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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.


Related in: MedlinePlus

Time course of the effect of PAA-10, astemizole, clotrimazole, and rutin on the junctional currents of Cx50 channels expressed in N2A cell pairs measured by dual whole-cell patch-clamp.
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Figure 1: Time course of the effect of PAA-10, astemizole, clotrimazole, and rutin on the junctional currents of Cx50 channels expressed in N2A cell pairs measured by dual whole-cell patch-clamp.

Mentions: To identify Cx50 inhibitors we first screened a small library of compounds containing known ion channel pharmacophores including the antihistamine astemizole, several psoralens and related heterocycles, benzothiazoles, triterpenes, and flavanoid glycosides as well as the antifungal agent clotrimazole. From this library we identified four novel low micromolar inhibitors of Cx50: Astemizole, rutin (a flavonoid glycoside), PAA-10 (an alkyl substituted dibenzazocinone), and clotrimazole (see Figure 1 for structures). All compounds produced significant inhibition of the Cx50 junctional current at a concentration of 10 μM (Figure 1). The inhibition of junctional currents caused by clotrimazole, astemizole and PAA-10 was completely reversible upon washout. In contrast, the effects of rutin were only partially reversible. Of these four hits, the triarylmethane clotrimazole seemed the most drug-like and attractive compound to us. Astemizole is known to affect many other ion channels including the cardiac K+ channel HERG (Kv11.1; Suessbrich et al., 1996), a liability not generally encountered with triarylmethanes (Toyama et al., 2008). We also discarded rutin as a template since preliminary experiments showed that the rutin aglycon, quercetin, had no effect on Cx50 at 10 μM (data not shown) demonstrating that the sugar moiety is essential for connexin inhibition. The dibenzazocinone PAA-10 would of course also have been a possible lead but we preferred to perform structure activity relationship (SAR) studies around clotrimazole since our laboratory had a library of 80 triarylmethanes that were immediately available for an SAR analysis on Cx50. These compounds had been previously synthesized for an SAR study to determine the structural requirements for inhibition of the intermediate-conductance calcium-activated potassium channel KCa3.1 (a.k.a. IKCa1, SK4). By using the so-called selective optimization of side activities (SOSA) approach, which allows for the selective optimization of the side activity of an old drug (Wermuth, 2004), our group successfully designed a triarylmethane, TRAM-34 (T34), that selectively blocked KCa3.1 channels without affecting cytochrome P450-dependent enzymes, the main target of clotrimazole (Wulff et al., 2000). This previous work had demonstrated that it is possible to achieve selectivity for different targets by appropriately modifying the triarylmethane (TRAM) pharmacophore, which was another reason for us to choose clotrimazole as a template for our current study on Cx50.


Triarylmethanes, a new class of cx50 inhibitors.

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

Time course of the effect of PAA-10, astemizole, clotrimazole, and rutin on the junctional currents of Cx50 channels expressed in N2A cell pairs measured by dual whole-cell patch-clamp.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368247&req=5

Figure 1: Time course of the effect of PAA-10, astemizole, clotrimazole, and rutin on the junctional currents of Cx50 channels expressed in N2A cell pairs measured by dual whole-cell patch-clamp.
Mentions: To identify Cx50 inhibitors we first screened a small library of compounds containing known ion channel pharmacophores including the antihistamine astemizole, several psoralens and related heterocycles, benzothiazoles, triterpenes, and flavanoid glycosides as well as the antifungal agent clotrimazole. From this library we identified four novel low micromolar inhibitors of Cx50: Astemizole, rutin (a flavonoid glycoside), PAA-10 (an alkyl substituted dibenzazocinone), and clotrimazole (see Figure 1 for structures). All compounds produced significant inhibition of the Cx50 junctional current at a concentration of 10 μM (Figure 1). The inhibition of junctional currents caused by clotrimazole, astemizole and PAA-10 was completely reversible upon washout. In contrast, the effects of rutin were only partially reversible. Of these four hits, the triarylmethane clotrimazole seemed the most drug-like and attractive compound to us. Astemizole is known to affect many other ion channels including the cardiac K+ channel HERG (Kv11.1; Suessbrich et al., 1996), a liability not generally encountered with triarylmethanes (Toyama et al., 2008). We also discarded rutin as a template since preliminary experiments showed that the rutin aglycon, quercetin, had no effect on Cx50 at 10 μM (data not shown) demonstrating that the sugar moiety is essential for connexin inhibition. The dibenzazocinone PAA-10 would of course also have been a possible lead but we preferred to perform structure activity relationship (SAR) studies around clotrimazole since our laboratory had a library of 80 triarylmethanes that were immediately available for an SAR analysis on Cx50. These compounds had been previously synthesized for an SAR study to determine the structural requirements for inhibition of the intermediate-conductance calcium-activated potassium channel KCa3.1 (a.k.a. IKCa1, SK4). By using the so-called selective optimization of side activities (SOSA) approach, which allows for the selective optimization of the side activity of an old drug (Wermuth, 2004), our group successfully designed a triarylmethane, TRAM-34 (T34), that selectively blocked KCa3.1 channels without affecting cytochrome P450-dependent enzymes, the main target of clotrimazole (Wulff et al., 2000). This previous work had demonstrated that it is possible to achieve selectivity for different targets by appropriately modifying the triarylmethane (TRAM) pharmacophore, which was another reason for us to choose clotrimazole as a template for our current study on Cx50.

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