A Substituted Anilino Enaminone Acts as a Novel Positive Allosteric Modulator of GABA_A Receptors in the Mouse Brain

Abstract

A small library of anilino enaminones was analyzed for potential anticonvulsant agents. We examined the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells (MC), in an olfactory bulb brain slice preparation using wholecell patch-clamp recording. These compounds are known to be effective in attenuating pentylenetetrazol-induced convulsions. Among the three compounds tested, 5-methyl-3-(4-trifluoromethoxy-phenylamino)-cyclohex-2-enone (KRS-5Me-4-OCF 3 ) showed potent inhibition of MC activity with an EC 50 of 24.5 M. It hyperpolarized the membrane potential of MCs accompanied by suppression of spontaneous firing. Neither ionotropic glutamate receptor blockers nor a GABA B receptor blocker prevented the KRS-5Me-4-OCF 3 -evoked inhibitory effects. In the presence of GABA A receptor antagonists, KRS-5Me-4-OCF 3 completely failed to evoke inhibition of MC spiking activity, suggesting that KRS-5Me-4-OCF 3 -induced inhibition may be mediated by direct action on GABA A receptors or indirect action through the elevation of tissue GABA levels. Neither vigabatrin (a selective GABA-T inhibitor) nor 1,2,5,6-tetrahydro-1-[2-[[(diphenylmethylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid hydrochloride (NNC-711) (a selective inhibitor of GABA uptake by GABA transporter 1) eliminated the effect of KRS-5ME-4-OCF 3 on neuronal excitability, indicating that the inhibitory effect of the enaminone resulted from direct activation of GABA A receptors. The concentration-response curves for GABA are left-shifted by KRS-5Me-4-OCF 3 , demonstrating that KRS-5Me-4-OCF 3 enhanced GABA affinity and acted as a positive allosteric modulator of GABA A receptors. The effect of KRS-5Me-4-OCF 3 was blocked by applying a benzodiazepine site antagonist, suggesting that KRS-5Me-4-OCF 3 binds at the classic benzodiazepine site to exert its pharmacological action. The results suggest clinical use of enaminones as anticonvulsants in seizures and as a potential anxiolytic in mental disorders.

6 Hz Active Anticonvulsant Fluorinated N-Benzamide Enaminones and Their Inhibitory Neuronal Activity

“…Data from our electrophysiology studies revealed that the lead analog KRS−5Me−4−OCF 3 (1) showed activity as a positive allosteric modulator of GABA at the GABA A benzodiazepine receptor site [8]. Using lead-based drug design methods, we proposed that optimizing the KRS−5Me−4−OCF 3 analog with an amide bridge functionality between the enaminone intermediate and the aromatic ring while maintaining the fluorinated group on the benzene ring (as seen in Figure 2) will allow for the analogs to retain anticonvulsant activity with better efficacy and little to no neurotoxicity.…”

Allosteric Modulation of GABAA Receptors by an Anilino Enaminone in an Olfactory Center of the Mouse Brain

“…Our hypothesis is that the substituted site in enaminones contributes to the mode of action of these compounds. Recent work determined the mechanism of anticonvulsant action of three enaminone compounds that have non- ortho -substituted cyclohexenone [ 18 ]. Specifically, the effects of three anilino enaminones on neuronal activity of output neurons, mitral cells, in an olfactory bulb brain slice preparation using whole-cell patch-clamp recording has been examined.…”

“…E139 is a para -bromo anilino enaminone derivative with a methyl ester substituted moiety at cyclohexenone and Compound 30 is the benzylamino analog. From [ 18 ].…”

“…In brain slices, mitral cells spontaneously generate action potentials at 1 to 6 Hz [ 14 ]. Bath application of either KRS-5Me-3Cl or KRS-5Me-4F modulates the spike rate and membrane potential of mitral cells ( Figure 6 ) [ 18 ]. KRS-5Me-4-OCF 3 shows a more potent inhibition of mitral cell activity compared to the other two compounds ( Figure 6 A,B).…”

Identification of Novel Proteasome Inhibitors from an Enaminone Library

“…Enaminones have also played an important role as building blocks for generating libraries of novel drug candidates (12)(13)(14)(15)(16). More recently, the therapeutic potential of the enaminone building block itself has become an active area of investigation (9,(17)(18)(19)(20).…”