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A tale of switched functions: from cyclooxygenase inhibition to M-channel modulation in new diphenylamine derivatives.

Peretz A, Degani-Katzav N, Talmon M, Danieli E, Gopin A, Malka E, Nachman R, Raz A, Shabat D, Attali B - PLoS ONE (2007)

Bottom Line: They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents.Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition.They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.

ABSTRACT
Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+) channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+) channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

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Inhibition of COX activity by compounds 7 and 15.The inhibition of COX enzyme activity was measured by the production of PGE2 in cultured mouse colon adenocarcinoma cells (CT26). While the COX activity is inhibited by the ester compound 15 (IC50 = 69 nM), it is completely unaffected by the amide compound 7. This typical experiment gave similar results in two additional trials. Shown are the chemical structures of compounds 7 and 15.
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pone-0001332-g001: Inhibition of COX activity by compounds 7 and 15.The inhibition of COX enzyme activity was measured by the production of PGE2 in cultured mouse colon adenocarcinoma cells (CT26). While the COX activity is inhibited by the ester compound 15 (IC50 = 69 nM), it is completely unaffected by the amide compound 7. This typical experiment gave similar results in two additional trials. Shown are the chemical structures of compounds 7 and 15.

Mentions: Considering the COX activity, it is striking to note that all the amide derivatives of diclofenac (compounds 1, 2, 4, 6 and 7) and meclofenamic acid (compounds 16–18) produced very weak or no inhibition of the COX enzymes in CT26 and D122 cells (IC50 >50 µM; Table 1 and Table S1). This virtual lack of inhibition of the COX activity was displayed by amides derivatives bearing ether as well as alkyl chains (e.g., compounds 2 and 7). While the issue of the COX selectivity was not directly addressed in this study, we tentatively assume that the lack of COX inhibition displayed by the various amide compounds involves both COX-1 and COX-2, since the D122 cell line is endowed with both COX-1 and COX-2 activities and the CT26 line expresses mainly though not exclusively COX-2 [16], [17]. These features contrast with the well known anti-COX-1/COX-2 properties of the parental templates [15]. In contrast, all ester compounds tested, produced a potent inhibition of the COX enzymes (IC50s∼1 to 230 nM) (Table 1 and Table S1). The prominent difference in COX inhibition between amide and ester derivatives is well illustrated with compounds 2 and 8 or with compounds 7 and 15 (Table 1, Table S1 and Figure 1). Comparison of compounds 8 and 13 suggests that the terminal hydroxyl functionality of the ester derivatives is not important for the anti-COX activity. Compound 13 is even more powerful than the parent diclofenac template. Similarly, a potent and non-selective inhibition of purified COX-1 and COX-2 enzymes was obtained with compounds 8 and 13 and diclofenac. Thus, at 1 µM concentration compounds 8, 13 and diclofenac respectively produced 58±1%, 59±1% and 64±2% inhibition of the purified ovine COX-1 enzyme (n = 3) and 85±1%, 83±1% and 72±2% inhibition of the human recombinant COX-2 enzyme (n = 3).


A tale of switched functions: from cyclooxygenase inhibition to M-channel modulation in new diphenylamine derivatives.

Peretz A, Degani-Katzav N, Talmon M, Danieli E, Gopin A, Malka E, Nachman R, Raz A, Shabat D, Attali B - PLoS ONE (2007)

Inhibition of COX activity by compounds 7 and 15.The inhibition of COX enzyme activity was measured by the production of PGE2 in cultured mouse colon adenocarcinoma cells (CT26). While the COX activity is inhibited by the ester compound 15 (IC50 = 69 nM), it is completely unaffected by the amide compound 7. This typical experiment gave similar results in two additional trials. Shown are the chemical structures of compounds 7 and 15.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2131780&req=5

pone-0001332-g001: Inhibition of COX activity by compounds 7 and 15.The inhibition of COX enzyme activity was measured by the production of PGE2 in cultured mouse colon adenocarcinoma cells (CT26). While the COX activity is inhibited by the ester compound 15 (IC50 = 69 nM), it is completely unaffected by the amide compound 7. This typical experiment gave similar results in two additional trials. Shown are the chemical structures of compounds 7 and 15.
Mentions: Considering the COX activity, it is striking to note that all the amide derivatives of diclofenac (compounds 1, 2, 4, 6 and 7) and meclofenamic acid (compounds 16–18) produced very weak or no inhibition of the COX enzymes in CT26 and D122 cells (IC50 >50 µM; Table 1 and Table S1). This virtual lack of inhibition of the COX activity was displayed by amides derivatives bearing ether as well as alkyl chains (e.g., compounds 2 and 7). While the issue of the COX selectivity was not directly addressed in this study, we tentatively assume that the lack of COX inhibition displayed by the various amide compounds involves both COX-1 and COX-2, since the D122 cell line is endowed with both COX-1 and COX-2 activities and the CT26 line expresses mainly though not exclusively COX-2 [16], [17]. These features contrast with the well known anti-COX-1/COX-2 properties of the parental templates [15]. In contrast, all ester compounds tested, produced a potent inhibition of the COX enzymes (IC50s∼1 to 230 nM) (Table 1 and Table S1). The prominent difference in COX inhibition between amide and ester derivatives is well illustrated with compounds 2 and 8 or with compounds 7 and 15 (Table 1, Table S1 and Figure 1). Comparison of compounds 8 and 13 suggests that the terminal hydroxyl functionality of the ester derivatives is not important for the anti-COX activity. Compound 13 is even more powerful than the parent diclofenac template. Similarly, a potent and non-selective inhibition of purified COX-1 and COX-2 enzymes was obtained with compounds 8 and 13 and diclofenac. Thus, at 1 µM concentration compounds 8, 13 and diclofenac respectively produced 58±1%, 59±1% and 64±2% inhibition of the purified ovine COX-1 enzyme (n = 3) and 85±1%, 83±1% and 72±2% inhibition of the human recombinant COX-2 enzyme (n = 3).

Bottom Line: They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents.Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition.They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.

ABSTRACT
Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+) channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+) channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

Show MeSH
Related in: MedlinePlus