Journal: Mediterranean Journal of Hematology and Infectious Diseases

Article Title: The Evolving Pharmacotherapeutic Landscape for the Treatment of Sickle Cell Disease

doi: 10.4084/MJHID.2020.010

Figure Lengend Snippet: Completed multicenter randomized double-blind placebo-controlled trials to prevent or treat sickle painful crises that failed, discontinued or terminated.

Article Snippet: Senicapoc , Pfizer , Gardos channel blocker, Preventive agent , Prevention of VOC , Phase II completed for SCD; Drug increased red cell survival and hematocrit and blood viscosity; Phase III trial failed , [ , ] .

Techniques: Polymer, Blocking Assay, Concentration Assay, Inhibition, Activation Assay, Viscosity, Adhesive

Journal: Current Neuropharmacology

Article Title: KCa3.1 Channel Modulators as Potential Therapeutic Compounds for Glioblastoma

doi: 10.2174/1570159X15666170630164226

Figure Lengend Snippet: Cartoon illustrating the binding sites of the triarylmethanes TRAM-34 and senicapoc in the inner pore and nifedipine in the fenestration region of K Ca 3.1. The tetramer of K Ca 3.1 alpha-subunits is shown as a grey ribbon. The channel associated calmodulin is shown in dark magenta. ( The color version of the figure is available in the electronic copy of the article ).

Article Snippet: Since senicapoc had been safe and well tolerated in humans, Icagen Inc. explored other therapeutic opportunities for senicapoc after this failure.

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Names, 2D structures and references of the compounds mentioned in the introduction.

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: IK blockers affect WM266−4 and Panc−1 viability regardless of IK channel expression. ( A ) Relative KCNN4 expression from RT-qPCR showing the difference in KCNN4 mRNA levels between WM266-4 and Panc-1 (N = 3). ( B ) Results from MTT viability assays after 72 h of exposure to 30 µM clotrimazole, 30 µM senicapoc or the corresponding amount of DMSO (N = 4 for all). Data are reported as absorbance (570 nm) ratio drug-/DMSO-treated cells (color code reported in the legend). ( C ) Exemplary pictures from experiments in ( B ): WM266-4 and Panc-1 cells treated for 72 h with 30 µM clotrimazole, 30 µM senicapoc or the corresponding amount of DMSO. Significance level is indicated by two asterisks ( p < 0.01).

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Clotrimazole and senicapoc decrease the migration of WM266-4 and Panc-1. ( A ) Migration rate from trans-well migration assays of cells exposed to 30 µM clotrimazole or 30 µM senicapoc with respect to DMSO-treated cells (N = 4 for all). Different cell lines and treatments are color coded as reported in the legend. ( B ) Relative increases of cell-covered areas at t = 24 h with respect to t = 0 from the same petri dish for WM266-4 (DMSO N = 9, clotrimazole N = 9, senicapoc N = 8) and Panc-1 (DMSO N = 6, clotrimazole N = 6, senicapoc N = 5). Data are significantly different from control for WM266-4, clotrimazole ( p = 0.0128), WM266-4, senicapoc ( p = 0.001), and Panc-1, senicapoc ( p = 0.0246). Significance level is indicated by an asterisk ( p < 0.05).

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Migration

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Ca 2+ −evoked whole-cell currents of WM266−4 cells. ( A ) Exemplary current traces of WM266-4 whole-cell currents when the patch pipette was filled with the Ca 2+ -free intracellular solution. ( B ) Distribution of current amplitudes in WM266-4 cells measured at 100 mV with 1 µM Ca 2+ in the pipette solution. ( C ) Exemplary WM266-4 current traces from recordings with 1 µM Ca 2+ in the intracellular solution; cells were perfused with standard bath solution (top) or with the same solution + 1 µM senicapoc (bottom). ( D ) Exemplary Panc-1 current traces from recordings with 1 µM Ca 2+ in the intracellular solution; cells were perfused with standard bath solution (top) or with the same solution + 1µM clotrimazole (bottom). ( E ) Average normalized IVs of WM266-4 cells before (blue circles) and after (green circles) application of 1 µM senicapoc (N = 4 cells). Currents are normalized to the value at 100 mV. ( F ) Average normalized IVs of WM266-4 cells before (blue circles) and after (red circles) application of 1 µM clotrimazole (N = 4). Currents are normalized to the value at 100 mV. ( G ) Background-subtracted currents (background was calculated from the mean of 4 cells measured in Ca 2+ -free conditions) in the presence/absence of senicapoc normalized to the currents measured in standard bath solution at the same voltage in the same cells (mean ± SE, N = 4). ( H ) Background-subtracted currents in the presence/absence of clotrimazole normalized for the currents measured in standard bath solution at the same voltage in the same cells (mean ± SE, N = 4). Significance level is indicated by two asterisks ( p < 0.01).

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Transferring

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Panc−1 cells lack functional IK channels. ( A ) Current traces measured from a typical Panc-1 cell with 1 µM Ca 2+ in the patch pipette in standard extracellular solution (top left), during perfusion with 1 µM senicapoc (top right), with 1 µM clotrimazole (bottom left) or 1 µM paxilline (bottom right). ( B ) Average normalized current voltage relationship in control conditions (turquoise symbols), in 1 µM senicapoc (green symbols, N = 7), 1 µM clotrimazole (red symbols, N = 7) and 1 µM paxilline (black symbols, N = 7) (currents are normalized to those measured in control conditions at 100 mV; error bars indicate SEM). ( C ) Average current density normalized to that measured in control conditions at 100 mV in the indicated conditions.

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Functional Assay, Transferring

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Senicapoc and clotrimazole do not alter the intracellular Ca 2+ concentration. ( A ) [Ca 2+ ] i over time from 32 WM266-4 cells (color code in the legend). ( B ) Mean Δ[Ca 2+ ] i from ( A ) with respect to bath solution (color-coded as in ( A ), N = 32). ( C ) Mean [Ca 2+ ] i over time from 32 Panc-1 cells (color-coded as in ( A )). ( D ) mean Δ[Ca 2+ ] i from ( C ) with respect to bath solution (color-coded as in ( C ), N = 32).

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration

doi: 10.3390/ijms242216285

Figure Lengend Snippet: Effect of IK blockers on F-actin organization. WM266-4 ( A ) and Panc-1 ( B ) cells incubated for 24 h in vehicle alone or with 30 µM clotrimazole, 30 µM senicapoc or the corresponding volume of DMSO, which have subsequently been labeled with phalloidin (red) and DAPI (blue) and processed for fluorescence microscopy (see ).

Article Snippet: Clotrimazole, senicapoc and paxilline were purchased from Merck (Milan, Italy)and dissolved in DMSO to prepare stock solutions according to the information provided from the manufacturer.

Techniques: Incubation, Labeling, Fluorescence, Microscopy

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: MPTP mouse model groups

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Control

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced loss of DA neurons. a – g WT or KCa3.1 −/− mice received sequential intraperitoneal injections of MPTP (20 mg/kg) with or without senicapoc (100 mg/kg, once daily, p.o.) treatment for 5 days as described in the “ ” section. Open field test ( b – e ) and the rotarod test ( f , g ) for bradykinesia were performed. Behavioral tests for MPTP-induced bradykinesia were conducted on the indicated days. Data are presented as mean ± SEM ( n = 10–15). b – e ** p < 0.01, *** p < 0.001, **** p < 0.0001, one-way ANOVA followed by the Dunnett’s multiple comparison test. f , g # p < 0.05, ## p < 0.01 compared to respective control. * p < 0.05, ** p < 0.01 compared to the MPTP group. Two-way ANOVA followed by the Bonferroni multiple comparison test. h The representative slices of the control and the treatment (MPTP, MPTP+Se, Se) mice. Scale bar 100 μm. i Quantitative analysis of TH-positive cells in SNpc. Data are presented as mean ± SEM, n = 5, * p < 0.05, ** p < 0.01, one-way ANOVA followed by the Dunnett’s multiple comparison test

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Comparison, Control

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuate MPTP-induced microgliosis. a , c Immunostaining for Iba1 in SNpc. Bar 50 μM. Quantitative analysis of Iba1 + cells in SNpc. Data are presented as mean ± SEM ( n = 5–8). * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by the Dunnett’s multiple comparison test. b , d Expression of iNOS, COX-2, TNF-a, IL-6, and IL-1β in SNpc was measured by qPCR. Data are presented as mean ± SEM ( n = 5). * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by the Dunnett’s multiple comparison test

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Immunostaining, Comparison, Expressing

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: Genetic KCa3.1 deletion and pharmacological blockade with senicapoc attenuated MPTP-induced ER stress. a , d Western blot analysis of GRP78 and CHOP protein levels in SNpc. b , c , e , f Data are presented as the mean ± SEM ( n = 5–6). Western blot was repeated three times and showed similar results. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control or WT group mice; * p < 0.05, *** p < 0.001 vs. MPTP or WT+MPTP group mice. One-way ANOVA followed by Dunnett’s multiple comparison test. WT, wild-type

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Western Blot, Control, Comparison

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: AKT modulation is crucial for KCa3.1-mediated ER stress in microglia. a , b Representative blots of p-AKT and total AKT in SNpc from a WT, WT+MPTP, KCa3.1 −/− , KCa3.1 −/− +MPTP group mice and from b control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. The OD value of p-AKT was normalized to that of AKT. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test. c Representative blots of p-mTOR, p-4EBP1, and total mTOR in SNpc from control, MPTP, MPTP+Se, Se group mice. Data are presented as the mean ± SEM ( n = 3–5). Western blot was repeated three times and showed similar results. * p < 0.05, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test. d Representative images of p-AKT (S473) and total AKT in microglia responses to 500 μM MPP + for 12 h with or without 1 μM senicapoc. Mean values of p-AKT relative to AKT. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated 3 times and showed similar results. * p < 0.05, one-way ANOVA followed by the Dunnett’s multiple comparison test. WT, wild-type

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Control, Western Blot, Comparison

Journal: Journal of Neuroinflammation

Article Title: The potassium channel KCa3.1 represents a valid pharmacological target for microgliosis-induced neuronal impairment in a mouse model of Parkinson’s disease

doi: 10.1186/s12974-019-1682-2

Figure Lengend Snippet: KCa3.1 involved in microglia SOCE and ER stress. a , b Representative images of GRP78, p-PERK, and p-eIF2α in KCa3.1 −/− microglia, responses to 500 μM MPP + ( a ) or 1 μM Tg ( b ) vs. WT cells. Mean values of GRP78, p-PERK, and p-eIF2α relative to β-actin. Data are presented as the mean ± SEM ( n = 3). Western blot was repeated three times and showed similar results. * p < 0.05, ** p < 0.01, unpaired, two-tailed Student’s t test. c Primary cultured microglia were treated with 500 μM MPP + for 12 h with or without pretreatment of 1 μM senicapoc or 10 μM 2-APB. Fluorescence intensities of [Ca 2+ ] i are shown. Fluorescence intensity was measured in the presence of 1 μM Tg with or without 2 mM Ca 2+ . Data are presented as the mean ± SEM ( n = 10). ### p < 0.001 vs. control, *** p < 0.001 vs. MPP + -treated cells. One-way ANOVA followed by Dunnett’s multiple comparison test. d Western blot analysis of KCa3.1 and Orai1 expression after 500 μM MPP + -treatment for 3, 6, 12 h. Data represent the mean ± SEM of KCa3.1 and Orai1 density normalized to β-actin values for n = 3 cultures. * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA followed by Dunnett’s multiple comparison test compared with control. e – g Levels of the dendritic marker MAP2 were compared between neurons cocultured with KCa3.1 −/− ( e , f ) or control microglia ( g ). e Neuron dendrites were immunostained with MAP2, and nuclei were stained with DAPI (blue). e , f Neuron cocultured with WT or KCa3.1 −/− microglia treated with 500 μM MPP + for 12 h. g Neuron cocultured with/without microglia treated with 500 μM MPP + for 12 h with pretreatment of 1 μM senicapoc for 30 min. Cell body area, neurite length, and branch point counts were analyzed by extended neurite outgrowth bioapplication software. Data represent mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by Dunnett’s multiple comparison test. Tg, thapsigargin; Se, senicapoc

Article Snippet: Senicapoc (ICA-17043; Target Molecule Corp., Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) to provide a 100-mM solution, which was diluted in medium for in vitro studies.

Techniques: Western Blot, Two Tailed Test, Cell Culture, Fluorescence, Control, Comparison, Expressing, Marker, Staining, Software