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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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Compound 15 inhibits firing of hippocampal and peripheral DRG neurons.(A) Representative rat hippocampal spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 15 and after washout (wash). (B) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 15 (n = 13; * p<0.01). (C) Representative solitary spike evoked in DRG neurons by 2 ms squared depolarizing current pulses (100–1100 pA in 100 pA increments) in the absence (control) or presence of 25 µM compound 15. (D) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in hippocampal and DRG neurons in the absence and presence of 25 µM compound 15 (n = 8; * p<0.01). (E) Rheobase current necessary to inject (2 ms) into DRG neurons to evoke a solitary spike in the absence and presence of 25 µM compound 15 (n = 12; * p<0.01).
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pone-0001332-g007: Compound 15 inhibits firing of hippocampal and peripheral DRG neurons.(A) Representative rat hippocampal spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 15 and after washout (wash). (B) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 15 (n = 13; * p<0.01). (C) Representative solitary spike evoked in DRG neurons by 2 ms squared depolarizing current pulses (100–1100 pA in 100 pA increments) in the absence (control) or presence of 25 µM compound 15. (D) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in hippocampal and DRG neurons in the absence and presence of 25 µM compound 15 (n = 8; * p<0.01). (E) Rheobase current necessary to inject (2 ms) into DRG neurons to evoke a solitary spike in the absence and presence of 25 µM compound 15 (n = 12; * p<0.01).

Mentions: We further characterized the properties of compound 15 on central and peripheral neurons, using the current-clamp mode of the whole-cell patch-clamp technique in rat primary cultures of hippocampal and DRG neurons (Figure 7). Repetitive spike discharges were evoked by depolarizing current injections. External application of compound 15 (25 µM) robustly and reversibly depressed the number of evoked action potentials in both hippocampal and DRG neurons (from 9.6±1.0 to 2.1±0.5, n = 8 and from 10.4±0.8 to 0.8±0.3, n = 8 respectively; 75–200 pA for 400 ms; p<0.01) (Figure 7A and D). Exposure to compound 15 (25 µM) hyperpolarized the resting membrane potential of DRG neurons by −8.3±0.2 mV (from −60.6±0.3 mV to −69.0±0.3 mV, n = 13; p<0.01; Figure 7B). A similar hyperpolarization was also obtained in hippocampal neurons exposed to compound 15 (−8.5 mV). Reflecting the dampening action of compound 15 on spike generation, the rheobase current needed to be injected into DRG neurons to generate a single spike, was increased upon exposure to the drug (for a 2 ms injection, from 400±41 pA to 577±38 pA, n = 12; p<0.01) (Figure 7C and E).


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)

Compound 15 inhibits firing of hippocampal and peripheral DRG neurons.(A) Representative rat hippocampal spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 15 and after washout (wash). (B) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 15 (n = 13; * p<0.01). (C) Representative solitary spike evoked in DRG neurons by 2 ms squared depolarizing current pulses (100–1100 pA in 100 pA increments) in the absence (control) or presence of 25 µM compound 15. (D) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in hippocampal and DRG neurons in the absence and presence of 25 µM compound 15 (n = 8; * p<0.01). (E) Rheobase current necessary to inject (2 ms) into DRG neurons to evoke a solitary spike in the absence and presence of 25 µM compound 15 (n = 12; * p<0.01).
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Related In: Results  -  Collection

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pone-0001332-g007: Compound 15 inhibits firing of hippocampal and peripheral DRG neurons.(A) Representative rat hippocampal spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 15 and after washout (wash). (B) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 15 (n = 13; * p<0.01). (C) Representative solitary spike evoked in DRG neurons by 2 ms squared depolarizing current pulses (100–1100 pA in 100 pA increments) in the absence (control) or presence of 25 µM compound 15. (D) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in hippocampal and DRG neurons in the absence and presence of 25 µM compound 15 (n = 8; * p<0.01). (E) Rheobase current necessary to inject (2 ms) into DRG neurons to evoke a solitary spike in the absence and presence of 25 µM compound 15 (n = 12; * p<0.01).
Mentions: We further characterized the properties of compound 15 on central and peripheral neurons, using the current-clamp mode of the whole-cell patch-clamp technique in rat primary cultures of hippocampal and DRG neurons (Figure 7). Repetitive spike discharges were evoked by depolarizing current injections. External application of compound 15 (25 µM) robustly and reversibly depressed the number of evoked action potentials in both hippocampal and DRG neurons (from 9.6±1.0 to 2.1±0.5, n = 8 and from 10.4±0.8 to 0.8±0.3, n = 8 respectively; 75–200 pA for 400 ms; p<0.01) (Figure 7A and D). Exposure to compound 15 (25 µM) hyperpolarized the resting membrane potential of DRG neurons by −8.3±0.2 mV (from −60.6±0.3 mV to −69.0±0.3 mV, n = 13; p<0.01; Figure 7B). A similar hyperpolarization was also obtained in hippocampal neurons exposed to compound 15 (−8.5 mV). Reflecting the dampening action of compound 15 on spike generation, the rheobase current needed to be injected into DRG neurons to generate a single spike, was increased upon exposure to the drug (for a 2 ms injection, from 400±41 pA to 577±38 pA, n = 12; p<0.01) (Figure 7C and E).

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