Discovery of a Small Molecule Activator of Slack (Kcnt1) Potassium Channels That Significantly Reduces Scratching in Mouse Models of Histamine‐Independent and Chronic Itch

Abstract Various disorders are accompanied by histamine‐independent itching, which is often resistant to the currently available therapies. Here, it is reported that the pharmacological activation of Slack (Kcnt1, KNa1.1), a potassium channel highly expressed in itch‐sensitive sensory neurons, has therapeutic potential for the treatment of itching. Based on the Slack‐activating antipsychotic drug, loxapine, a series of new derivatives with improved pharmacodynamic and pharmacokinetic profiles is designed that enables to validate Slack as a pharmacological target in vivo. One of these new Slack activators, compound 6, exhibits negligible dopamine D2 and D3 receptor binding, unlike loxapine. Notably, compound 6 displays potent on‐target antipruritic activity in multiple mouse models of acute histamine‐independent and chronic itch without motor side effects. These properties make compound 6 a lead molecule for the development of new antipruritic therapies targeting Slack.

Presented are data from published single-cell RNA-seq studies.A) Expression pattern in mouse DRG neurons (6-8 week old). [1]B) Expression pattern in mouse DRG neurons (embryonic day 11.5 to postnatal day 42) presented as a force-directed layout. [2]Downloaded from:  (indicated as increased F/F(Baseline) ratio) was detected after incubation with both loxapine and vehicle (FluxOR assay buffer containing 0.03% DMSO).As Slack is activated by Na + , the vehicle-induced Slack activation was most likely mediated by Na + present in the FluxOR assay buffer and in the vehicle.B) In a Na + -free buffer (with replacement of NaCl by choline chloride), Slack activation was observed after incubation with 30 µM loxapine and 140 mM NaCl, but not after incubation with vehicle (Na + -free buffer with 2 mM Ca 2+ , 2 mM Mg 2+ and 0.03% DMSO).C) DMSO at a concentration of 0.03-3% did not activate Slack in a Na + -free buffer with 2 mM Ca 2+ and 2 mM Mg 2+ .D) Concentration-response experiments with loxapine in a Na + -free buffer with 2 mM Ca 2+ , 2 mM Mg 2+ and 0.03% DMSO yielded an EC50 value of 23.45 µM in the first preliminary experiments.E,F) As Slack is inhibited by bivalent cations, further control experiments with loxapine in a Na + and Ca 2+ free buffer (E) and in a Na + , Ca 2+ and Mg 2+ free buffer (F) containing 0.03% DMSO were conducted, which however did not result in appropriate concentration-response curves.G) Concentration-response experiments with a Slack inhibitor (compound 31 from ref [5] ) in a Na + -free buffer with 2 mM Ca 2+ , 2 mM Mg 2+ and 0.03% DMSO and pre-stimulation with 25 µM loxapine revealed that compound 31 inhibited the F/F(Baseline) ratio in a concentration-dependent manner (IC50 = 2.1 µM), confirming that the readout depends on Slack.Therefore, all further experiments with the FluxOR assay in this study, which are presented in Figure 1A, were performed using a Na + -free buffer with 2 mM Ca 2+ , 2 mM Mg 2+ and 0.03% DMSO.All conditions were measured at least in duplicate and data are shown as mean ± SD.H,I) Representative traces of (H) loxapine and (I) compound 6 from FluxOR experiments that are presented in Figure 1A.
In (I), loxapine (50 µM) is included for better comparability.that allows for simultaneous investigation of cellular viability, nuclear morphology, mitochondrial mass and membrane integrity [6] was performed in HEK293 cells after 6 h incubation of new compounds at different concentrations.Cell counts were normalized against cells treated with 0.1 % DMSO (100%).A) None of the compounds decreased the cell viability by more than 50%, which is deemed a relevant impairment. [6]B) A decrease of normalized cell count to more than 50% based on Hoechst High Intensity objects, which indicates compound precipitation, [7] was only detected for compound 1 at 100 µM.C) No alterations in membrane integrity were detected.D) A decrease in mitochondrial mass by more than 50%, indicating cellular stress, [6] was only observed for compound 8 at 100 µM.E) In HEK-Slack cells, none of the compounds significantly impaired the confluence after 24 h incubation.Data are shown as mean ± SEM of biological duplicates.

Figure S6
. Target binding of compound 6 and loxapine.A) Slack was purified from HEK293F cells transiently expressing Slack via affinity purification and size-exclusion chromatography.B) Melting temperatures of purified Slack incubated with an increasing concentration of compound 6, loxapine and a Slack inhibitor (compound 31 from ref [5] ) that was used as positive control.Purified Slack in buffer without any compound was used as a   were tested at a concentration of 10 µM in binding and enzyme and uptake assays of 44 targets (mostly human; except BZD, NMDA, MAO-A, Ca 2+ channel, KV channel, and Na + channel, which were obtained from rat specimens).Compound binding was calculated as a % inhibition of the binding of a radioactively labeled ligand (agonist or antagonist, as indicated in brackets) specific for each target.Compound enzyme inhibition effect was calculated as % inhibition of control enzyme activity.Results showing an inhibition (or stimulation for assays run in basal conditions) higher than 50% are considered to represent significant effects of the test compounds and are presented in red.Results showing an inhibition or stimulation between 25% and 50% (indicative of weak to moderate effects) and those lower than 25% (considered mostly attributable to variability of the signal around the control level) are presented in grey.Measurements were performed in duplicate.Table S3.Histamine H 1 receptor affinity of compound 6 and loxapine.The corresponding lactam (1.0 equiv) was dissolved in freshly distilled phosphorus oxychloride (0.5 M) and N,N-dimethylaniline (0.6 equiv) was added.The resulting mixture was heated to reflux in an oil bath for 5 h.Thereafter, excess phosphorus oxychloride was removed under reduced pressure and the residue obtained was taken up in toluene and washed once with cold water.The organic phase was dried over magnesium sulfate, filtered, and evaporated to give the crude product, which was immediately used in the next step without further purification.

GP7: Formation of loxapine derivatives
The corresponding amine (2.0 equiv) was added to a solution of the appropriate imidoylchloride (1.0 equiv) in p-xylene (0.15 M regarding imidoylchloride).The resulting mixture was heated in an oil bath at 140 °C for 5 h.Solvents were evaporated, and the crude substance purified by preparative HPLC or flash chromatography.If unsubstituted piperazine or homopiperazine was employed, then alkylation of these derivatives was obtained by GP8

GP8: Alkylation of loxapine derivatives
A mixture of loxapine derivative (1 equiv), corresponding alkyl chloride (2 equiv) and triethylamine (10 equiv) in acetonitrile (0.15 M) was heated to reflux in an oil bath for 16 h.
After completion, the reaction mixture was evaporated, and the residue purified by preparative HPLC or flash chromatography.

Characterization of compounds
Methyl 2-methoxy-5-(trifluoromethyl)benzoate (9 A solution of 2-methoxy-5-(trifluoromethyl)benzoic acid (1.00 g, 4.41 mmol) in MeOH (10 mL) was treated with concentrated sulfuric acid (235 µL, 4.41 mmol) and heated to reflux in an oil bath overnight.The reaction mixture was concentrated under reduced pressure and the residue was taken up in water.The aqueous phase was extracted three times with ethyl acetate and the combined organic phases were dried over magnesium sulfate and filtered.

Figure S1 .
Figure S1.Expression of Kcnt1 and critical itch receptors across sensory neuron subsets.

Figure S2 .
Figure S2.Chemical synthesis scheme of new compounds.A) General synthesis of lactam starting from methyl esters of salicylic acid and 2-fluoronitrobenzene derivatives.B) General synthesis of lactam starting from 2-fluorobenzoic acids and 2-aminophenol derivatives.C) General synthesis of loxapine derivatives starting from lactam.

Figure S3 .
Figure S3.Establishing a modified version of the FluxOR assay.Cultured HEK293 cells stably expressing human Slack (HEK-Slack cells) were incubated with compounds in different buffers.A) In the buffer provided with the FluxOR assay kit, Slack activation

Figure S4 .
Figure S4.New compounds evoke Slack-mediated potassium currents.A) Currentvoltage (I-V) curves from patch-clamp experiments that are presented in Figure 1B.Wholecell voltage recordings on HEK-Slack cells were performed at baseline and after incubation with a new compound (50 µM), loxapine (50 µM) or vehicle (external solution containing 0.03% DMSO).Loxapine was incubated in each series of experiments as a positive control.n = 5-24 cells per group.Data are shown as mean ± SEM.B) Representative original current traces evoked by a voltage step to +80 mV recorded before (black) and after the application of loxapine (red) or the tested new compounds (blue).Data are from patch-clamp experiments pooled in Figure 1B.

Figure S5 .
Figure S5.Cytotoxicity of new compounds.A-D) A live-cell phenotypic screening assay negative control.The melting temperature (Tm in °C) was determined with differential scanning fluorimetry (DSF) and plotted against the concentration of the compounds used.The dashed line indicates the Tm of purified Slack without compound.n = 3 experiments.Data are shown as mean ± SD.

Figure S7 .
Figure S7.Slack-mediated potassium currents evoked by compound 6 are increased in presence of intracellular sodium.A) Current-voltage (I-V) curves from whole-cell voltage recordings on HEK-Slack cells at baseline and after incubation with compound 6 (25 µM).Recordings were performed with a Na + -free pipette solution and with 10 mM Na + in the pipette solution.B) Fold increase of current densities shown in (A) relative to baseline at a voltage of +80 mV.Data show that compound 6 is more potent in the presence of intracellular Na + .* P < 0.05, one-tailed unpaired Student's t test.n = 8-9 cells per group.Data are shown as mean ± SEM.

Figure S8 .
Figure S8.In vitro pharmacology screening of compound 6 and loxapine.Compounds

Figure S9 .
Figure S9.Sex-related effects of compound 6 in acute itch models.Breakdown of results in male and female mice from (A) Figure 3C, (B) Figure 3E, (C) Figure 3F, (D) Figure 3G, and (E) Figure 3H.No obvious sex-related differences were observed in any assay.Statistical significance was assessed by one-way-ANOVA with Dunnett's correction.Data represent the mean ± SEM (n = 2-5).

Figure S10 .
Figure S10.Sex-related effects of compound 6 and correlation of behavioral outcomes in chronic itch models.A-D) Breakdown of results in male and female mice from (A) Figure 4B, (B) Figure 4C, (C) Figure 4E, and (D) Figure 4F.There was a tendency toward reduced chronic itch behavior in males compared to females (head shaking in the DNFB model and scratching in the MC903 model), albeit not significant.Statistical significance was assessed by one-way-ANOVA with Dunnett's correction.Data represent the mean ± SEM (n = 1-6).E-G) Correlation of number of head shakes with number of scratching bouts (E) in the DNFB model (Figure 4B,C), (F) in the MC903 model (Figure 4E,F) and (G) in both models.Statistical significance was assessed by a Pearson correlation.

Figure S11 .
Figure S11.Potential side effects of compound 6.A,B) Pulse oximetry on non-anesthetized mice using a MouseOX Plus device revealed that i.p. delivery of compound 6 or vehicle did not alter (A) the heart rate or (B) the breath rate, whereas morphine significantly lowered both parameters (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle, two-way MC ANOVA and Dunnett test).C,D) Sensing of (C) mechanical stimuli (von Frey filament test) and (D) heat stimuli (Hargreaves test) was not affected after i.p. delivery of compound 6 or vehicle (n = 12).Data represent the mean ± SEM.

Figure S12 .
Figure S12.Tetrodotoxin-resistant Na + currents in IB4-binding DRG neurons are not affected by compound 6.Representative recordings of whole-cell INa superfused with 250 nM tetrodotoxin under control conditions (black) and after addition of (A) 50 µM compound 6 (red), (B) vehicle (external solution containing 0.03% DMSO; grey), or (C) 50 µM lidocaine (green), which was used as a positive control.INa was elicited after a 700 ms prepulse in 10 mV depolarizing steps from -80 mV to +40 mV for 50 ms.Recordings were performed in 3 IB4-binding DRG neurons per group.
[ 3 H]-Spiperone competition binding assays were performed using a cell membrane preparation of CHO cells stably expressing the human D2s and D3 receptor.Ki data represent mean with the 95% confidence interval (CI 95%) of n independent experiments, each performed in triplicate using seven appropriate concentrations of test compound.Haloperidol was used as a reference compound and to determine non-specific binding at 10 μM. 5 mg/kg) Cmax (ng/mL) tmax (min) AUC0-inf (ng*min/mL) t1/2 (min) [ 3 H]-Pyrilamine competition binding assays were performed using cell membrane preparation of CHO cells stably expressing the human H1 receptor.Ki data represent mean with the 95% confidence interval (CI 95%) of n independent experiments, each performed in triplicate using seven appropriate concentrations of test compound.Chlorphenamine maleate was used as reference compound.Non-specific binding was determined in the presence of 10 µM chlorphenamine maleate.Supplemental protocols for synthesis of compounds (A) General synthesis of lactam starting from methyl esters of salicylic acid and 2fluoronitrobenzene derivatives.GP1: Formation of the diarylether Potassium carbonate (1.5 equiv) was added to a solution of the corresponding methyl salicylate (1.5 equiv) and the corresponding 2-fluoronitrobenzene (1.0 equiv) in DMF (1.5 M regarding 2-fluoronitrobenzene).The resulting solution was heated in an oil bath to 120 °C overnight.Solvents were evaporated under reduced pressure and the residue taken into water and extracted three times with ethyl acetate.The combined organic phases were dried over magnesium sulfate, filtered, and evaporated.The resulting crude product was purified by flash chromatography.GP2: Reduction of the nitro groupA solution of SnCl2•2H2O (4.0 equiv) in conc.HCl (3 M) was added to a solution of the corresponding methyl 2-(2-nitrophenoxy)benzoate derivative (1.0 equiv) in a mixture of ethanol/conc.HCl 1:1 (0.5 M).The resulting solution was stirred at rt overnight.After that time, the temperature was set to 0 °C and the pH of the reaction solution made slightly basic by addition of sodium carbonate.The resulting solution was then extracted three times with ethyl acetate.The combined organic phases were dried over magnesium sulfate, filtered, and evaporated.The resulting crude product was purified by flash chromatography.GP3: Formation of the lactam ring via intramolecular condensationA solution of the corresponding methyl 2-(2-aminophenoxy) benzoate derivative (1.0 equiv) in DMF (0.2 M) was treated with concentrated sulfuric acid (1.3 equiv) and heated in an oil bath at 120 °C overnight.After that time, the reaction was cooled to 0 °C and a few millilitres of water were added.The precipitated product was then filtered off and dried under vacuum to obtain the crude product, which was used in the next step without further purification.(B) General synthesis of lactam starting from 2-fluorobenzoic acids and 2-aminophenol derivatives.GP4: Formation of the amideTo a solution of the corresponding 2-fluorobenzoic acid (1.0 equiv) in THF (1 M) freshly distilled thionylchloride (2.0 equiv) was added and heated to reflux in an oil bath for 2 h.Thereafter, excess thionylchloride and THF were removed under reduced pressure and the residue obtained was taken up in THF (2.5 M) again.This solution was added dropwise to a solution of the corresponding 2-aminophenol derivative (1.0 equiv) and triethylamine (2 equiv) in THF (2.5 M referred to 2-aminophenol derivative) at 0 °C and the reaction mixture was stirred overnight at rt.After that time, the reaction mixture was concentrated under reduced pressure and the residue taken up in ethyl acetate.The organic phase was first washed with an aq HCl solution (2 M), water and saturated aq NaCl solution, then dried over magnesium sulfate, filtered, and evaporated.The resulting crude product was purified by flash chromatography.GP5: Formation of the lactam ring via intramolecular nucleophilic aromatic substitutionA solution of the corresponding 2-fluoro-N-(2-hydroxyphenyl)benzamide derivative (1.0 equiv) in DMF (0.25 M) was treated with freshly powdered NaOH (1.0 equiv) and heated in an oil bath at 150 °C for 5 h.After that time, the reaction was cooled to 0 °C and a few millilitres of water were added.The precipitated product was then filtered off and dried under vacuum to obtain the crude product, which was used in the next step without further purification.(C)General synthesis of loxapine derivatives starting from lactam GP6: Reaction with phosphorus oxychloride