Kenneth N. Mewett

GABAC Receptors as Drug Targets

GABA(C) receptors are the least studied of the three major classes of GABA receptors. The physiological roles of GABA(C) receptors are still being unravelled and the pharmacology of these receptors is being developed. A range of agents has been described that act on GABA(C) receptors with varying degrees of specificity as agonists, partial agonists, antagonists and allosteric modulators. Pharmacological differences are known to exist between subtypes of cloned GABA(C) receptors that have been cloned from mammalian sources. There is evidence for functional GABA(C) receptors in the retina, spinal cord, superior colliculus, pituitary and gastrointestinal tract. Given the lower abundance and less widespread distribution of GABA(C) receptors in the CNS compared to GABA(A) receptors, GABA(C) receptors may be a more selective drug target than GABA(A) receptors. The major indications for drugs acting on GABA(C) receptors are in the treatment of visual, sleep and cognitive disorders. The most promising leads are THIP, a GABA(C) receptor antagonist in addition to its well known activity as a GABA(A) receptor partial agonist, which is being evaluated for sleep therapy, and CGP36742, an orally active GABA(B) and GABA(C) receptor antagonist, which enhances cognition. Analogues of THIP and CGP36742, such as aza-THIP, that are selective for GABA(C) receptors are being developed. TPMPA and related compounds such as P4MPA, PPA and SEPI are also important leads for the development of systemically active selective GABA(C) receptor antagonists.

Modulation of Ionotropic GABA Receptors by Natural Products of Plant Origin

Publisher Summary This chapter discusses the modulation of ionotropic γ‐aminobutyric acid (GABA) receptors by natural products of plant origin. There is an impressive array of natural products that are known to influence the function of ionotropic receptors for GABA, the major inhibitory neurotransmitter in the brain. The major chemical classes of such natural products are flavonoids, terpenoids, phenols, and polyacetylenic alcohols. The interaction of flavonoids with benzodiazepine modulatory sites on GABAA receptors lead to the great interest in flavonoids as positive modulators of such receptors, many of the interactions between flavonoids and GABAA receptors do not involve classical flumazenil‐sensitive benzodiazepine sites. There are significant synergistic interactions between some of these positive modulators such as between substances isolated from Valeriana officinalis. Thus, the sleep inducing effects of hesperidin are potentiated by 6‐methylapigenin, while the sedating and sleep inducing effects of valerenic acid are potentiated when co‐administered with the flavonoid glycoside linarin. The discovery of second order positive modulators adds new dimension to the concept of the allosteric modulation of GABAA receptors. Second order positive modulators act only in conjunction with a specific first order positive modulator.

GABAC receptor antagonists differentiate between human ρ1 and ρ2 receptors expressed in Xenopus oocytes

The selective GABA(C) receptor antagonist, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA), is eight times more potent against human recombinant p receptors than p2 receptors expressed in Xenopus oocytes. (3-Aminopropyl)methylphosphinic acid (CGP35024), the methylphosphinic acid analogue of GABA, and [(E)-3-aminopropen-1-yl]methylphosphinic acid (CGP44530), an open chain analogue of TPMPA, were five and four times, respectively, more potent as antagonists of p1 receptors than as antagonists of p2 receptors. Isoguvacine was a weak partial agonist at both p1 and p2 receptors with intrinsic activities (calculated as a percentage of the maximum whole cell current produced by a maximum dose of GABA) of 45 and 68%, respectively, of the maximum response produced by GABA. In agreement with other workers, it was found that imidazole-4-acetic acid was a partial agonist at both p1 and p2 receptors, showing higher intrinsic activity at p2 than at p1 receptors. The p1 receptor antagonist, trans-4-amino-2-methylbut-2-enoic acid (2-MeTACA), was a partial agonist at p2 receptors with an intrinsic activity of 34%. 2-MeTACA may be useful in differentiating between homo-oligomeric p1 and p2 receptors in native systems. These studies reveal significant differences in the antagonist profile of human recombinant p1 and p2 GABA(C) receptors.

(+)‐ and (‐)‐cis‐2‐Aminomethylcyclopropanecarboxylic Acids Show Opposite Pharmacology at Recombinant ρ1 and ρ2 GABAC Receptors

Abstract: The effects of the enantiomers of (±)‐CAMP and(±)‐TAMP [(±)‐cis‐ and(±)‐trans‐2‐aminomethylcyclopropanecarboxylic acids,respectively], which are cyclopropane analogues of GABA, were tested onGABAA and GABAC receptors expressed in Xenopuslaevis oocytes using two‐electrode voltage clamp methods. (+)‐CAMP wasfound to be a potent and full agonist at homooligomeric GABACreceptors (KD∼40 μM andImax∼100% at ρ1;KD∼17 μM and Imax∼100% at ρ2) but a very weak antagonist atα1β2γ2L GABAAreceptors. In contrast, (‐)‐CAMP was a very weak antagonist at bothα1β2γ2L GABAAreceptors and homooligomeric GABAC receptors (IC50∼900 μM at ρ1 and ∼400 μM atρ2). Furthermore, (+)‐CAMP appears to be a superior agonist tothe widely used GABAC receptor partial agonistcis‐4‐aminocrotonic acid (KD∼74μM and Imax∼78% at ρ1;KD∼70 μM and Imax∼82% at ρ2). (‐)‐TAMP was the most potent of thecyclopropane analogues on GABAC receptors (KD∼9 μM and Imax∼40% atρ1; KD∼3 μM andImax∼50‐60% at ρ2), but it was also amoderately potent GABAA receptor partial agonist(KD∼50‐60 μM and Imax∼50% at α1β2γ2LGABAA receptors). (+)‐TAMP was a less potent partial agonist atGABAC receptors (KD∼60 μM andImax∼40% at ρ1; KD∼30 μM and Imax∼60% atρ2) and a weak partial agonist atα1β2γ2L GABAAreceptors (KD∼500 μM andImax∼50%). None of the isomers of (±)‐CAMP and(±)‐TAMP displayed any interaction with GABA transport at theconcentrations tested. Molecular modeling based on the present resultsprovided new insights into the chiral preferences for either agonism orantagonism at GABAC receptors.

A new synthesis resolution and in vitro activities of (R)- and (S)-β-Phenyl-Gaba

Abstract β-Phenyl-GABA (2) was resolved by separation by crystallization and/or h.p.l.c. of the diastereoisomeric (R)-(-)-pantolactone esters of the N-phthalimido protected β-phenyl-GABA. The absolute stereochemical assignments obtained from chiroptical studies of the enantiomers(8a) and(8b) and an X-ray crystallographic study of the diastereoisomer(7a) were supported by the activities of the enantiomers(8a) and(8b) in binding and electrophysiological studies. Details of synthesis, binding, electrophysiological, chiroptical and X-ray crystallographic studies are reported.

trans-4-Amino-2-methylbut-2-enoic acid (2-MeTACA) and (±)-trans-2-aminomethylcyclopropanecarboxylic acid ((±)-TAMP) can differentiate rat ρ3 from human ρ1 and ρ2 recombinant GABAC receptors

This study investigated the effects of a number of GABA analogues on rat ρ3 GABAC receptors expressed in Xenopus oocytes using 2‐electrode voltage clamp methods. The potency order of agonists was muscimol (EC50=1.9±0.1 μM) (+)‐trans‐3‐aminocyclopentanecarboxylic acids ((+)‐TACP; EC50=2.7±0.9 μM) trans‐4‐aminocrotonic acid (TACA; EC50=3.8±0.3 μM) GABA (EC50=4.0±0.3 μM) > thiomuscimol (EC50=24.8±2.6 μM) > (±)‐cis‐2‐aminomethylcyclopropane‐carboxylic acid ((±)‐CAMP; EC50=52.6±8.7 μM) > cis‐4‐aminocrotonic acid (CACA; EC50=139.4±5.2 μM). The potency order of antagonists was (±)‐trans‐2‐aminomethylcyclopropanecarboxylic acid ((±)‐TAMP; KB=4.8±1.8 μM) (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA; KB=4.8±0.8 μM) > (piperidin‐4‐yl)methylphosphinic acid (P4MPA; KB=10.2±2.3 μM) 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP; KB=10.2±0.3  μM) imidazole‐4‐acetic acid (I4AA; KB=12.6±2.7 μM) > 3‐aminopropylphosphonic acid (3‐APA; KB=35.8±13.5 μM). trans‐4‐Amino‐2‐methylbut‐2‐enoic acid (2‐MeTACA; 300 μM) had no effect as an agonist or an antagonist indicating that the C2 methyl substituent is sterically interacting with the ligand‐binding site of rat ρ3 GABAC receptors. 2‐MeTACA affects ρ1 and ρ2 but not ρ3 GABAC receptors. In contrast, (±)‐TAMP is a partial agonist at ρ1 and ρ2 GABAC receptors, while at rat ρ3 GABAC receptors it is an antagonist. Thus, 2‐MeTACA and (±)‐TAMP could be important pharmacological tools because they may functionally differentiate between ρ1, ρ2 and ρ3 GABAC receptors in vitro.

Inhibition of high affinity l-glutamic acid uptake into rat cortical synaptosomes by the conformationally restricted analogue of glutamic acid, cis-1-aminocyclobutane-1,3-dicarboxylic acid

The action of two cyclobutane derivatives of L-glutamic acid on the high affinity uptake of L-glutamic acid was investigated using a preparation of synaptosomes from rat cerebral cortex. cis-1-Aminocyclobutane-1,3-dicarboxylic acid (also known as trans-2,4-methanoglutamic acid) potently inhibited L-glutamic acid uptake (IC50 30 microM), whereas trans-1-aminocyclobutane-1,3-dicarboxylic acid (also known as cis-2,4-methanoglutamic acid), a potent N-methyl-D-aspartate (NMDA) agonist, was inactive. Analysis of the kinetics of L-glutamic acid uptake in the presence and absence of cis-1-aminocyclobutane-1,3-dicarboxylic acid (CACB) suggests that it may act as a competitive inhibitor (Ki 8 microM). CACB may be substrate for the L-glutamic acid high-affinity uptake carrier since preincubation of CACB with the synaptosomal preparation increased its potency in inhibiting L-glutamic acid uptake. The conformationally restricted structure of CACB may be indicative of the conformations of L-glutamic acid that interact with the high affinity uptake carrier.

Neurochemicals for the Investigation of GABAC Receptors

GABAC receptors are being investigated for their role in many aspects of nervous system function including memory, myopia, pain and sleep. There is evidence for functional GABAC receptors in many tissues such as retina, hippocampus, spinal cord, superior colliculus, pituitary and the gut. This review describes a variety of neurochemicals that have been shown to be useful in distinguishing GABAC receptors from other receptors for the major inhibitory neurotransmitter GABA. Some selective agonists (including (+)-CAMP and 5-methyl-IAA), competitive antagonists (such as TPMPA, (±)-cis-3-ACPBPA and aza-THIP), positive (allopregnanolone) and negative modulators (epipregnanolone, loreclezole) are described. Neurochemicals that may assist in distinguishing between homomeric ρ1 and ρ2 GABAC receptors (2-methyl-TACA and cyclothiazide) are also covered. Given their less widespread distribution, lower abundance and relative structural simplicity compared to GABAA and GABAB receptors, GABAC receptors are attractive drug targets.

R-(−)-β-phenyl-GABA is a full agonist at GABAB receptors in brain slices but a partial agonist in the ileum

R-(-)-beta-phenyl-GABA has been compared at GABAB receptors using cortical and ileal preparations. R-(-)-beta-phenyl-GABA (EC50 = 25 microM) was a less potent full agonist than R,S-(+/-)-baclofen (EC50 = 2.5 microM), in depressing CA1 population spikes of rat hippocampal slices, and 5 times less potent in attenuating the spontaneous discharges of rat neocortex. However, R-(-)-beta-phenyl-GABA (100-400 microM) was only a weak partial agonist in the ileum. All these actions were sensitive to CGP 35348 (3-aminopropyl-(P-diethoxymethyl)-phosphinic acid) and therefore mediated by GABAB receptors.

Medicinal chemistry of ρ GABAC receptors

The inhibitory neurotransmitter, GABA, is a low-molecular-weight molecule that can achieve many low-energy conformations, which are recognized by GABA receptors and transporters. In this article, we assess the structure-activity relationship profiles of GABA analogs at the ionotropic ρ GABA(C) receptor. Such studies have significantly contributed to the design and development of potent and selective agonists and antagonists for this subclass of GABA receptors. With these tools in hand, the role of ρ GABA(C) receptors is slowly being realized. Of particular interest is the development of selective phosphinic acid analogs of GABA and their potential use in sleep disorders, inhibiting the development of myopia, and in improving learning and memory.