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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 inhibition for aminothiazole and aminopyrimdine substituted triarylmethanes with various functional groups in ortho-position on one of the phenyl rings. 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 6: Table showing the structures and IC50 values for Cx50 inhibition for aminothiazole and aminopyrimdine substituted triarylmethanes with various functional groups in ortho-position on one of the phenyl rings. 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: Since the alcohol T3, which is the first-step intermediate for the heterocyclic substituted triarylmethanes, was also found to reduce Cx50 currents with an IC50 of 2 μM we further synthesized and tested several triarylmethane alcohols, amines, nitriles, and ureas on Cx50 (Table A1 in Appendix). While several of the alcohols including the p-chloro substituted T1, the m-chlorosubstituted T2 as well as the non-substituted triphenylmethanol and triphenylamine (T162) exhibited IC50 values for Cx50 in the 1–2 μM range (Table A1 in Appendix), all these compounds lacked selectivity over KCa3.1 and were further found to inhibit other connexins like Cx43, Cx46 at similar concentrations as Cx50 (data not shown). In addition, the inhibition produced by these compounds was often enhanced by a prior application of the compounds. We therefore did not study these compounds further and instead concentrated our synthetic efforts on the heterocyclic substituted triarylmethanes and explored the substitution position of the chlorine atom on one of the phenyl rings by moving it from the ortho- to the meta- or para-position or completely removing it (Figure 5). All four imidazole ring containing compounds (T97, clotrimazole, T143, T144) but only the o-chloro and m-chloro substituted pyrazole derivatives (T34 and T142) inhibited Cx50 channels with IC50s of 5–8 μM. In the 2-aminothiazole and 2-aminopyrimidine series only the o-chloro substituted T66 and T68 were active, while the other regio-isomers or the unsubstituted analogs showed no effect at 10 μM. Because the imidazole and pyrazole-substituted triarylmethanes were poorly selective for Cx50 over KCa3.1, we studied the effect of modifications of the o-chloro substituent in the 2-aminothiazole and 2-aminopyrimdine series. Specifically, we replaced the o-chloro substituent with other halogens (F, Br, I), the more lipophilic CF3 or OCF3 groups or electron-donating methyl or methoxy groups. All 14 compounds exhibited IC50 values in the low micromolar range (Figure 6) with the methoxy-substituted T122 (IC50 1.2 μM) and the iodo-substituted T136 (IC50 2.4 μM) being the most potent (Figure 6).


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 inhibition for aminothiazole and aminopyrimdine substituted triarylmethanes with various functional groups in ortho-position on one of the phenyl rings. 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 6: Table showing the structures and IC50 values for Cx50 inhibition for aminothiazole and aminopyrimdine substituted triarylmethanes with various functional groups in ortho-position on one of the phenyl rings. 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: Since the alcohol T3, which is the first-step intermediate for the heterocyclic substituted triarylmethanes, was also found to reduce Cx50 currents with an IC50 of 2 μM we further synthesized and tested several triarylmethane alcohols, amines, nitriles, and ureas on Cx50 (Table A1 in Appendix). While several of the alcohols including the p-chloro substituted T1, the m-chlorosubstituted T2 as well as the non-substituted triphenylmethanol and triphenylamine (T162) exhibited IC50 values for Cx50 in the 1–2 μM range (Table A1 in Appendix), all these compounds lacked selectivity over KCa3.1 and were further found to inhibit other connexins like Cx43, Cx46 at similar concentrations as Cx50 (data not shown). In addition, the inhibition produced by these compounds was often enhanced by a prior application of the compounds. We therefore did not study these compounds further and instead concentrated our synthetic efforts on the heterocyclic substituted triarylmethanes and explored the substitution position of the chlorine atom on one of the phenyl rings by moving it from the ortho- to the meta- or para-position or completely removing it (Figure 5). All four imidazole ring containing compounds (T97, clotrimazole, T143, T144) but only the o-chloro and m-chloro substituted pyrazole derivatives (T34 and T142) inhibited Cx50 channels with IC50s of 5–8 μM. In the 2-aminothiazole and 2-aminopyrimidine series only the o-chloro substituted T66 and T68 were active, while the other regio-isomers or the unsubstituted analogs showed no effect at 10 μM. Because the imidazole and pyrazole-substituted triarylmethanes were poorly selective for Cx50 over KCa3.1, we studied the effect of modifications of the o-chloro substituent in the 2-aminothiazole and 2-aminopyrimdine series. Specifically, we replaced the o-chloro substituent with other halogens (F, Br, I), the more lipophilic CF3 or OCF3 groups or electron-donating methyl or methoxy groups. All 14 compounds exhibited IC50 values in the low micromolar range (Figure 6) with the methoxy-substituted T122 (IC50 1.2 μM) and the iodo-substituted T136 (IC50 2.4 μM) being the most potent (Figure 6).

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