Journal: ACS Medicinal Chemistry Letters

Article Title: Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4)

doi: 10.1021/acsmedchemlett.9b00274

Figure Lengend Snippet: Spirocarbamate lead. (a) TRPV4 IC50 values are averages of at least two measurements. (b) DMPK properties are averages of measurements taken in at least two Sprague–Dawley rats. (c) Rat PPB fu values are averages of at least two measurements.

Article Snippet: 8 , 9 GlaxoSmithKline (GSK) previously reported TRPV4 antagonists series, quinolines and benzimidazoles, failed to deliver clinical drug candidates necessitating the need to identify an alternative series.

Techniques:

Journal: ACS Medicinal Chemistry Letters

Article Title: Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4)

doi: 10.1021/acsmedchemlett.9b00274

Figure Lengend Snippet: Early methyl-cyclohexane N-aryl analog. (a) TRPV4 IC50 values are averages of at least two measurements. (b) DMPK properties are an average of measurements taken in at least two Sprague–Dawley rats.

Article Snippet: 8 , 9 GlaxoSmithKline (GSK) previously reported TRPV4 antagonists series, quinolines and benzimidazoles, failed to deliver clinical drug candidates necessitating the need to identify an alternative series.

Techniques:

Journal: ACS Medicinal Chemistry Letters

Article Title: Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4)

doi: 10.1021/acsmedchemlett.9b00274

Figure Lengend Snippet: Potent TRPV4 inhibitor with desirable rat PK. (a) TRPV4 IC50 is an average of at least two measurements. (b) DMPK properties are an average of measurements taken in two Sprague–Dawley rats. (c) Rat PPB fu is an average of at least two measurements.

Article Snippet: 8 , 9 GlaxoSmithKline (GSK) previously reported TRPV4 antagonists series, quinolines and benzimidazoles, failed to deliver clinical drug candidates necessitating the need to identify an alternative series.

Techniques:

Journal: ACS Medicinal Chemistry Letters

Article Title: Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4)

doi: 10.1021/acsmedchemlett.9b00274

Figure Lengend Snippet: GSK2798745 TRPV4 oral candidate. (a) TRPV4 IC50 is an average of at least two measurements. (b) DMPK properties are an average of measurements taken in two Sprague–Dawley rats. (c) Rat PPB fu is an average of at least two measurements.

Article Snippet: 8 , 9 GlaxoSmithKline (GSK) previously reported TRPV4 antagonists series, quinolines and benzimidazoles, failed to deliver clinical drug candidates necessitating the need to identify an alternative series.

Techniques:

Journal: ACS Medicinal Chemistry Letters

Article Title: Discovery of GSK2798745: A Clinical Candidate for Inhibition of Transient Receptor Potential Vanilloid 4 (TRPV4)

doi: 10.1021/acsmedchemlett.9b00274

Figure Lengend Snippet: N -Aryl Methylcyclohexane TRPV4 Potency SAR

Article Snippet: 8 , 9 GlaxoSmithKline (GSK) previously reported TRPV4 antagonists series, quinolines and benzimidazoles, failed to deliver clinical drug candidates necessitating the need to identify an alternative series.

Techniques:

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Cyclic compression increases F508 Del CFTR expression in ciliated human airway epithelium

doi: 10.1152/ajplung.00020.2019

Figure Lengend Snippet: Cyclic compressive stress (CCS) increases CFTR maturation in human airway epithelial cells, an effect requiring cilia, transient receptor potential vanilloid (TRPV4), and nitric oxide synthase (NOS). A: F508Del homozygous human airway cells [air-liquid interface 1 (ALI 1)] underwent CCS (see Fig. 2C) and immunoblotting for CFTR. CCS increased partially (B band) and fully (C band) mature CFTR. B: 9 replicates of experiment in A (ALI 1 and 2); **P < 0.001 by t-test and ***P < 0.001 (for nonparametric distribution) rank-sum test for B and C bands, respectively. C and D: nonciliated F508Del-transfected (C; CFBE41o−) or wild-type (WT)-transfected CFBE41o− cells (D) studied as in A. CFTR did not change with CCS (P = NS), suggesting that cilia, not present in CFBE41o− cells, are required for the CCS effect. E: ciliated F508Del homozygous human airway cells (ALI 2) exposed as in A–D to CCS showed that the B and C bands increase (relative to actin) with CCS was inhibited by the TRPV4 inhibitor HC067047 [both B and C band, *P < 0.01, CCS vs control; CCS/H067047 did not differ from unventilated (P = NS); n = 4 each). F: in WT ciliated airway epithelial cells (ALI 5 and 6), B and C bands increased with CCS, an effect partly blocked by nitro-l-arginine methyl ester [l-NAME; *P < 0.01, CCS vs control; CCS/l-NAME did not differ from unventilated (P = NS); n = 9]. Inset: S-nitrosothiols during CCS were decreased by l-NAME (n = 3 each; *P < 0.05). G: lungs from eNOS −/− mice expressed less B-band CFTR than those from WT. eNOS, endothelial nitric oxide synthase.

Article Snippet: In selected experiments, CCS was also performed after a 2-h preincubation with the TRPV4 inhibitor HC067047 (500 nM) or the NOS inhibitor nitro- l -arginine methyl ester (10 mM l -NAME; Cayman Chemical, Ann Arbor, MI).

Techniques: Western Blot, Transfection

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: Trpv4 mRNA expression in renal cells. A: Trpv4 mRNA was detected by RT-PCR in a mIMCD-3 RNA sample treated with reverse transcriptase (RT) but not in an untreated sample. PCR products were subjected to electrophoresis on a 4% agarose gel stained with ethidium bromide. The 79-bp PCR product migrated a bit more slowly than the 80-bp marker, but its identity was confirmed with sequencing. Trpv4 mRNA was also detected in RT reactions from 3 additional, independent mIMCD-3 cultures (data not shown). B: as measured by RT-qPCR, expression of Trpv4 mRNA was similar in ciliated (176-5) and nonciliated (176-5Δ) renal cells, regardless of which reference gene was used: succinate dehydrogenase complex flavoprotein subunit A (Sdha) or B2m (P > 0.5, t-test). For B, n = 3 experiments.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Electrophoresis, Agarose Gel Electrophoresis, Staining, Marker, Sequencing, Quantitative RT-PCR

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: Hyperosmolality-induced expression of Akr1b3 (aldose reductase) and Slc6a12 [betaine/GABA transporter (Bgt1)] mRNA is affected by the presence of cilia and by stimulation of TRPV4 or TRPM3. A and B: ciliated (176-5) and nonciliated (176-5Δ) renal epithelial cells were treated for 16 h with 0.1% DMSO, a TRPV4 agonist [100 nM GSK1016790A (GSK)], or a TRPM3 agonist [100 μM pregnenolone sulfate (PSS)] at 300 mOsm/kg or 500 mOsm/kg [adjusted with NaCl (N)]. RT-qPCR was performed to determine mRNA expression relative to the reference gene B2m. Hyperosmolal stress induced Akr1b3 and Slc6a12 expression in both cell lines, but the response was greater in ciliated than nonciliated cells. The TRPV4 agonist abrogated hyperosmolality-induced Akr1b3 and Slc6a12 expression in both cell lines (although abrogation of Akr1b3 was not significant for the nonciliated cells). The TRPM3 agonist attenuated hyperosmolality-induced Akr1b3 and Slc6a12 expression only in ciliated cells. C and D: to examine a second cell line, expression of Akr1b3 and Slc6a12 was measured in Orpk cilia(+) and cilia(−) renal epithelial cells exposed to isosmolal (300 mOsm/kg) and hyperosmolal [500 mOsm/kg; adjusted with NaCl (N)] conditions. Hyperosmolality-induced expression of Akr1b3 was enhanced by the presence of cilia. A trend toward increased hyperosmolality-induced Slc6a12 expression was observed in Orpk cilia(+) cells compared with cilia(−) cells, but the increase did not reach statistical significance. Expression is relative to the reference gene Sdha. ND, not detectable. E: to examine another osmolyte, expression of Akr1b3 was measured in Orpk cilia(+) and cilia(−) renal epithelial cells with osmolality increased to 500 mOsm/kg by addition of mannitol (M), rather than NaCl. Mannitol-induced expression of Akr1b3 was enhanced by the presence of cilia. Expression is relative to the reference gene Sdha. %Significantly different (P < 0.05, by Student-Newman-Keuls method) from all other conditions. &Significantly different from all conditions except each other. No other comparisons were significantly different. n = 3 (A, B, and E) and 4 (C and D) experiments.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Expressing, Quantitative RT-PCR

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: qPCR primers

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Sequencing

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: TRPM3 and TRP vanilloid-4 (TRPV4) cellular localization in 176-5Δ and 176-5 cells. A: immunofluorescence was performed using primary antibodies directed against acetylated α-tubulin (green) and TRPM3 (red), and images were overlaid (Merged). Images were obtained using a Zeiss LSM710 confocal microscope and analyzed using Imaris Scientific 3D/4D Image Analysis software. Top: in 176-5 cells, arrows indicate the presence of cilia (left), ciliary TRPM3 (middle), and colocalization (right). Scale bars = 5 µm. Middle: magnified view of the area within the white box in top row. Bottom: 176-5Δ cells were devoid of primary cilia (green, left), and TRPM3 expression was observed diffusely throughout the cells (middle and right). Scale bars = 10 µm. B: immunofluorescence was performed using primary antibodies directed against acetylated α-tubulin (green) and TRPV4 (red), and images were overlaid (Merged). Images were obtained and analyzed as described in A. Top: primary cilia were observed in 176-5 cells (green, left); cell body (possibly plasma membrane) expression of TRPV4 expression was apparent (red, middle), but no ciliary expression of TRPV4 was observed (right). Middle: magnified view of the area within the white box in top row. Bottom: 176-5Δ cells were devoid of primary cilia (green, left); cell body expression of TRPV4 resembled that of 176-5 cells (middle and right). Scale bars = 10 µm.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Immunofluorescence, Microscopy, Software, Expressing, Clinical Proteomics, Membrane

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: TRPM3 (A–C) and TRPV4 (D–F) cellular localization in mIMCD-3 cells. A and B: immunofluorescence was performed using primary antibodies directed against acetylated α-tubulin (A, green) and TRPM3 (B, red). Images were obtained using a Nikon A1 inverted confocal microscope and analyzed using Imaris Scientific 3D/4D Image Analysis software. C: overlaid image of A and B demonstrates TRPM3 coexpression with acetylated α-tubulin (yellow), indicating localization on primary cilia. D and E: immunofluorescence was performed using primary antibodies directed against acetylated α-tubulin (D, green) and TRPV4 (E, red). Images were obtained and analyzed as described in A and B. F: overlaid image of D and E demonstrating no coexpression of TRPV4 with acetylated α-tubulin.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Immunofluorescence, Microscopy, Software

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: TRPV4 protein expression in 176-5Δ and 176-5 cells. A: Western blot of TRPV4 expression in ciliated 176-5 and nonciliated 176-5Δ cells (lanes 3 and 4) and in Orpk cilia(−) cells with and without stable expression of a TRPV4-targeted shRNA construct (lanes 1 and 2) as an antibody control (54). A band for TRPV4 was observed just below 100 kDa, which is consistent with the predicted molecular weight (48). A higher-molecular-weight band was observed between 100 and 150 kDa, which is consistent with a glycosylated form of TRPV4 (49). Expression of both TRPV4 bands was eliminated in Orpk cilia(−) cells expressing the TRPV4-targeted construct. B: densitometric analysis of TRPV4 expression in 176-5 and 176-5Δ cells normalized to GAPDH levels. *P < 0.05. n = 5 experiments.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Expressing, Western Blot, shRNA, Construct, Control, Molecular Weight

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: TRPV4-dependent currents (I) in an excised inside-out patch from the apical membrane (A) and in an excised single primary cilium (B), both from mIMCD-3 cells. Recordings were obtained under each of 3 conditions: control (black trace), in the presence of the TRPV4 agonist (GSK1016790A, 100 nM; red trace), and in the presence of both the agonist (100 nM) and a TRPV4 antagonist (HC-067047, 1 µM; blue trace). Reagents were applied to the cytoplasmic face of the membrane.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Membrane, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Primary cilia regulate the osmotic stress response of renal epithelial cells through TRPM3

doi: 10.1152/ajprenal.00465.2015

Figure Lengend Snippet: Hyperosmolality-induced tonicity element-binding protein (TonEBP) reporter activity is affected by expression of cilia and stimulation of TRPV4 or TRPM3. A: ciliated (176-5) and nonciliated (176-5Δ) pSEAP2TonE/pcDNA3.1-transfected cells in 96-well plates were treated with a TRPV4 agonist [100 nM GSK1016790A (GSK)] or a TRPM3 agonist [100 μM pregnenolone sulfate (PSS)] at 300 mOsm/kg or 500 mOsm/kg [adjusted with NaCl (N)]. Vehicle control groups were treated with 0.1% DMSO. TonEBP activity (normalized to control) was significantly induced in 176-5 cells only. This significant induction was not observed in groups treated with the TRPV4 agonist and the TRPM3 agonist in these cells. A405, absorbance at 405 nm. B: mIMCD-3 cells transfected with pSEAP2TonE/pcDNA3.1 were treated with a TRPV4 agonist (25 nM GSK1016790A) or a TRPM3 agonist (100 μM pregnenolone sulfate) at 300 mOsm/kg or 500 mOsm/kg (adjusted with NaCl). Vehicle control groups were treated with 0.1% DMSO. Hyperosmolality significantly induced TonEBP reporter activity, which was not observed in groups treated with the TRPV4 agonist and the TRPM3 agonist. C and D: agonist-mediated attenuation of hyperosmolality-induced TonEBP reporter activity was competed away by cotreatment with the TRPV4 antagonist HC-067047 and by the TRPM3 antagonist isosakuranetin (Isosak). E: in mIMCD-3 cells, increasing osmolality to 500 mOsm/kg [adjusted with mannitol (M)] produced a statistically significant increase in TonEBP reporter activity that was not significant with concurrent treatment with the TRPM3 agonist. Treatment groups were compared with controls using Friedman’s test with Dunn’s correction for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001. ns, Not significant. n = 10 (A), 6 (B–D), and 9 (E) experiments.

Article Snippet: Cells harboring the reporter construct were then seeded into tissue culture-treated 96-well plates (Becton Dickinson, Franklin Lakes, NJ), and, after confluence for 1 wk with FBS concentration reduced to 5% to maximize ciliation, cells were exposed to hyperosmolality (500 mOsm/kg, achieved by supplementation of the culture medium with NaCl or mannitol to raise osmolality an additional 200 mOsm/kg) or maintained at basal osmolality (300 mOsm/kg) for 12 h. Cells were concurrently treated with a TRPV4 agonist (GSK1016790A), a TRPM3 agonist (pregnenolone sulfate sodium salt), or vehicle control (0.1% DMSO) under isosmolal and hyperosmolal conditions. mIMCD-3 cells were also treated concurrently for 12 h under hyperosmolal conditions with the TRPV4 antagonist HC-067047 and the TRPM3 antagonist isosakuranetin (Extrasynthese, Lyon, France), in combination with their respective agonists.

Techniques: Binding Assay, Activity Assay, Expressing, Transfection, Control, Produced