Limits...
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.

Show MeSH

Related in: MedlinePlus

Chemical structures of NSAIDs lacking the diphenylamine moiety but still bearing a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen and naproxen.Chemical structures of N-phenylanthranilic acid drugs containing the diphenylamine moiety like mefenamate, tolfenamate and flufenamate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2131780&req=5

pone-0001332-g003: Chemical structures of NSAIDs lacking the diphenylamine moiety but still bearing a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen and naproxen.Chemical structures of N-phenylanthranilic acid drugs containing the diphenylamine moiety like mefenamate, tolfenamate and flufenamate.

Mentions: The diphenylamine moiety itself appears to be important for activating the M-channels. We found that NSAID compounds lacking this group but still holding a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen or naproxen, do not activate Kv7.2/3 channels, while those bearing both functionalities (diphenylamine and carboxylate) such as N-phenylanthranilic acid drugs (mefenamate, tolfenamate, or flufenamate) are all M-channel openers (Figure 3). Noteworthy, our data indicate that a terminal hydroxyl group in the ether or alkyl chain is absolutely required to obtain an active M-channel opener. For example, the compounds 10, 12, 19 and 20 whose terminal hydroxyl function has been replaced by methyl or isobutyl groups are totally inactive vis-à-vis Kv7.2/3 channels (Table 1, Table S1 and Figure 2). Remarkably, in the diclofenac series we obtained potent M-channel blockers with compounds 11, 13 and 14 where the terminal hydroxyl has been replaced by isobutyl, ethylamine and 2,3-epoxypropyl groups, respectively (Table 1 and Table S1). When tested on recombinant Kv7.2/3 channels that were heterologously expressed in CHO cells, compound 13 potently and dose-dependently produced a blockade of the K+ currents (IC50 = 11 µM). At 25 µM compound 13 produced more than 90 % inhibition of the Kv7.2/3 current at a wide range of membrane potentials (from −60 to +50 mV; Figure 4A and B). Recent work suggested that M-currents play a key role in controlling the excitability of sensory dorsal root ganglion (DRG) neurons and may therefore represent a therapeutic target for the treatment of pain [18]. Hence, measuring the effects of novel M-channel modulators on DRG neuronal excitability is of important value, considering their potential impact on nociceptive signaling pathways. We examined in the current-clamp configuration the effects of compound 13 on spike activity of cultured rat DRG neurons (Figure 4C). A single spike discharge pattern was evoked by injecting a minimal depolarizing current pulse (∼10 pA, 400 ms). Within 2 min, external application of 1 µM compound 13 depolarized the DRG membrane potential (ΔV = +7±1 mV) and potently increased the number of evoked spikes (∼10–20 pA for 400 ms, from 1±0 to 12±2; n = 5, p<0.01) (Figure 4C). The hyperexcitability profile of compound 13 (1 µM) on spike generation was reflected by a decrease in rheobase current of about 300 pA that is needed to generate a single spike (for a 2 ms injection, from 650±71 pA to 347±44 pA, n = 6; p<0.01). The hyperexcitability discharge pattern induced by this M-channel blocker was so strong that in some cases, it could lead the DRG neurons to fire spontaneously, with no need of injecting depolarizing current (Figure 4D).


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)

Chemical structures of NSAIDs lacking the diphenylamine moiety but still bearing a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen and naproxen.Chemical structures of N-phenylanthranilic acid drugs containing the diphenylamine moiety like mefenamate, tolfenamate and flufenamate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001332-g003: Chemical structures of NSAIDs lacking the diphenylamine moiety but still bearing a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen and naproxen.Chemical structures of N-phenylanthranilic acid drugs containing the diphenylamine moiety like mefenamate, tolfenamate and flufenamate.
Mentions: The diphenylamine moiety itself appears to be important for activating the M-channels. We found that NSAID compounds lacking this group but still holding a carboxylate function like ibuprofen, flurbiprofen, ketoprofen, fenoprofen or naproxen, do not activate Kv7.2/3 channels, while those bearing both functionalities (diphenylamine and carboxylate) such as N-phenylanthranilic acid drugs (mefenamate, tolfenamate, or flufenamate) are all M-channel openers (Figure 3). Noteworthy, our data indicate that a terminal hydroxyl group in the ether or alkyl chain is absolutely required to obtain an active M-channel opener. For example, the compounds 10, 12, 19 and 20 whose terminal hydroxyl function has been replaced by methyl or isobutyl groups are totally inactive vis-à-vis Kv7.2/3 channels (Table 1, Table S1 and Figure 2). Remarkably, in the diclofenac series we obtained potent M-channel blockers with compounds 11, 13 and 14 where the terminal hydroxyl has been replaced by isobutyl, ethylamine and 2,3-epoxypropyl groups, respectively (Table 1 and Table S1). When tested on recombinant Kv7.2/3 channels that were heterologously expressed in CHO cells, compound 13 potently and dose-dependently produced a blockade of the K+ currents (IC50 = 11 µM). At 25 µM compound 13 produced more than 90 % inhibition of the Kv7.2/3 current at a wide range of membrane potentials (from −60 to +50 mV; Figure 4A and B). Recent work suggested that M-currents play a key role in controlling the excitability of sensory dorsal root ganglion (DRG) neurons and may therefore represent a therapeutic target for the treatment of pain [18]. Hence, measuring the effects of novel M-channel modulators on DRG neuronal excitability is of important value, considering their potential impact on nociceptive signaling pathways. We examined in the current-clamp configuration the effects of compound 13 on spike activity of cultured rat DRG neurons (Figure 4C). A single spike discharge pattern was evoked by injecting a minimal depolarizing current pulse (∼10 pA, 400 ms). Within 2 min, external application of 1 µM compound 13 depolarized the DRG membrane potential (ΔV = +7±1 mV) and potently increased the number of evoked spikes (∼10–20 pA for 400 ms, from 1±0 to 12±2; n = 5, p<0.01) (Figure 4C). The hyperexcitability profile of compound 13 (1 µM) on spike generation was reflected by a decrease in rheobase current of about 300 pA that is needed to generate a single spike (for a 2 ms injection, from 650±71 pA to 347±44 pA, n = 6; p<0.01). The hyperexcitability discharge pattern induced by this M-channel blocker was so strong that in some cases, it could lead the DRG neurons to fire spontaneously, with no need of injecting depolarizing current (Figure 4D).

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