MBC 3320 GABA Systems

GABA synthesis

g-Amino butyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. It is found in almost every region of brain, and is formed through the activity of the enzyme glutamic acid decarboxylase (GAD).

GAD catalyzes the formation of GABA from glutamic acid. The synthesis of GABA is linked to the Kreb's cycle. GAD requires vitamin B6 (pyridoxal phosphate) as a cofactor, which can be used to regulate the levels of GABA. GABA can be metabolized by a transamination reaction with a-ketoglutarate, catalyzed by GABA-transaminase. Compounds such as the competitive GAD inhibitor allylglycine, inhibit GABA formation and cause convulsions due to the lack of GABA activity.

Sodium valproate (or valproic acid) on the other hand, blocks GABA transaminase activity, thereby elevating GABA levels, and thus alleviating seizures. Sodium valproate is useful in the treatment of epilepsy and bipolar mood disorders. Another strategy is to block GABA-transaminase with g-acetylenic GABA, thereby increasing the concentration of GABA at the synapse.

GABA receptors

GABA receptors can be divided into three classes. GABA_A and GABA_C receptors are ligand-gated ion channels, while GABA_B receptors are G protein-coupled receptors.

GABA_A receptors belong to the superfamily of ligand-gated ion channel receptors that also includes the nicotinic and glycine receptors. The GABA_A receptor consists of three separate subunits with an a₂,b₂,g arrangement similar to that found for neuronal nicotinic receptors. The ligand binding site is located at the interface between the a and b subunits. The benzodiazepine binding site is located at a similar level at the interface between the a and g subunits. Each subunit is comprised of four hydrophobic sequences which span the membrane, with a large extracellular amino terminus containing the binding site and a carboxyl terminus located intracellularly.

GABA binds to GABA_A receptors in the extended form as demonstrated by the activity of trans 4-aminocrotonoic acid and the lower activity of cis 4-aminocrotonoic acid.

Related compounds with activity on GABA systems include nipecotic acid and isonipecotic acid. Nipecotic acid is a GABA reuptake inhibitor which enhances GABA levels at the synapse. Isonipecotic acid acts directly as an agonist on GABA_A receptors.

Muscimol is a naturally occurring compound isolated from Amanita muscaria and acts as a GABA_A agonist. The isoxazole ring also is found in the GABA_A agonist tetrahydroisoxazolopyridinol (THIP).

Bicucullin acts as a direct antagonist at the GABA_A receptor as does SR 42641. Binding of GABA to GABA_A receptors is regulated by the benzodiazepines and results in the opening of a Cl^- ion channel. Muscimol is active at GABA_A receptors.

Baclofen is direct agonist at GABA_B receptors, which are coupled to G proteins. GABA_B receptors may regulate Ca2+ and K+ influx through the Gi/o family of G proteins and act presynaptically to inhibit the release of excitatory amino acids such as glutamate. Baclofen is orally active as a muscle relaxant and has been used in the treatment of rigidity and spasticity of cerebral palsy. Phaclofen is a weak partial agonist, while saclofen is an antagonist at GABA_B receptors.

Two isoforms of GABA_B receptors exist: GABA_B1A and GABA_B1B. Recent studies indicate that GABA_B receptors form heterodimers that exhibit functional activity.

Anxiolytics: Benzodiazepines

Benzodiazepines represent a class of compounds collectively referred to as anxiolytics. Benzodiazepines modulate the binding of GABA to the GABA_A receptor. Benzodiazepines increase the binding of GABA to GABA_A receptors and promote Cl^- influx.

Diazepam (Valium) is one of the most widely prescribed drugs on the market. Important features of the benzodiazepines include: one electronegative group on the first ring, generally free substitution on the second ring nitrogen and carbon, and a phenyl group with halogenation only in the ortho position (as in Lorazepam, a muscle relaxant).

As noted above, benzodiazepines derivatives with substituents on the second ring also exhibit anxiolytic activity. Note the imidazole ring on midazolam. Flurazepam and chlordiazepam also are anxiolytic.

Benzodiazepines exhibit anxiolytic activity due to their ability to promote GABA binding. The act as indirect agonists. Several ligands can block the actions of benzodiazepines, including flumazenil, a benzodiazepine antagonist. Benzodiazepine antagonists have no action by themselves but prevent the enhancement of GABA activity by the benzodiazepines. Ro-15-4513 is able to act (apparently through the GABA receptor) to block Cl^- conductance associated with ethanol. Ro-15-4513 does not alter the effects of muscimol or pentobarbital on Cl^- flux so its action is independent of GABA binding per se. Ro-15-1788 is able to reverse the effects of Ro-15-4513 yet does not affect Cl- conductance on its own.

Benzodiazepines possess anticonvulsant activity in addition to their anxiolytic action. Ro-15-4513 is proconvulsive in that it lowers the seizure threshold to bicuculline and lowers the efficacy of both sodium pentobarbital and ethanol as anticonvulsants. Moreover, Ro-15-4513 completely reverses the anticonvulsant effects of benzodiazepines.

Recent efforts have focused on the development of compounds that exhibit selectivity for different combinations of subunits. Zolpidem and alpidem discriminate between GABA_A receptors with different subunit composition.

b-Carbolines antagonize the anxiolytic, anticonvulsant and sedative effects of the benzodiazepines. These compounds also can differentiate between the anticonvulsant and anxiolytic actions of the benzodiazepines. Early studies suggested that the b-carbolines may be produced endogenously to modulate GABA receptors in vivo although a physiolgical role of the b-carbolines has been refuted.

Within the class of b-carbolines, molecules with varying potency and efficacy have been developed for benzodiazepine binding sites. Both methyl- and ethyl-b-carboline-3-carboxylate are proconvulsant. They block the binding of benzodiazepines and act as inverse agonists since they produce the opposite effect of diazepam.

Propyl-b-carboline-3-carboxylate also binds at the benzodiazepine site on the GABA receptor but acts as a benzodiazepine antagonist in the same fashion as flumazenil. Both compounds are effective blockers of benzodiazepine action and prevent the anticonvulsant effects of diazepam. Both propyl-b-carboline-3-carboxylate and flumazenil block the convulsant action of methyl- and ethyl-b-carboline-3-carboxylate.

The b-carboline derivative 3-(methoxycarbonyl)amino-b-carboline (CMC) blocks the sedative properties of diazepam and the convulsant properties of methyl-b-carboline-3-carboxylate, but does not block either the aniconvulsant or anxiolytic properties of diazepam. The development of new ligands within the series of b-carbolines may yield new compounds with novel properties useful in the treatment of epilepsy and anxiety.

Anticonvulsant drugs

Anticonvulsants are used to treat the various forms of epilepsy, which is characterized by excessive neuronal firing in the cortical and temporal lobe regions of the brain. Electroencephalograms (EEG) can pick up the rhythmic discharge of neurons from electrodes placed on the scalp. Rhythmic discharges of spikes and slow waves characterize the EEG during seizures.

"Grand mal" epilepsy comprises the most severe form of convulsions, followed by "petit mal" seizures, and then psychomotor "absences" which exhibit little convulsant activity. Seizure activity can be limited as in partial seizures to one area of brain. The symptoms depend on the area involved and the degree of spread of electrical activity. Generalized seizures involving more than one focus usually involve loss of conciousness, and varying degrees of motor disturbances. Absences are characterized by short periods of loss of consciousness, usually not remembered following the episode. Postictal confusion is generally not encountered.

The benzodiazepines have an anticonvulsant action in addition to the anxiolytic activity. Recent work has helped distinguish between the two roles for the benzodiazepines. Picrotoxin is a convulsant which interacts with the GABA receptor complex and blocks the Cl- ionophore. A variety of hypnotic, anesthetic and anticonvulsant drugs interact with a dihydropicrotoxin binding site, and may act as Cl- ionophore agonists. Barbiturates such as pentobarbital and phenobarbital enhance GABA binding in proportion to general anesthetic activity.

Benzodiazepines are also used clinically as anticonvulsants, although they do not interact with the dihydropicrotoxin binding site directly. Instead, the depressant action of the benzodiazepines may be on the Cl^--channel through benzodiazepine binding sites, which modulate GABA binding.

Diphenylhydantoin (phenytoin) is useful in the treatment of epilepsy. Phenytoin stabilizes the neuron against the excitation associated with seizure activity, without producing sedation. High-frequency stimulation of neurons in some regions of the brain (e.g., hippocampus) can produce a phenomenon called post-tetanic potentiation. After presentation of a stimulus, a response may be recorded in a neuron or from a population of neurons. Following repetitive stimulation at high frequency (100 Hz), the response elicited by the same stimulus is enhanced. Phenytoin blocks post-tetanic potentiation, possibly through reduction of the intracellular concentration of sodium by lowering sodium permeability at rest.

While phenytoin is effective against some generalized (grand mal) seizures and partial seizures. Other generalized seizure states (e.g., absences) must be treated with other drugs such as the oxazolidinediones, since phenytoin can increase seizure frequency. Phenytoin is taken up in the duodenum, metabolized in the liver and excreted from the urine as the glucuronic acid conjugate.

The oxazolidinedione trimethadione is effective in treating absences, but ineffective against grand mal siezures. Trimethadione seems to act by direct stimulation of neurons in the thalamus. The oxazolidinediones decrease synaptic transmission by increasing the duration of the refractory period in neurons involved in repetitive discharges. Side effects of trimethadione include nephrosis, aplastic anemia, and bone marrow depression. Succinimides (e.g., ethosuxamide) are less toxic than trimethadione, and are believed to act in a similar manner. Ethosuximide is useful in the treatment of absences and some generalized seizures, and blocks sodium and certain classes of calcium channels.

Other anticonvulsants include carbamazepine, which is useful in the treatment of grand mal seizures. Carbamazepine exerts its action through blockade of sodium channels. Lamotrigine also blocks voltage-sensitive sodium channels, and exhibits anticonvulsant activity. Through blocking sodium channels, labotrigine decreases excitatory amino acid neurotransmitter release. It possesses very few CNS side effects, and is useful in reducing the incidence of refractory partial seizures and generalized seizures not controlled by other anticonvulsants.

References

1. Principles of Medicinal Chemistry. by Foye, W.O., T.L. Lemke and D.A. Williams. Williams & Wilkins. Fourth Edition, 1995.
2. The RBI Handbook of Receptor Classification and Signal Transduction. K.J. Watling. RBI. Third Edition, 1998.