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Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Deering-Rice CE, Mitchell VK, Romero EG, Abdel Aziz MH, Ryskamp DA, Križaj D, Gopal VR, Reilly CA - Pharmacol Res Perspect (2014)

Bottom Line: The antispasmodic agent drofenine was identified as a new TRPV3 agonist.Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin.Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Utah, 30 S. 2000 E., Room 201 Skaggs Hall, Salt Lake City, UT 84112, USA.

ABSTRACT
Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology, as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 over-expressing HEK-293 cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

No MeSH data available.


Mechanism of TRPV3 activation by drofenine. (A) Chemical structures of drofenine and 2-APB. (B) Mutation of the 2-APB “binding site” residue H426 blocks drofenine induced calcium flux at low concentrations. HEK-293 cells were transiently transfected with TRPV3-WT, TRPV3-H426N, and TRPV3-R696K and calcium flux was measured via fluorescence microscopy using 100 μmol/L carvacrol, 100 μmol/L 2-APB, and 100 and 200 μmol/L drofenine. Data are blank subtracted and expressed as the percentage of maximum cellular fluorescence elicited by ionomycin (10 μmol/L) and normalized to TRPV3-WT transfected cells. Data are the mean and SEM (n ≥ 3) and asterisks indicate a statistical difference between TRPV3-WT and TRPV3-H426N or R696K using two-way ANOVA with Bonferroni multiple comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA, analysis of variance.
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fig04: Mechanism of TRPV3 activation by drofenine. (A) Chemical structures of drofenine and 2-APB. (B) Mutation of the 2-APB “binding site” residue H426 blocks drofenine induced calcium flux at low concentrations. HEK-293 cells were transiently transfected with TRPV3-WT, TRPV3-H426N, and TRPV3-R696K and calcium flux was measured via fluorescence microscopy using 100 μmol/L carvacrol, 100 μmol/L 2-APB, and 100 and 200 μmol/L drofenine. Data are blank subtracted and expressed as the percentage of maximum cellular fluorescence elicited by ionomycin (10 μmol/L) and normalized to TRPV3-WT transfected cells. Data are the mean and SEM (n ≥ 3) and asterisks indicate a statistical difference between TRPV3-WT and TRPV3-H426N or R696K using two-way ANOVA with Bonferroni multiple comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA, analysis of variance.

Mentions: The chemical structure of drofenine is similar to the known nonspecific TRPV3 agonist 2-APB (Fig. 4A). The contributions of the residues previously shown to determine 2-APB sensitivity were evaluated as determinants of drofenine sensitivity by comparing calcium flux elicited by carvacrol, 2-APB, and drofenine in HEK-293 cells transiently transfected with wild-type human TRPV3 (TRPV3-WT), TRPV3-H426N, or TRPV3-R696K. The TRPV3-H426N mutant exhibited reduced activation relative to TRPV3-WT using both drofenine and 2-APB at a concentration of 100 μmol/L (Fig. 4B). Mutation of H426 had no effect on calcium flux induced by carvacrol (100 μmol/L), as has been previously reported for the structurally similar monoterpenoid agonist camphor (Hu et al. 2009). At 200 μmol/L drofenine, the H426N mutation did not reduce TRPV3 activation, indicating only a shift in the binding of drofenine to TRPV3 or that drofenine may potentially interact with TRPV3 similar to, but not identically to 2-APB. The TRPV3-R696K mutant displayed diminished-to-no-function using all three agonists. Of significance, based on structural similarity and screening results, the 2-APB and drofenine analogs dicyclomine (Bentyl; anticholinergic, antispasmodic), was neither a TRPV3 agonist nor antagonist, while diphenhydramine was a nonselective TRPV3 inhibitor.


Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Deering-Rice CE, Mitchell VK, Romero EG, Abdel Aziz MH, Ryskamp DA, Križaj D, Gopal VR, Reilly CA - Pharmacol Res Perspect (2014)

Mechanism of TRPV3 activation by drofenine. (A) Chemical structures of drofenine and 2-APB. (B) Mutation of the 2-APB “binding site” residue H426 blocks drofenine induced calcium flux at low concentrations. HEK-293 cells were transiently transfected with TRPV3-WT, TRPV3-H426N, and TRPV3-R696K and calcium flux was measured via fluorescence microscopy using 100 μmol/L carvacrol, 100 μmol/L 2-APB, and 100 and 200 μmol/L drofenine. Data are blank subtracted and expressed as the percentage of maximum cellular fluorescence elicited by ionomycin (10 μmol/L) and normalized to TRPV3-WT transfected cells. Data are the mean and SEM (n ≥ 3) and asterisks indicate a statistical difference between TRPV3-WT and TRPV3-H426N or R696K using two-way ANOVA with Bonferroni multiple comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA, analysis of variance.
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fig04: Mechanism of TRPV3 activation by drofenine. (A) Chemical structures of drofenine and 2-APB. (B) Mutation of the 2-APB “binding site” residue H426 blocks drofenine induced calcium flux at low concentrations. HEK-293 cells were transiently transfected with TRPV3-WT, TRPV3-H426N, and TRPV3-R696K and calcium flux was measured via fluorescence microscopy using 100 μmol/L carvacrol, 100 μmol/L 2-APB, and 100 and 200 μmol/L drofenine. Data are blank subtracted and expressed as the percentage of maximum cellular fluorescence elicited by ionomycin (10 μmol/L) and normalized to TRPV3-WT transfected cells. Data are the mean and SEM (n ≥ 3) and asterisks indicate a statistical difference between TRPV3-WT and TRPV3-H426N or R696K using two-way ANOVA with Bonferroni multiple comparison posttest. *P < 0.05, **P < 0.01, ***P < 0.001. ANOVA, analysis of variance.
Mentions: The chemical structure of drofenine is similar to the known nonspecific TRPV3 agonist 2-APB (Fig. 4A). The contributions of the residues previously shown to determine 2-APB sensitivity were evaluated as determinants of drofenine sensitivity by comparing calcium flux elicited by carvacrol, 2-APB, and drofenine in HEK-293 cells transiently transfected with wild-type human TRPV3 (TRPV3-WT), TRPV3-H426N, or TRPV3-R696K. The TRPV3-H426N mutant exhibited reduced activation relative to TRPV3-WT using both drofenine and 2-APB at a concentration of 100 μmol/L (Fig. 4B). Mutation of H426 had no effect on calcium flux induced by carvacrol (100 μmol/L), as has been previously reported for the structurally similar monoterpenoid agonist camphor (Hu et al. 2009). At 200 μmol/L drofenine, the H426N mutation did not reduce TRPV3 activation, indicating only a shift in the binding of drofenine to TRPV3 or that drofenine may potentially interact with TRPV3 similar to, but not identically to 2-APB. The TRPV3-R696K mutant displayed diminished-to-no-function using all three agonists. Of significance, based on structural similarity and screening results, the 2-APB and drofenine analogs dicyclomine (Bentyl; anticholinergic, antispasmodic), was neither a TRPV3 agonist nor antagonist, while diphenhydramine was a nonselective TRPV3 inhibitor.

Bottom Line: The antispasmodic agent drofenine was identified as a new TRPV3 agonist.Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin.Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Utah, 30 S. 2000 E., Room 201 Skaggs Hall, Salt Lake City, UT 84112, USA.

ABSTRACT
Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology, as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 over-expressing HEK-293 cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

No MeSH data available.