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

Compound 6 enhances Kv7.2/3 currents and inhibits firing of peripheral DRG neurons.(A) Representative traces recorded from the same CHO cell before (left panel) and after (right panel) external application 100 µM compound 6. The membrane potential was stepped from −90 mV (holding potential) to +40 mV for 1.5 s pulse duration in 10mV increments, followed by a repolarizing step to −60 mV. (B) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 25 µM (solid squares) and 100 µM (empty circles) compound 6-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (C) The potency of compound 6 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 6 concentration and fitted by a sigmoidal function yielding an EC50 value of 14±2 µM (n = 5). (D) Representative rat DRG spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 6 and after washout (wash). (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 6 (n = 8; * p<0.01). (F) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in DRG neurons in the absence and presence of 25 µM compound 6 (n = 12; * p<0.01). (G) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 6 (n = 7; * p<0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2131780&req=5

pone-0001332-g009: Compound 6 enhances Kv7.2/3 currents and inhibits firing of peripheral DRG neurons.(A) Representative traces recorded from the same CHO cell before (left panel) and after (right panel) external application 100 µM compound 6. The membrane potential was stepped from −90 mV (holding potential) to +40 mV for 1.5 s pulse duration in 10mV increments, followed by a repolarizing step to −60 mV. (B) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 25 µM (solid squares) and 100 µM (empty circles) compound 6-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (C) The potency of compound 6 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 6 concentration and fitted by a sigmoidal function yielding an EC50 value of 14±2 µM (n = 5). (D) Representative rat DRG spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 6 and after washout (wash). (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 6 (n = 8; * p<0.01). (F) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in DRG neurons in the absence and presence of 25 µM compound 6 (n = 12; * p<0.01). (G) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 6 (n = 7; * p<0.01).

Mentions: As mentioned above, we succeeded to design an amide derivative of the diclofenac series, which displays a potent M-channel activity but does not inhibit COX-1/COX-2 enzymes. We characterized further its action on recombinant Kv7.2/3 channels expressed in CHO cells and its properties on primary cultured neurons. Superfusion of compound 6 enhanced Kv7.2/3 currents at all voltages. Like compound 15, the activating effects of compound 6 became weaker when membrane voltage was stepped to more positive potentials (Figure 9A). At −50 mV, a physiologically relevant subthreshold potential, compound 6 (25 µM) increased Kv7.2/3 current amplitude by 9.4±1.2 fold (n = 5). The activating effect of compound 6 mostly originated from a left-shift of the Kv7.2/3 activation curve. For example, 10 µM and 100 µM of compound 6 produced left-shifts of −13 mV and −31 mV, respectively, compared to control (Figure 9B and C). Compound 6 dose-dependently activated Kv7.2/3 channels, a feature quantified by the concentration dependent left-shift (ΔV50), yielding an EC50 of 14 µM (Figure 9C).


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 6 enhances Kv7.2/3 currents and inhibits firing of peripheral DRG neurons.(A) Representative traces recorded from the same CHO cell before (left panel) and after (right panel) external application 100 µM compound 6. The membrane potential was stepped from −90 mV (holding potential) to +40 mV for 1.5 s pulse duration in 10mV increments, followed by a repolarizing step to −60 mV. (B) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 25 µM (solid squares) and 100 µM (empty circles) compound 6-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (C) The potency of compound 6 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 6 concentration and fitted by a sigmoidal function yielding an EC50 value of 14±2 µM (n = 5). (D) Representative rat DRG spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 6 and after washout (wash). (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 6 (n = 8; * p<0.01). (F) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in DRG neurons in the absence and presence of 25 µM compound 6 (n = 12; * p<0.01). (G) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 6 (n = 7; * p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001332-g009: Compound 6 enhances Kv7.2/3 currents and inhibits firing of peripheral DRG neurons.(A) Representative traces recorded from the same CHO cell before (left panel) and after (right panel) external application 100 µM compound 6. The membrane potential was stepped from −90 mV (holding potential) to +40 mV for 1.5 s pulse duration in 10mV increments, followed by a repolarizing step to −60 mV. (B) The normalized conductance (G/Gmax) was plotted as a function of the test voltages, for control (open squares), 25 µM (solid squares) and 100 µM (empty circles) compound 6-treated cells. The activation curves were fitted using one Boltzmann function (n = 5). (C) The potency of compound 6 was determined by the extent of left-shift (ΔV50), plotted as a function of compound 6 concentration and fitted by a sigmoidal function yielding an EC50 value of 14±2 µM (n = 5). (D) Representative rat DRG spiking discharge, evoked by a squared depolarizing current pulse (100 pA for 400 msec) before (control), during exposure to 25 µM compound 6 and after washout (wash). (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 6 (n = 8; * p<0.01). (F) Number of spikes evoked by injecting squared depolarizing current pulses (75–200 pA for 400 ms) in DRG neurons in the absence and presence of 25 µM compound 6 (n = 12; * p<0.01). (G) Resting membrane potential of DRG neurons before (control) and following exposure to 25 µM compound 6 (n = 7; * p<0.01).
Mentions: As mentioned above, we succeeded to design an amide derivative of the diclofenac series, which displays a potent M-channel activity but does not inhibit COX-1/COX-2 enzymes. We characterized further its action on recombinant Kv7.2/3 channels expressed in CHO cells and its properties on primary cultured neurons. Superfusion of compound 6 enhanced Kv7.2/3 currents at all voltages. Like compound 15, the activating effects of compound 6 became weaker when membrane voltage was stepped to more positive potentials (Figure 9A). At −50 mV, a physiologically relevant subthreshold potential, compound 6 (25 µM) increased Kv7.2/3 current amplitude by 9.4±1.2 fold (n = 5). The activating effect of compound 6 mostly originated from a left-shift of the Kv7.2/3 activation curve. For example, 10 µM and 100 µM of compound 6 produced left-shifts of −13 mV and −31 mV, respectively, compared to control (Figure 9B and C). Compound 6 dose-dependently activated Kv7.2/3 channels, a feature quantified by the concentration dependent left-shift (ΔV50), yielding an EC50 of 14 µM (Figure 9C).

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