D Reviews • BIOTREND Reviews • BIOTREND Reviews • BIOTREND Reviews • BIOTREND Reviews Review No.7 / 1-2011 GABA receptors Wolfgang Froestl , CNS & Chemistry Expert, AC Immune SA, PSE Building B - EPFL, CH-1015 Lausanne, Phone: +41 21 693 91 43, FAX: +41 21 693 91 20, E-mail: [email protected] GABA Activation of the GABA A receptor leads to an influx of chloride GABA ( -aminobutyric acid; Figure 1) is the most important and ions and to a hyperpolarization of the membrane. 16 subunits with γ most abundant inhibitory neurotransmitter in the mammalian molecular weights between 50 and 65 kD have been identified brain 1,2 , where it was first discovered in 1950 3-5 . It is a small achiral so far, 6 subunits, 3 subunits, 3 subunits, and the , , α β γ δ ε θ molecule with molecular weight of 103 g/mol and high water solu - and subunits 8,9 . π bility. At 25°C one gram of water can dissolve 1.3 grams of GABA. 2 Such a hydrophilic molecule (log P = -2.13, PSA = 63.3 Å ) cannot In the meantime all GABA A receptor binding sites have been eluci - cross the blood brain barrier. It is produced in the brain by decarb- dated in great detail. The GABA site is located at the interface oxylation of L-glutamic acid by the enzyme glutamic acid decarb- between and subunits. Benzodiazepines interact with subunit α β oxylase (GAD, EC 4.1.1.15). It is a neutral amino acid with pK = combinations ( ) ( ) , which is the most abundant combi - 1 α1 2 β2 2 γ2 4.23 and pK = 10.43. GABA interacts with three receptors: GABA , nation to form a pentamer, or with combinations of , 2 A α2βγ 2 α3βγ 2 GABA and GABA . and . They bind at the interface between and subunits 10 . B C α5βγ 2 α γ O Other allosteric binding sites are the barbiturate site, the site of general anesthetics and the site for the channel blocking agents H2N C OH TBPS and picrotoxin(in) (Figure 3). Figure 1. -Aminobutyric acid (GABA). γ GABA A Receptors GABA site Barbiturate GABA GABA receptor is an ion channel receptor of the Cys-loop receptor muscimol A site isoguvacine barbiturates bicuculline superfamily together with the nicotinic acetylcholine receptor, the etomidate SR95531 6 etazolate 5-HT 3 receptor and the strychnine-sensitive glycine receptor . It was cloned in collaboration between the groups of Eric A. Barnard Benzodiazepine site agonists of the MRC in Cambridge, UK and Peter H. Seeburg of Genentech benzodiazepines {antagonists General non-benzodiazepines inverse agonists in 1987 7. It is a pentamer consisting of five subunits with a large anesthetics propofol Subsynaptic membrane extracellular N-term, 4 transmembrane domains and a short extra - steroids halothane cellular C-term arranged in such a way that the transmembrane ethanol Picrotoxine domains 2 form the inner walls of a chloride channel (Figure 2). site bicyclophosphates TBPS tetrazoles Figure 3. Different binding sites at the GABA A receptor (courtesy of Prof. N. G. Bowery). The best known GABA agonists are GABA (Figure 1), muscimol, and isoguvacine (Figure 4). THIP (Gaboxadol) 11,12 , a hypnotic drug, however, is interacting with subunits at extrasynaptic sites α4β3δ (as do the general anesthetics and ethanol). Figure 2. The architecture of the GABA A receptor (taken from Wikipedia). GABA receptors NH O N N GABAA Receptor Agonists + O Cl N Cl N HO O HN O- N OH NH2 N HN O OH Muscimol (BN0352) Isoguvacine (BN0277) THIP, Gaboxadol Chlordiazepoxide (Librium) Diazepam (Valium) (BN0520) (BG0424) GABAA Receptor Antagonists Figure 5. O Classical benzodiazepine N receptor agonists O O N (Bold text denotes N N N compound available O N from BIOTREND H H N NH with catalogue N N O number). O2N Cl O O F Cl O O OH Bicuculline (BN0106) Gabazine (SR-95531) (BN0507) Flunitrazepam (Rohypnol) Triazolam (Halcion) (BG0423) (BG0425) S O O The benzodiazepine receptor antagonist Flumazenil (Anexate; Ro 15-1788) was discovered at Roche and marketed in 1987 19 O O H O N (Figure 6). It is used as an antidote for overdoses of benzo- H NH O diazepines. A selective inhibitor of 1-containing GABA A receptors OH β NH H H was discovered by Merck UK scientists, salicylidene salicyl- HO 20 HO hydrazide (SCS; Figure 6) . HO OH O H Figure 6. Benzodiazepine receptor antagonists Thiocolchicoside (BG0522) R 5135 (Bold text denotes compound available O N from BIOTREND with catalogue number). Figure 4. GABA A receptor agonists and antagonists N O (Bold text denotes compound available from BIOTREND OH with catalogue number). H N N F N 13 O OH The best known GABA antagonists are bicuculline , gabazine O (SR-95531) 14 , thiocolchicoside, a semisynthetic derivative of Flumazenil (Anexate) Salicylidene salicylhydrazide (SCS) colchicoside used as muscle relaxant with anti-inflammatory and (BG0396) (BN0477) analgesic action 15 and the steroid derivative 3 -hydroxy-16-imino- α 5 -17-aza-androstan-11-one (R-5135) 16 (Figure 4). Partial inverse agonists, such as Ro 15-4513 and Ro 19-4603 β are structurally related to flumazenil. They selectively block the The biggest boost to GABA A receptor research came in 1957 with effects of ethanol on locomotor behavior and suppress ethanol the discovery of the benzodiazepines by Leo H. Sternbach and intake 21,22 (Figure 7). Other partial inverse agonists are the -car - β colleagues of Roche, Nutley 17 . In fact, chlordiazepoxide had been boline derivatives FG-7142 (ZK-31906), which improves memory synthesized already in 1955, was somehow forgotten and sent to retention in animal studies 23 and DMCM (methyl 6,7-dimethoxy- biological testing in 1957 after a clean-up of the lab. Three years 4-ethyl- -carboline-3-carboxylate), which displays anxiogenic β later the drug was on the US market launched as Librium (Figure 5). and convulsant properties 24 . Roche Basel biologists established that the benzodiazepines act O O N N as positive allosteric modulators (PAMs) of GABA receptors 18 . Diazepam (Valium) was launched in 1963 and became the top- N O N O selling drug in the USA from 1969 to 1982 (Figure 5). Other N N “classical” benzodiazepine receptor agonists are Flunitrazepam N N S N (Rohypnol; Figure 5) and Triazolam (Halcion; Figure 5). O O Ro 15-4513 (BN0442) Ro 19-4603 (BN0443) O O O N O H N O N N N H H FG-7142 (ZK-31906) (BN0637) DMCM Figure 7. Non-subtype selective benzodiazepine receptor inverse agonists 2 (Bold text denotes compound available from BIOTREND with catalogue number). GABA receptors Many valuable compounds without a 1,4-benzodiazepine structure Although benzodiazepines are very valuable drugs, they also have (“ non-classical ” benzodiazepines ”) bind also to benzodiazepine several severe side effects such as tolerance, addiction and po- receptors, such as the imidazo[1,2-a]pyridine Zolpidem (Stilnox, tentiation of its effects by ethanol 32-35 . Christian Lüscher and col - Ambien), a widely prescribed hypnotic drug 25 (Figure 8). Zopiclone leagues in Geneva found out, that the addictive effects of benzodi - (Imovane), now replaced by Eszopiclone (Lunesta), is a short azepines can be attributed to -containing GABA receptors in α1 A acting non-benzodiazepine sedative hypnotic 26,39 . Indiplon (Figure 8) the ventral tegmental area of mice 36 . For the use as anxiolytics displays a ten-fold selectivity for the subunit-containing GABA as well as for cognition enhancing drugs, it would be desirable to α1 A receptors 27 . GBLD-345 is an imidazo-pyridazine anxiolytic 28 . eliminate the sedative effects of benzodiazepines. Ro 19-8022 (Figure 8) is a partial agonist at benzodiazepine recep- tors with no sedative or motor-impairing effects 29 . Loreclezole Sustained efforts, in particular by Hanns Möhler and colleagues (Figure 8) is a sedative and anticonvulsant drug, which interacts in Zürich 37 and by Ruth M. McKernan and her team at Merck UK 38 , selectively with and subunit containing GABA receptors 30 . made it possible to elucidate the pharmacology of the different β2 β3 A subtype combinations. Point mutations on (H101R), α1 (H101R), (H126R) and (H105R) in knock-in mice allowed α2 α3 α5 N the study of the effects of benzodiazepines on isolated GABA A N N O receptor subtypes showing that agonists at GABA receptors α1βγ 2 A N mediate sedation, amnesia and anticonvulsive effects, agonists N O O at GABA receptors mediate anxiolytic and myorelaxant N N α2βγ 2 A O effects, agonists at GABA receptors mediate anxiolytic and α3βγ 2 A N N analgetic actions and inverse agonists at GABA receptors α5βγ 2 A improve learning and memory. Zolpidem (Stilnox, Ambien) Eszopiclone (Lunesta) (BG0430) (BG0417) Zopiclone (Imovane) (BG0418) Cl Figure 9 and subtype selective benzo- α2βγ 2 α3β2γ2 N O diazepine receptor agonists. (Bold text denotes compound available from BIOTREND O with catalogue number). O F H N N N N N N F N H2N N NC N N N S F GBLD-345 (BN0218) N O O Figure 8. L-838,417 F N F Indiplon (BG0536) Non-classical benzodiazepine receptor agonists (Bold text denotes compound available from N N TP-003 O BIOTREND with catalogue number). N HO F F N N N N Cl Cl N N HO N N N N N N F N O Cl O Cl O MRK-409 (MK-0343) TPA023 (MK-0777) Ro 19-8022 Loreclezole (BN0309) N N N N N Benzodiazepines act as anxiolytics, hypnotics, anesthetics, muscle N relaxants, analgetic and antiepileptic drugs. Currently there are NH O 50 drugs on the market 31 (alpidem, alprazolam, bentazepam, HO 2 N N HO bromazepam, brotizolam, camazepam, chlordiazepoxide, O cinolazepam, clobazam, clonazepam, clorazepate, clotiazepam, N N S cloxazolam, delorazepam, diazepam, estazolam, eszopiclon, ethyl- NC N loflazepate, etizolam, fludiazepam, flumazenil (a benzodiazepine TPA-023B SB-205,384 (BN0457) receptor antagonist), flunitrazepam, flurazepam, flutoprazepam, halozepam, haloxazolam, 123 I-iomazenil (a single photon emission F tomography [SPECT] ligand), ketazolam, loprazolam, lorazepam, F lormetazepam, medazepam, metaclazepam, mexazolam, Big efforts were made by several companies to find selective midazolam, nimetazepam, nitrazepam, nordazepam, oxazepam, agonists for GABA receptors mediating anxiolytic effects α2βγ 2 A oxazolam, pinazepam, prazepam, quazepam, temazepam, without sedation.