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Triarylmethanes, a new class of cx50 inhibitors.

Bodendiek SB, Rubinos C, Trelles MP, Coleman N, Jenkins DP, Wulff H, Srinivas M - Front Pharmacol (2012)

Bottom Line: We initially screened a library of common ion channel modulating pharmacophores for their inhibitory effects on Cx50 GJ channels, and identified four new classes of compounds.The SAR studies also indicated that the TRAM pharmacophore required for connexin inhibition is significantly different from the pharmacophore required for blocking the calcium-activated KCa3.1 channel.In addition, our results indicate that a similar approach may be used to find specific inhibitors of other connexin subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California Davis, CA, USA.

ABSTRACT
The paucity of specific pharmacological agents has been a major impediment for delineating the roles of gap junction (GJ) channels formed by connexin proteins in physiology and pathophysiology. Here, we used the selective optimization of side activities (SOSA) approach, which has led to the design of high affinity inhibitors of other ion channels, to identify a specific inhibitor for channels formed by Cx50, a connexin subtype that is primarily expressed in the lens. We initially screened a library of common ion channel modulating pharmacophores for their inhibitory effects on Cx50 GJ channels, and identified four new classes of compounds. The triarlymethane (TRAM) clotrimazole was the most potent Cx50 inhibitor and we therefore used it as a template to explore the structure activity relationship (SAR) of the TRAMs for Cx50 inhibition. We describe the design of T122 (N-[(2-methoxyphenyl)diphenylmethyl]-1,3-thiazol-2-amine) and T136 (N-[(2-iodophenyl)diphenylmethyl]-1,3-thiazol-2-amine), which inhibit Cx50 with IC(50)s of 1.2 and 2.4 μM. Both compounds exhibit at least 10-fold selectivity over other connexins as well as major neuronal and cardiac voltage-gated K(+) and Na(+) channels. The SAR studies also indicated that the TRAM pharmacophore required for connexin inhibition is significantly different from the pharmacophore required for blocking the calcium-activated KCa3.1 channel. Both T122 and T136 selectively inhibited Cx50 GJ channels in lens epithelial cells, suggesting that they could be used to further explore the role of Cx50 in the lens. In addition, our results indicate that a similar approach may be used to find specific inhibitors of other connexin subtypes.

No MeSH data available.


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Connexin-selectivity of T122 and T136. (A) Concentration dependence of T122 and T136 on Cx50 gap junction channels. Each point represents the mean ± SEM of gj (% of the initial conductance) values obtained from 4 to 10 cell pairs. The solid line is a fit of the data points to the Hill equation (see Materials and Methods). The EC50 and Hill slope values are indicated in the text. Each cell pair was exposed to only a single concentration. (B) Bar graph illustrating that T122 (10 μM) and T136 (10 μM) has no significant effect on Cx26, Cx32, Cx43, Cx45, and Cx46 gap junction channels. Each bar represents the mean ± SEM of four to six cell pairs.
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Figure 7: Connexin-selectivity of T122 and T136. (A) Concentration dependence of T122 and T136 on Cx50 gap junction channels. Each point represents the mean ± SEM of gj (% of the initial conductance) values obtained from 4 to 10 cell pairs. The solid line is a fit of the data points to the Hill equation (see Materials and Methods). The EC50 and Hill slope values are indicated in the text. Each cell pair was exposed to only a single concentration. (B) Bar graph illustrating that T122 (10 μM) and T136 (10 μM) has no significant effect on Cx26, Cx32, Cx43, Cx45, and Cx46 gap junction channels. Each bar represents the mean ± SEM of four to six cell pairs.

Mentions: The effects of T122 and T136 on Cx50 junctional channels were further characterized using a five-point dose response curve (Figure 7A). Non-linear least-squares fit of the individual data points to the Hill equation (see Materials and Methods) yielded IC50 values of 1.2 and 2.4 μM for the inhibition of Cx50 GJ channels by T122 and T136, respectively. In both cases, the Hill coefficients were ≈ 2 (1.6 for T122, 1.7 for T136), indicating that binding of two TRAM molecules was required to inhibit Cx50 GJ channels. Both compounds exhibited high selectivity for Cx50 channels over other connexin subtypes. The effects of T122 and T136 on GJ channels formed by several other connexins, including Cx26, Cx32, Cx40, Cx43, and Cx46 are illustrated in Figure 7B. At a concentration of 10 μM, sufficient to cause near-maximal decreases in Cx50 junctional current, T122 and T136 did not significantly inhibit Cx26, Cx32, Cx46, or Cx43 GJ channel currents. The reduction of junction conductance was less than 20% in each of these cases. These results demonstrate that inhibition of Cx50 GJ channels by T122 and T136 is highly connexin-selective.


Triarylmethanes, a new class of cx50 inhibitors.

Bodendiek SB, Rubinos C, Trelles MP, Coleman N, Jenkins DP, Wulff H, Srinivas M - Front Pharmacol (2012)

Connexin-selectivity of T122 and T136. (A) Concentration dependence of T122 and T136 on Cx50 gap junction channels. Each point represents the mean ± SEM of gj (% of the initial conductance) values obtained from 4 to 10 cell pairs. The solid line is a fit of the data points to the Hill equation (see Materials and Methods). The EC50 and Hill slope values are indicated in the text. Each cell pair was exposed to only a single concentration. (B) Bar graph illustrating that T122 (10 μM) and T136 (10 μM) has no significant effect on Cx26, Cx32, Cx43, Cx45, and Cx46 gap junction channels. Each bar represents the mean ± SEM of four to six cell pairs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368247&req=5

Figure 7: Connexin-selectivity of T122 and T136. (A) Concentration dependence of T122 and T136 on Cx50 gap junction channels. Each point represents the mean ± SEM of gj (% of the initial conductance) values obtained from 4 to 10 cell pairs. The solid line is a fit of the data points to the Hill equation (see Materials and Methods). The EC50 and Hill slope values are indicated in the text. Each cell pair was exposed to only a single concentration. (B) Bar graph illustrating that T122 (10 μM) and T136 (10 μM) has no significant effect on Cx26, Cx32, Cx43, Cx45, and Cx46 gap junction channels. Each bar represents the mean ± SEM of four to six cell pairs.
Mentions: The effects of T122 and T136 on Cx50 junctional channels were further characterized using a five-point dose response curve (Figure 7A). Non-linear least-squares fit of the individual data points to the Hill equation (see Materials and Methods) yielded IC50 values of 1.2 and 2.4 μM for the inhibition of Cx50 GJ channels by T122 and T136, respectively. In both cases, the Hill coefficients were ≈ 2 (1.6 for T122, 1.7 for T136), indicating that binding of two TRAM molecules was required to inhibit Cx50 GJ channels. Both compounds exhibited high selectivity for Cx50 channels over other connexin subtypes. The effects of T122 and T136 on GJ channels formed by several other connexins, including Cx26, Cx32, Cx40, Cx43, and Cx46 are illustrated in Figure 7B. At a concentration of 10 μM, sufficient to cause near-maximal decreases in Cx50 junctional current, T122 and T136 did not significantly inhibit Cx26, Cx32, Cx46, or Cx43 GJ channel currents. The reduction of junction conductance was less than 20% in each of these cases. These results demonstrate that inhibition of Cx50 GJ channels by T122 and T136 is highly connexin-selective.

Bottom Line: We initially screened a library of common ion channel modulating pharmacophores for their inhibitory effects on Cx50 GJ channels, and identified four new classes of compounds.The SAR studies also indicated that the TRAM pharmacophore required for connexin inhibition is significantly different from the pharmacophore required for blocking the calcium-activated KCa3.1 channel.In addition, our results indicate that a similar approach may be used to find specific inhibitors of other connexin subtypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of California Davis, CA, USA.

ABSTRACT
The paucity of specific pharmacological agents has been a major impediment for delineating the roles of gap junction (GJ) channels formed by connexin proteins in physiology and pathophysiology. Here, we used the selective optimization of side activities (SOSA) approach, which has led to the design of high affinity inhibitors of other ion channels, to identify a specific inhibitor for channels formed by Cx50, a connexin subtype that is primarily expressed in the lens. We initially screened a library of common ion channel modulating pharmacophores for their inhibitory effects on Cx50 GJ channels, and identified four new classes of compounds. The triarlymethane (TRAM) clotrimazole was the most potent Cx50 inhibitor and we therefore used it as a template to explore the structure activity relationship (SAR) of the TRAMs for Cx50 inhibition. We describe the design of T122 (N-[(2-methoxyphenyl)diphenylmethyl]-1,3-thiazol-2-amine) and T136 (N-[(2-iodophenyl)diphenylmethyl]-1,3-thiazol-2-amine), which inhibit Cx50 with IC(50)s of 1.2 and 2.4 μM. Both compounds exhibit at least 10-fold selectivity over other connexins as well as major neuronal and cardiac voltage-gated K(+) and Na(+) channels. The SAR studies also indicated that the TRAM pharmacophore required for connexin inhibition is significantly different from the pharmacophore required for blocking the calcium-activated KCa3.1 channel. Both T122 and T136 selectively inhibited Cx50 GJ channels in lens epithelial cells, suggesting that they could be used to further explore the role of Cx50 in the lens. In addition, our results indicate that a similar approach may be used to find specific inhibitors of other connexin subtypes.

No MeSH data available.


Related in: MedlinePlus