinoline derivatives for their potential activity on the K_Ca3.1 channel (Fig. 1). Two of the five quinoline derivatives, SKA-346 and NKV-104, augmented K_Ca3.1 currents with EC₅₀ values 1.9 μM and 1.7 μM, respectively (Fig. 1A and E). We therefore prioritized these two compounds for more extensive characterization.

Fig. 1 Patch-clamp recordings of the five quinoline derivatives in activating K_Ca3.1 channel. Representative current recordings and concentration response curves of the five quinoline derivatives towards the K_Ca3.1 channel are shown in the left and right columns, respectively; (A) SKA-346, (B) NKV-101, (C) NKV-102, (D) NKV-103, (E) NKV-104. Concentrations of each quinoline derivatives (between 300 nM to 30 μM) were log transformed and plotted against the normalized current values derived from at least three individual cells to generate the concentration–response curves. The EC₅₀ values in activating K_Ca3.1 were computationally determined via GraphPad Prism v10.1.2. EC₅₀, effective concentration 50%; EC₅₀: >30 μM, EC₅₀ could not be achieved at the highest tested concentration of 30 μM. Data are mean ± SEM of at least three independent experiments.

2.3. SKA-346 shows selectivity for the K_Ca3.1 channel

There are multiple K⁺ and other ion channels that are differentially expressed on various cell types and tissues.²⁴ Non-specific activation of ion channels may lead to potential detrimental side effects, underscoring the need for drug selectivity testing. We therefore examined SKA-346 and NKV-104 on three closely related calcium-activating K⁺ channels [K_Ca1.1, expressed mainly in brain, smooth muscle, and skeletal muscle cells; K_Ca2.2, expressed mainly in neuronal and skeletal muscle cells; and K_Ca2.3, expressed mainly in neurons and vascular endothelial cells] and two voltage-gated K⁺ channels [K_V1.3, expressed in T-cells and various immune cell types including macrophages; and hERG/K_V11.1, a cardiac channel required for the assessment of cardiac safety]²⁵ (Fig. 2A–E). NKV-104, at a concentration of 30 μM, inhibited the hERG/K_V11.1 channel (Fig. 2B, right panel) and activated the K_Ca2.2 and K_Ca2.3 channels with EC₅₀ values of 11.9 μM and 29.3 μM, respectively (Fig. 2D and E, right panels), showing its lack of selectivity towards K_Ca3.1. SKA-346 did not activate any of the above channels (Fig. 2, left panels), highlighting its selectivity for K_Ca3.1. We therefore focused SKA-346 for further analysis.

Fig. 2 Patch-clamp recordings of SKA-346 and NKV-104 selectivity in activating various K⁺ channels. Representative current recordings and concentration response curves of SKA-346 and NKV-104 for various K⁺ channels are shown in the left and right columns, respectively; (A) K_V1.3, (B) hERG/K_V11.1, (C) K_Ca1.1, (D) K_Ca2.2, and (E) K_Ca2.3. For testing of activation effect, each cell served as its own maximal control and the increases in cell conductance were normalized to the effect obtained with a saturating activator concentration (30 μM SKA-346/NKV-104) applied during the last liquid periods before the final washout. For channels that were not activated by quinoline derivatives, cell currents were normalized to the last vehicle period applied to the cell. Increasing concentrations of SKA-346 and NKV-104 were log transformed and plotted against the normalized current values derived from at least three individual cells to generate the concentration–response curves. Note: NKV-104, at a concentration of 30 μM, caused inhibition to K_V11.1 current. EC₅₀, effective concentration 50%. EC₅₀: >30 μM, EC₅₀ could not be achieved at the highest tested concentration of 30 μM. Data are mean ± SEM of at least three independent experiments.

Finally, we tested SKA-346 on 9 additional ion channels – (1) K_V1.1, expressed in brain, heart, smooth muscle and distal convoluted tubule; (2) K_V1.2, expressed in neurons; (3) K_V1.4, expressed in brain, heart and pancreatic β-cells; (4) K_V1.5, expressed in heart, brain, smooth muscle and pancreatic β-cells; (5) K_V1.7, expressed in neuronal adrenal gland, brain and smooth muscle cells; (6) K_V3.1, expressed in neuronal cells; (7) K_V4.2, expressed in the heart and in neurons; (8) Na_V1.5 expressed in heart, neurons, and skeletal muscle cells; and (9) Kir2.1 expressed in smooth muscle, neurons and endothelial cells.²⁴ SKA-346 did not impact any of these ion channels at concentrations up to 30 μM (Fig. 3), confirming its selectivity towards K_Ca3.1 channel over 14 other ion channels.

Fig. 3 Patch-clamp recordings of SKA-346 selectivity in activating various ion channels. Representative current recordings and concentration response curves of SKA-346 towards 9 different ion channels are shown in the left and right columns, respectively. (A) K_V1.1, (B) K_V1.2, (C) K_V1.4, (D) K_V1.5, (E) K_V1.7, (F) K_V3.1, (G) K_V4.2, (H) Na_V1.5, and (I) Kir2.1. Concentrations of SKA-346 (between 300 nM to 30 μM) were log transformed and plotted against the normalized current values derived from at least three individual cells to generate the concentration–response curves. Note: SKA-346 did not significantly affect any of the tested channels. Data are mean ± SEM of at least three independent experiments.

2.4. Molecular docking of SKA-346

The interface between the calmodulin N-lobe and the S₄₅A helix in the S4–S5 linker of K_Ca3.1 is intimately involved in Ca²⁺-calmodulin-dependent activation of the K_Ca3.1 channel and has been suggested to be a binding pocket for activators of the channel.^25,26 SKA-346 was docked into this critical pocket via RosettaLigand computer-aided docking.^27,28 Ligand docking using the RosettaLigand method involves three stages. First, the ligand is randomly placed within a 10 Å sphere of the binding site. Conformers are generated and scored for shape compatibility with the protein, and the best models are filtered and selected for further stages. In the second stage, the ligand's position and orientation are fine-tuned using Monte Carlo minimization, with protein side chains repacked and optimized. The full-atom Rosetta energy function, including softened van der Waals interactions, is used for scoring. The final stage applies a stricter gradient-based minimization with a hard-repulsive van der Waals potential to refine the ligand and protein interactions, improving discrimination between native and non-native binding modes.^29,30 A total of 10 000 models were generated, taken through the above three stages of RosettaLigand docking, followed by the selection of the top 1000 models that attained the lowest total energy score. The models were further sieved to identify the top 10 models which were inspected for ligand/channel interaction and convergence. RosettaLigand predicted a single, well-converged pose for SKA-346 (Fig. 4A).

Fig. 4 In silico analysis of the docking and binding affinities of SKA-346 to the binding site of K_Ca3.1. (A) RosettaLigand docking models of the lowest energy-binding poses of SKA-346 in the binding site of K_Ca3.1. Van der Waals forces and hydrogen bonds of the quinoline derivative upon docking within the binding site of K_Ca3.1 are shown in the left and right columns, respectively. (B) SKA-346 was prepared in Schrodinger ligand prep Wizard and XP (extra precision) module prior to in silico analysis by GLIDE. The binding affinity between SKA-346 and the binding site of K_Ca3.1 are computationally predicted that gives an arbitrary number as a docking score, whereby a more negative value suggests a more ‘nature-like’ fitting of the quinoline derivative within the binding site.

We next performed a secondary in silico analysis via the GLIDE program (open state −1, pdb: 6CNN) that empirically predicts the binding affinity between the compound and binding site upon docking. For this, SKA-346 was prepared in Schrodinger ligand prep Wizard and XP (extra precision) module (Schrödinger, Inc.). Various orientations of SKA-346 inputted into the binding pocket in the calmodulin/K_Ca3.1 interface were computationally determined and labelled as a docking score (Fig. 4B). We found a high negative docking score of −6.285, suggesting a ‘nature-like’ fitting of SKA-346 within the binding site.

2.5. Preclinical pharmacology of SKA-346

To access preclinical pharmacology of SKA-346, we first characterized its physicochemical properties by using pkCSM (https://biosig.lab.uq.edu.au/pkcsm)³¹ and Chemdraw 22.2.0. SKA-346, molecular formula C₁₁H₉N₃S, has a molecular weight of 215.27 g mol⁻¹ with calculated partition coefficient (log P) of 2.74 (computed using pkCSM) and 2.53 (computed using Chemdraw 22.2.0). The compound contains 1 hydrogen-bond donor and 4 hydrogen-bond acceptors. The molar refractivity of SKA-346 is 64.29 with topological polar surface area (TPSA) of 90.579 (computed using pkCSM) and 80.04 (computed using Chemdraw 22.2.0). All these parameters satisfy the generally accepted Lipinski's rule of five,³² suggesting SKA-346 to be a drug-like compound. SKA-346 solubility in dimethyl sulfoxide (DMSO) was calculated to be 40.6 mg mL⁻¹ or 188.64 mM (ESI Fig. 6†) Notably, in vitro treatment of human primary T-cells (isolated from healthy volunteers' blood using standard protocol)³³ with SKA-346 concentrations up to 10 μM (∼5 times of EC₅₀) were non-cytotoxic (ESI Fig. 7†).

We (co-author Wulff's research group) have previously shown that up to 30 mg kg⁻¹ of the benzothiazole compound SKA-31 is well-tolerated in Sprague-Dawley rats and in mice when administered intraperitoneally (i.p.).²² Using this dataset as a reference and to minimize animal usage, we assessed whether the same single dose of SKA-346 (30 mg kg⁻¹ in 100 μL per animal) administered into female C57BL/6 mice (n = 6 each group) via i.p. route was tolerated over a short-term period of 14 consecutive days (Fig. 5A). When compared to the vehicle control group, we did not detect any obvious clinical sign of toxicity due to SKA-346 administration, as there was no significant mouse body weight loss (Fig. 5B). SKA-346 treatment did not cause any significant change in the levels of serum aspartate aminotransferase (AST, Fig. 5C), indicating that there was no major damage or injury to the liver. AST is a commonly used marker for liver function as any insult or damage to the liver or even skeletal and heart tissues will cause this enzyme to be released into the bloodstream resulting in its significantly elevated levels in the serum.³⁴ Since the study was designed to provide a preliminary assessment of SKA-346 safety in vivo, we limited our study to a single dose of 30 mg kg⁻¹ for 14 consecutive days treatment. It would be important to test both lower and higher doses of SKA-346 over extended periods and perform assessments using multiple biomarkers to fully characterize potential dose-dependent sub-chronic and chronic toxicity and further establish its safety profile.

Fig. 5 In vivo tolerability of SKA-346. SKA-346 solution was prepared in 3% DMSO + 97% peanut oil to achieve 6 mg mL⁻¹ concentration. (A) Timeline of SKA-346 toxicity studies. SKA-346 (30 mg kg⁻¹, total volume 100 μL) was administered daily into female C57BL/6 mice (n = 6 for each group) via i.p. for 14 days. (B) Body weights of control and SKA-346-treated mice recorded every 2–4 days. (C) The serum biochemical indicator ast (mU/mL) of control and SKA-346-treated mice. (D) Mice brain, kidney, and liver tissues were harvested upon termination and analysed for SKA-346 biodistribution by LC-MS. (E) H&E staining of mice tissue samples (kidney, liver, brain, heart, thymus, and spleen) harvested upon termination at day 14. Representative images are shown. The full images of the tissue sections are provided as ESI Fig. 9–14.† H&E, hematoxylin and eosin; i.p., intraperitoneal; LC-MS, liquid chromatography-mass spectrometry; nd, not detected; ns, non-significant.

LC-MS analysis of harvested organ samples showed substantial amounts of SKA-346 in the kidney (60.7 pg mg⁻¹) and liver (22.5 pg mg⁻¹), but not in the brain (Fig. 5D). Moreover, the boiled-egg model analysis from SwissADME (http://www.swissadme.ch/), that utilizes the TPSA and log P values to evaluate the likelihood of the molecule in passing through the blood–brain barrier (BBB),³⁵ indicated SKA-346 being unable to cross the BBB (ESI Fig. 8†). It is possible that SKA-346 distributes into other tissues such as heart, thymus, spleen, lungs, and bones, which could not be analyzed in the current study. When compared to the vehicle control, no detectable change in histological morphologies was observed in vital organs (kidney, liver, heart, brain, thymus, and spleen) of mice due to SKA-346 administration (Fig. 5E). These findings collectively demonstrate that SKA-346 did not cause any unwanted toxicity effect to the vital organs.

Finally, we determined the circulating half-life (t_1/2) of SKA-346 (30 mg kg⁻¹) administered by i.p. injection in female BALB/c mice (n = 4 each). Analysis of blood plasma samples, harvested at multiple time-points over the course of 24 h, by LC-MS (Fig. 6A) revealed that SKA-346 was absorbed rapidly into the blood, reaching its peak concentration in the blood plasma within 0.25 h (Fig. 6B, ESI Fig. 15†). An estimated circulated half-life (t_1/2) of SKA-346 was 2.84 h with the peak plasma concentration (C_max) of 6.29 μg mL⁻¹ (29.2 μM) at 15 min, which is about 15 times higher than the EC₅₀ for K_Ca3.1 activation. We also noted mean residence time from time of dosing to time of last measurable concentration (MRTlast) to be 4.82 h and the area under the curve from the time of dosing to the time of the last measurable concentration (AUClast) to be 45 487.06 h*ng mL⁻¹ (Fig. 6C; ESI Table 1†).

Fig. 6 In vivo pharmacological analysis of SKA-346. SKA-346 solution was prepared in 3% DMSO + 97% peanut oil to achieve 6 mg mL⁻¹ concentration. (A) Workflow of SKA-346 treatment and sample collection over 24 h. Female BALB/c mice (n = 4/group, 2 groups) were administered at 30 mg kg⁻¹; 100 μL of SKA-346 preparation via i.p. Blood plasma was taken from each group of mice alternately at 8 different time-points over the course of 24 h; 0 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. (B) SKA-346 concentrations in the mice blood plasma, as determined by LC-MS, were plotted against time over a period of 24 h. The circulating half-life (t_1/2) of SKA-346 was calculated to be 2.8 h. (C) Key pharmacological parameters obtained from SKA-346-treated mice are tabulated. Detailed list of pharmacological parameters is provided as ESI Table 1.† i.p., intraperitoneal; LC-MS, liquid chromatography-mass spectrometry.

3 Conclusions

In summary, our current study identified SKA-346 as a novel selective and safe activator of the K_Ca3.1 channel. SKA-346 constitutes a promising novel therapeutic for use in conditions that require increasing K_Ca3.1 activity.

4 Materials and methods

4.1. Ion channel electrophysiology analysis

All the five quinoline derivatives were dissolved in DMSO to prepare 10 mM stock solutions and stored at −80 °C until use. For patch-clamp analysis, fixed-ratio dilutions of the above five compounds were made in extracellular solution buffers, specific for each channel as detailed below. Patch-clamp recordings were captured using an automated QPatch-HTX electrophysiology platform (Sophion Biosciences) equipped with Sophion QPatch software. Each current slope was the average of five measurements taken at the end of the respective liquid period. Data fitting to the Hill equation to obtain EC₅₀ values was performed with GraphPad Prism v10.1.2. For testing of activation effect, each cell served as its own maximal control and the increases in cell conductance were normalized to the effect obtained with a saturating activator concentration (30 μM SKA-346) applied during the last liquid periods before the final washout.

4.1.1. Patch-clamp of K_Ca3.1, K_Ca2.2, and K_Ca2.3 channels. K_Ca3.1 and K_Ca2.2 channels were stably expressed in the human kidney-derived cell line HEK293, and K_Ca2.3 channels were stably expressed in African green monkey kidney fibroblast-like cell line COS-7.³⁶ Channel currents were recorded at a holding potential of −80 mV, stepping to −120 mV for 20 ms, then ramping from −120 mV to 40 mV over 200 ms, and stepping back to −120 mV for 20 ms at every 10 seconds interval. The external solution consisted of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The internal buffer contained 120 mM KCl, 5.96 mM CaCl₂, 1.75 mM MgCl₂, 10 mM EGTA, 10 mM HEPES, and 4 mM Na₂ATP, pH 7.2.

4.1.2. Patch-clamp of K_V1.3 and K_V3.1 channels. K_V1.3 and K_V3.1 channels were stably expressed in mouse fibroblast cell line L929.³⁷ Channel currents were recorded at a holding potential of −90 mV, then depolarized to 40 mV for 200 ms at every 45 seconds interval. The external solution consisted of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES, pH 7.2. The internal buffer contained 160 mM KF, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.2.

4.1.3. Patch-clamp of K_V1.1, K_V1.4, K_V4.2, K_V1.2, and K_V1.7 channels. K_V1.1 channels were stably expressed in mouse L929 cells, K_V1.4 and K_V4.2 channels were stably expressed in the mouse cell line LTK, K_V1.2 channels were stably expressed in the mouse cell line B82, and K_V1.7 channels were stably expressed on the Chinese hamster lung-derived cell line CHL.^37,38 Channel currents were recorded at a holding potential of −80 mV, then depolarized to 40 mV for 200 ms. Interpulse intervals were every 10 seconds for K_V1.1 channels, every 15 seconds for K_V4.2 channels, and every 30 seconds for K_V1.4, K_V1.2 and K_V1.7 channels. The external solution consisted of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES, pH 7.2. The internal buffer contained 160 mM KF, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.2.

4.1.4. Patch-clamp of K_V1.5 channels. K_V1.5 channels were stably expressed in the murine erythroleukemia MEL cell line.³⁷ Channel currents were recorded at a holding potential of −80 mV, then depolarized to 60 mV for 200 ms at every 30 seconds interval. The external solution consisted of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES, pH 7.2. The internal buffer contained 160 mM KF, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.2.

4.1.5. Patch-clamp of K_Ca1.1 channels. K_Ca1.1 channels were stably expressed in human HEK293 cells.³⁹ Channel currents were recorded at a holding potential of −80 mV, then depolarized to 60 mV for 200 ms at every 10 seconds interval. The external solution consisted of 160 mM NaCl, 4.5 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, and 10 mM HEPES, pH 7.4. The internal buffer contained 160 mM KF, 2 mM MgCl₂, 10 mM EGTA, and 10 mM HEPES, pH 7.4.

4.1.6. Patch-clamp of hERG/K_V11.1 channels. hERG channels were stably expressed in human HEK293 cells (obtained from ICE Bioscience Inc., China). Channel currents were recorded at a holding potential of −80 mV, stepping to −50 mV for 80 ms, then depolarizing to 30 mV for 4.8 seconds, followed by a 5 seconds tail step to −50 mV, and then stepping back to −80 mV for 3.1 seconds at every 15 seconds interval. The external solution consisted of 145 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The internal buffer contained 115 mM K-Aspartic acid, 20 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP, pH 7.2.

4.1.7. Patch-clamp of Kir2.1 channels. Kir2.1 channels were stably expressed in human HEK293 cells (obtained from ICE Bioscience Inc., China). Channel currents were recorded at a holding potential of −80 mV, stepping to −150 mV for 5 ms, then ramping from −150 mV to 50 mV over 200 ms, and stepping back to −80 mV for 20 ms at every 10 seconds interval. The external solution consisted of 145 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The internal buffer contained 115 mM K-Aspartic acid, 20 mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM HEPES, and 2 mM Na₂ATP, pH 7.2.

4.1.8. Patch-clamp of Na_v1.5 channels. Na_V1.5 channels were stably expressed in the Chinese hamster epithelial cell line CHO (obtained from ICE Bioscience Inc., China). Channel currents were recorded at a holding potential of −120 mV, depolarized to 0 mV for 20 ms, stepped to a V-half voltage of −70 mV for 8 seconds, followed by stepping back to −120 mV for 20 ms before a depolarizing voltage step to 0 mV for 20 ms, and then back to −120 mV at every 20 seconds interval. The external solution consisted of 145 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The internal buffer contained 120 mM CsF, 5 mM NaCl, 2 mM MgCl₂, 30 mM CsCl, 10 mM EGTA, and 5 mM HEPES, pH 7.2.

4.2. Solubility of SKA-346

SKA-346 solubility in DMSO was determined using UV-Vis spectrophotometer (Varioskan LUX Multimode Microplate Reader, ThermoFisher Scientific, USA), as described earlier.⁴⁰ Briefly, SKA-346 was weighed in a 2 mL glass tube and dissolved in DMSO to achieve a stock concentration of 0.5 mg mL⁻¹. The solution was serially diluted to get concentrations ranging from 0.5 to 0.007 mg mL⁻¹. The absorption spectrum of 200–800 nm showed the absorption peak for SKA-346 at 345 nm (λ_max). Accordingly, a calibration curve was generated by plotting the absorbance at 345 nm (after blank correction) against known concentrations. To determine SKA-346 solubility, excess amount of SKA-346 (50 mg) was added to 500 μL DMSO in a 2 mL glass tube, which was then placed on a shaker at 1000 rpm for 24 h to ensure saturation. The suspension was filtered using a 0.22 μm syringe filter to remove undissolved particles and the filtrate was diluted 100-fold in DMSO. The optical density at the absorption peak was used to determine the solubility of SKA-346 with reference to the calibration curve.

4.3. SKA-346 tolerability assessment in mice

All protocols for animal treatment and care were in accordance with the guidelines for Responsible Care and Use of Laboratory Animals and were reviewed and approved by the Animal Ethics Committee of Singapore Health Services, SingHealth Singapore (IACUC #2018/SHS/1417). DMSO is a polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents, including various oils. For administering SKA-346 in mice, we dissolved it in 3% DMSO + 97% peanut oil (Sigma-Aldrich, USA) to wet the surface and break up the SKA-346 crystals to allow for better dissolution of this highly lipophilic compound in peanut oil, as used earlier with a slight modification.^41–43 Goal was to reduce the amount of DMSO to a minimum possible volume. Briefly, peanut oil was heated to a temperature of ∼60 °C in a sterile glass container with a mini-stir bar on a magnetic hotplate stirrer. SKA-346 in DMSO was added to oil with constant stirring to obtain the final preparation containing 6 mg per mL SKA-346 (DMSO: peanut oil = 3 : 97). The final SKA-346 solution was allowed to cool to room temperature, and it appeared light brownish in colour. The SKA-346 preparation was then aliquoted into autoclaved 2 mL glass vials and stored at −20 °C until use. Prior to injection, the SKA-346 preparation was thawed, heated to ∼60 °C with stirring and then cooled to room temperature. Female C57BL/6 mice (aged 6–7 weeks at the start of the treatment, n = 6) were injected (i.p.) with ∼100 μL of 6 mg mL⁻¹ preparation (i.e., 30 mg kg⁻¹) of SKA-346 once daily for 14 consecutive days. Similarly, control mice (n = 6) were injected with a solution of 3% DMSO in 97% peanut oil. Both groups (vehicle and SKA-346 treated) consisted of mice of similar age and weight ranges to ensure that any observed effects could be attributed primarily to the treatment rather than differences in these biological factors. Mice at the SingHealth facility (Singapore) were housed under standard conditions on a 12 h/12 h light/dark cycle with regulated temperature and humidity and fed with certified standard chow and tap water ad libitum. Mice were monitored daily by trained technicians throughout the study to check any signs of weight loss, intractable diarrhoea, or infection. Body weights were recorded every 2–4 days throughout the experiment. At the end of the treatment on day 14, mice were euthanized, and blood and organs (kidney, liver, brain, heart, thymus, and spleen) were harvested for biochemical and histological analysis.

AST levels in the mice serum samples were determined using commercially available colorimetric kits from Abcam (UK) in accordance with the instructions provided.

Harvested organs were fixed, embedded in paraffin, sectioned, and then mounted on glass slides. Tissue sections slides were placed in a 60 °C incubator for 1 h to remove excess paraffin. The section slides were stained with the histological stains hematoxylin and eosin using Leica ST5010 Autostainer (Leica, Germany). Cover glass slides were mounted onto the H&E-stained tissue sections and dried in the fume hood overnight. Slides were imaged using Zeiss Axioscan 7 microscope slide scanner (Zeiss, Germany) and presented.

4.4. In vivo pharmacokinetics profiling

Animal experiments for in vivo pharmacokinetics assessments of SKA-346 were conducted as per the protocol approved by the Animal Ethics Committee of Biological Resource Centre, A*STAR, Singapore (IACUC #201561). Female BALB/c mice (aged 7–9 weeks at the start of the treatment, n = 4 for each time-point) were injected intraperitoneally with SKA-346 at the dose of 30 mg kg⁻¹/10 mL using 3% DMSO + 97% peanut oil formulation. Post-administration of SKA-346, blood samples were collected at 0, 0.25, 0.5, 1, 2, 4, 8, and 24 h. Plasma was separated from the blood samples and subsequently analyzed by LC-MS for SKA-346 quantification.

4.5. LC-MS analysis of SKA-346 in mice blood plasma and organ samples

The LC-MS analysis was performed using Waters equipment coupled with Xevo TQ-S MS system (Waters Corporation, USA). Mice blood plasma samples (20 μL each) were prepared by rapid mixing with 60 μL LC-MS grade methanol: dichloromethane (1 : 1) on a vortex mixer. Tissue samples were prepared by homogenization of pre-weighed organs in 500 μL LC-MS grade methanol: dichloromethane (1 : 1) using a tissue homogenizer. These samples were incubated at 4 °C for 30 min, centrifuged at 14 000 rpm at 4 °C for 10 min, and then injected into LC-MS system for analysis. The UHPLC system was equipped with a BEH C18 column (2.1 mm × 100 mm, 1.7 μm, Waters Corporation, USA). The column temperature was kept at 40 °C. The mice blood plasma and organs samples were eluted at a flow rate of 0.3 mL min⁻¹. Water containing 0.1% formic acid (Solvent A) and acetonitrile containing 0.1% formic acid (Solvent B) were used as mobile phases. For blood plasma samples, the LC was operated with the gradient conditions as follows: 0 min, 20% B; 0.5 min, 20% B; 5 min, 95% B; 7 min, 95% B; 7.1 min, 20% B; 10 min, 20% B. For organ samples, the LC was operated with the gradient conditions as follows: 0 min, 20% B; 5 min, 95% B; 6 min, 95% B; 7 min, 20% B; 10 min, 20% B. The total chromatographic run time was 10 min. Mass spectrometer conditions were set as follows: capillary voltage at 0.5 kV, desolvation temperature at 350 °C, cone gas flow rate at 150 L h⁻¹, desolvation gas flow rate at 800 L h⁻¹, source temperature at 150 °C. The mass spectrometer was operated with an electrospray ionization source (ESI) in the positive mode and performed in the multiple reaction monitoring (MRM) mode. Data acquisition, integration and quantification were performed using MassLynx and TargetLynx (Waters Corporation, USA).

4.6. Statistical analysis

Experiments were replicated independently at least three times. Depending on specific experiments, comparison between two groups was made by paired Student's t-test and comparisons of multiple groups within a single experiment were made by one-way ANOVA. Prism 9.5.0 (GraphPad) was used to make the graphs and statistical analyses.

Ethical statement

All animal procedures were performed in accordance with the Guidelines for Responsible Care and Use of Laboratory Animals and as per the protocol approved by the Animal Ethics Committee of Singapore Health Services, SingHealth Singapore (IACUC #2018/SHS/1417) as well as the Biological Resource Centre, A*STAR, Singapore (IACUC #201561). Experiments utilizing human peripheral blood samples were performed in accordance with institutional guidelines and the protocol approved by the Institutional Review Board of Nanyang Technological University Singapore (IRB-2018-05-034).

Data availability

The data supporting this article have been included in the main article and the ESI.†

Author contributions

Conceptualization, B. H. S. W. and N. K. V.; patch clamp analysis, B. H. S. W., S. S. M. G. and S. T. O.; SKA-346 synthesis, H. W. and H. S.; molecular docking, H. S.; animal experiments, data acquisition and analysis, N. A. B. M. T., K. X. Y. C., K. L., D. H., C. K. O. and T. S. V.; pharmacology, T. S. V., B. H. S. W., D. H. and Y. W.; LC-MS analysis, Y. C. C. and Y. W.; formal analysis, B. H. S. W., R. D. W., V. G. S., N. K. V. and H. W.; investigation, B. H. S. W. and N. K. V.; resources, N. K. V. and C. K. O.; data curation, B. H. S. W., S. T. O. and H. S.; writing—original draft, B. H. S. W. and N. K. V.; writing—review & editing, B. H. S. W., S. T. O., D. H., C. K. O., Y. W., H. W., R. D. W., V. G. S. and N. K. V.; supervision, N. K. V., R. D. W. and V. G. S.; project administration, N. K. V.; funding acquisition, N. K. V. All authors have seen and approved the final version of the manuscript.

Conflicts of interest

There is no conflict of interest to declare.

Acknowledgements

Authors acknowledge Professor K. George Chandy for reading, editing, and critiquing this manuscript, and Sai Life Sciences for their help in chemical synthesis. Authors also acknowledge Dr Chang Jun Jie and Dr Loh Xian Jun at Institute of Materials Research and Engineering (IMRE), A*STAR for their assistance in determining the drug solubility. This research was supported, in part, by the Singapore Ministry of Education (MOE) under its MOE Academic Research Fund (AcRF) Tier 2 Grant (MOE2017-T2-2-004), MOE AcRF Tier 1 Grant (RG94/22), and the National Research Foundation Singapore under its Open Fund Large Collaborative Grant (OFLCG-23May0039) and administered by the Singapore Ministry of Health’s National Medical Research Council (NMRC). B. H. S. W. was provided with PhD fellowship by HealthTech NTU.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4ra07330d

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