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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

Table showing the structures and IC50 values for Cx50 and KCa3.1 inhibition of heterocyclic substituted triarylmethanes. Concentrations of triarylmethanes that caused a half-maximal inhibition (IC50) values were obtained by fitting the data to the Hill equation, as described in the methods. Means of current inhibition and SD were determined by application of two or three concentrations of each triarylmethane to multiple cells (n ranging from 3 to 8 per concentration). The SD values are not shown for clarity; SD values typically ranged between 5 and 15%.
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Figure 4: Table showing the structures and IC50 values for Cx50 and KCa3.1 inhibition of heterocyclic substituted triarylmethanes. Concentrations of triarylmethanes that caused a half-maximal inhibition (IC50) values were obtained by fitting the data to the Hill equation, as described in the methods. Means of current inhibition and SD were determined by application of two or three concentrations of each triarylmethane to multiple cells (n ranging from 3 to 8 per concentration). The SD values are not shown for clarity; SD values typically ranged between 5 and 15%.

Mentions: Using clotrimazole as a template we explored the SAR of the triphenylmethane scaffold according to the synthetic strategy shown in Figure 3. In a Grignard reaction mono-substituted benzophenones and bromobenzenes were reacted in anhydrous diethyl ether to yield the corresponding triphenylmethanols. These alcohols were then either ammonolyzed, cyanated with copper cyanide or chlorinated using acetyl chloride. The triphenylmethane chlorides were further reacted in a nucleophilic substitution to give the respective triphenylmethane derivatives (further details on exact conditions and quantities are given in the see Materials and Methods). We first substituted the imidazole ring of clotrimazole with several other heterocycles, differently substituted carbocycles or aliphatic functional groups while keeping the 2-chlorophenyldiphenyl methane basic structure (Figure 3). Except for T44 (pyrrol), T69 (2-aminopyridine), T89 (4-methyl-2-phenylimidazole) and the bicyclic T71 (phthalimide) and T103 (2-aminobenzothiazole), most of the heterocyclic derivatives blocked Cx50 in the low micromolar range. Spacer linked carbocycles (T102, T104, T106, T107, T109, T150) in contrast showed no effect on Cx50 at concentrations of 10 μM with the exception of T106, which was found to be a weak blocker with an IC50 10 μM (Figure 4). We next tested the heterocyclic substituted triarylmethanes for selectivity over KCa3.1 (Figure 4). As previously reported (Wulff et al., 2000), clotrimazole and T34 (= TRAM-34) are nanomolar KCa3.1 blockers, that exhibit IC50 values of 70 and 20 nM, respectively, and are therefore not useful as Cx50 inhibitors. In contrast, the aminothiazole and aminopyrimidine substituted T66 and T68 were found to be 15- to 200-fold less potent on KCa3.1 and T66 even exhibited a moderate three-fold selectivity for Cx50 over KCa3.1.


Triarylmethanes, a new class of cx50 inhibitors.

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

Table showing the structures and IC50 values for Cx50 and KCa3.1 inhibition of heterocyclic substituted triarylmethanes. Concentrations of triarylmethanes that caused a half-maximal inhibition (IC50) values were obtained by fitting the data to the Hill equation, as described in the methods. Means of current inhibition and SD were determined by application of two or three concentrations of each triarylmethane to multiple cells (n ranging from 3 to 8 per concentration). The SD values are not shown for clarity; SD values typically ranged between 5 and 15%.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Table showing the structures and IC50 values for Cx50 and KCa3.1 inhibition of heterocyclic substituted triarylmethanes. Concentrations of triarylmethanes that caused a half-maximal inhibition (IC50) values were obtained by fitting the data to the Hill equation, as described in the methods. Means of current inhibition and SD were determined by application of two or three concentrations of each triarylmethane to multiple cells (n ranging from 3 to 8 per concentration). The SD values are not shown for clarity; SD values typically ranged between 5 and 15%.
Mentions: Using clotrimazole as a template we explored the SAR of the triphenylmethane scaffold according to the synthetic strategy shown in Figure 3. In a Grignard reaction mono-substituted benzophenones and bromobenzenes were reacted in anhydrous diethyl ether to yield the corresponding triphenylmethanols. These alcohols were then either ammonolyzed, cyanated with copper cyanide or chlorinated using acetyl chloride. The triphenylmethane chlorides were further reacted in a nucleophilic substitution to give the respective triphenylmethane derivatives (further details on exact conditions and quantities are given in the see Materials and Methods). We first substituted the imidazole ring of clotrimazole with several other heterocycles, differently substituted carbocycles or aliphatic functional groups while keeping the 2-chlorophenyldiphenyl methane basic structure (Figure 3). Except for T44 (pyrrol), T69 (2-aminopyridine), T89 (4-methyl-2-phenylimidazole) and the bicyclic T71 (phthalimide) and T103 (2-aminobenzothiazole), most of the heterocyclic derivatives blocked Cx50 in the low micromolar range. Spacer linked carbocycles (T102, T104, T106, T107, T109, T150) in contrast showed no effect on Cx50 at concentrations of 10 μM with the exception of T106, which was found to be a weak blocker with an IC50 10 μM (Figure 4). We next tested the heterocyclic substituted triarylmethanes for selectivity over KCa3.1 (Figure 4). As previously reported (Wulff et al., 2000), clotrimazole and T34 (= TRAM-34) are nanomolar KCa3.1 blockers, that exhibit IC50 values of 70 and 20 nM, respectively, and are therefore not useful as Cx50 inhibitors. In contrast, the aminothiazole and aminopyrimidine substituted T66 and T68 were found to be 15- to 200-fold less potent on KCa3.1 and T66 even exhibited a moderate three-fold selectivity for Cx50 over KCa3.1.

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