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Inverse agonism and its therapeutic significance Gurudas Khilnani, Ajeet Kumar Khilnani1

ABSTRACT

A large number of G-protein-coupled receptors (GPCRs) show varying degrees of basal or constitutive activity. This constitutive activity is usually minimal in natural receptors but is markedly observed in wild type and mutated (naturally or induced) receptors. According to conventional two-state interaction model, binding of a may initiate activity ( with varying degrees of positive ) or prevent the effect of an agonist (antagonist with zero intrinsic activity). Inverse bind with the constitutively active receptors, stabilize them, and thus reduce the activity (negative intrinsic activity). Receptors of many classes (- and -, , GABAergic, serotoninergic, opiate, and angiotensin receptors) have shown basal activity in suitable in vitro models. Several that have been conventionally classified as antagonists (-blockers, antihistaminics) have shown effects on corresponding constitutively active Department of , receptors. Nearly all H and H antihistaminics (antagonists) have been shown to be inverse RNT Medical College, Udaipur, 1 2 agonists. Among the -blockers, and bucindolol demonstrate low level of inverse 1Department of ENT, JLN agonism as compared to propranolol and nadolol. Several antipsychotic drugs (D receptors Medical College Hospital, Ajmer, 2 Rajasthan, India antagonist), antihypertensive (AT1 receptor antagonists), antiserotoninergic drugs and antagonists have significant inverse agonistic activity that contributes partly or wholly to RReceived:eceived: 18-06-2010 their therapeutic value. Inverse agonism may also help explain the underlying mechanism of RRevised:evised: 10-01-2011 beneficial effects of carvedilol in congestive failure, -induced withdrawal syndrome AAccepted:ccepted: 01-07-2011 in opioid dependence, in psychosis, and candesartan in cardiac hypertrophy. Understanding inverse agonisms has paved a way for newer drug development. It is now CCorrespondenceorrespondence to:to: possible to develop agents, which have only desired therapeutic value and are devoid Dr. Gurudas Khilnani, of unwanted . (ACP-103), a highly selective 5-HT2A inverse E-mail: [email protected] agonist, attenuates psychosis in patients with Parkinson’s disease with psychosis and is devoid of extrapyramidal side effects. This dissociation is also evident from the development of anxioselective devoid of habit-forming potential. Hemopressin is a peptide ligand that acts as an antagonist as well as inverse agonist. This agent acts as an antinociceptive agent in different in vivo models of pain. Treatment of obesity by drugs

having inverse agonist activity at CB1/2 receptors is also underway. An exciting development is evaluation of -blockers in chronic bronchial asthma—a condition akin to congestive heart failure where -blockade has become the standard mode of therapy. Synthesis and evaluation of selective agents is underway. Therefore, inverse agonism is an important aspect of drug–receptor interaction and has immense untapped therapeutic potential.

KKEYEY WORDS:WORDS: Constitutive activity, G-protein-coupled receptors, inverse agonism, inverse agonists

AAccessccess thisthis articlearticle onlineonline Introduction QQuickuick ResponseResponse Code:Code: WWebsite:ebsite: www.ijp-online.com Drug–receptor interaction has fascinated researchers DDOI:OI: 10.4103/0253-7613.84947 since antiquity. It has been evolved from a concept of “Lock and Key” to the use of Furchgott’s method (1966) to quantify dissociation constants and relative of agonist– receptor complexes. In the last 40 years, great strides in developments in molecular pharmacology have resulted in

492 Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 Khilnani and Khilnani: Therapeutic implications of inverse agonism the accumulation of a vast amount of knowledge. This article G-proteins or the alternative pathways. In addition, wild-type briefly summarizes the recent developments in drug–receptor or experimentally mutated receptors also exhibit varying interactions with focus on inverse agonism and its potential degrees of spontaneous activation. Under physiological therapeutic utility. conditions, this activity is usually low because the fraction of unoccupied receptors in active conformational state is Inverse Agonism minimal but when the population of constitutive receptors is The two-state receptor model (binary model) shows that overexpressed in the in vitro systems, such as when expressed a receptor remains in an inactive (Ri) or active (R*) state in high amounts in cultured cells, they exhibit significant and and there is an equilibrium, which can be shifted by ligands measurable spontaneous activity.[2] Interestingly, when certain that bind to receptors [Figure 1]. The profile of a ligand, ligands bind these constitutively activated receptors in suitable traditionally, endogenous or synthetic, is characterized by its experimental settings, the overall effects are opposite to pure “affinity” toward the receptor and intrinsic activity or “.” or full agonists. There is a shift of equilibrium from activation It expresses the degree to which different ligands produce (R*) to quiescence (Ri), as shown in Figures 2-4. Such ligands varying biological responses while occupying same number of are called as “inverse agonists.” Inverse agonists preferentially receptors. The drug or a ligand avidly binds with the receptor bind and stabilize receptors in the inactive (Ri) state, and thus (affinity) and produces conformational changes, which shift the have negative intrinsic activity [Figures 2 and 5]. This results balance toward active state [Figure 1]. Thus, a drug-activated in a reduction in spontaneous receptor activity. If receptors do receptor complex may initiate an activity (intrinsic activity or not exhibit constitutive activity, the same inverse agonist may efficacy), resulting in a response. The ultimate response can behave as competitive antagonist. Neutral antagonists have be stimulatory or inhibitory depending on the activation of equal preferences for both Ri and R* states, lack any intrinsic subcellular effector mechanisms. activity, and are able to block actions produced by either According to this hypothesis, receptors are quiescent at rest agonists or inverse agonists. Many conventional antagonists, (Ri) and are stimulated by drugs (R*, receptors are switched on) such as antihistaminics are now considered to be inverse known as agonists. Antagonists do not have intrinsic activity and agonists.[1,3] As described above, a ligand must recognize at therefore oppose the effects of agonists by preventing agonist least two receptor conformational species as being identical: binding and activation (receptors are switched off). On the R* and Ri. For constitutively active receptors (which couple other hand, partial agonists have varying degrees of affinity and with G-proteins at rest), a three-state model [Figure 4] is best intrinsic activities and produce submaximal tissue responses suited to explain this interaction, that is, R#G (constitutively at any degree of receptor occupancy (usually less than 100%). activated GPCR), R*G (agonist-activated GPCR), and RiG (resting This simple mechanism of “on–off switch” remained elusive or inactive state), any of these 3 states are available for ligand for a longer period of time to pharmacologists who could (L) binding and forming ternary complexes as R#G–L, R*G–L, or not correlate certain discrepant experimental findings in cell RiG–L. The degree of observed inverse agonism depends on the systems, using isolated and mutated receptors with this model of relative affinity of the inverse agonist for the various receptor drug–receptor interaction. For example, experimental evidence species and the degree of constitutive activity in the system. indicates that all agonists do not produce the same active state Therefore, there are partial inverse agonists and full inverse of G-protein-coupled receptors (GPCRs).[1] The receptors for benzodiazepines, opiates, , , , Figure 2: Intrinsic activities of full agonist, antagonist, and inverse and a variety of other (heptahelical) GPCRs show activity even agonists when not occupied by agonists, that is, they have constitutive activity independent of ligand activation [Figure 2]. This means that a proportion of the receptor population spontaneously undergoes a conformational change that can bind and activate

Figure 1: Conventional two-state model of drug–receptor interaction

Two-state receptor model and inverse agonism Receptors are generally considered to be quiet (Ri). An agonist shifts the activity toward (R*). R# represents constitutively active receptors. An inverse agonist binds with R# and shifts the conformational state to (Ri). According to ternary complex models of receptor activation, the DR*G complex is in equilibrium with DR + G. A receptor remains in dynamic equilibrium in two states Ri and R*. A ligand that Neutral antagonists have equal preferences for both Ri and R* states, lack any binds and shifts the equilibrium to R* state is called agonist. A ligand that binds intrinsic activity, and are able to block actions produced by either agonists or but does not shift the equilibrium to active state is called antagonist. Ligand inverse agonists. Inverse agonists have higher affi nity for R#, shift R#G (or DR#G) binding is an equilibrium process. toward Ri + G (or DRi + G), decreasing the spontaneous activity of receptors.

Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 493 Khilnani and Khilnani: Therapeutic implications of inverse agonism

Figure 3: Constitutively active receptor and inverse agonism Figure 4: Proposed drug–receptor model to explain inverse agonism

A constitutively active receptor remains active at basal state, even without ligand binding. Therefore, there is a basal activity in the form of increased GTPase formation with subsequent second messenger activation. An agonist can still bind (hatched arrow) and produce conformational change to R* state. However, an inverse agonist can bind with constitutively activate receptor (thick arrow) but renders the receptor in Ri state. This results in reduced (reversed) activation of second messenger.

Figure 5: Inverse agonist-receptor interaction

R# Constitutively activated Ri Resting inactive Multiple activation states of a receptor (R* Active), (Ri quiescent or inactive) and # R* Agonist-Activated (R Constitutionally active) Diagrammatic expression of inverse agonism. Spontaneous or induced mutations (a) Unliganded receptor state in the receptors make them constitutively active (R#). An agonist which binds with (b- i and ii) Agonists shift equilibrium to activated state R# reduces the basal activity and shifts the equilibrium toward Ri. In contrast, (c) Antagonists shift equilibrium to Ri # a pure agonist shifts the equilibrium toward R* increasing subcellular activity. (d) Inverse agonists act to reduce spontaneous activity of R . agonists [Figure 5].[1-3] agonists increase basal effectors activity. Receptor inverse agonists, such as G-protein antagonists and GTPase inhibitors, G-Protein-Coupled Receptors and Inverse Agonism decrease spontaneous G-protein activity. It is interesting to The GPCRs have the ability to undergo conformational note that neutral antagonists block the effects of inverse changes upon ligand binding. The properties include existence agonists. A term “protean agonist” is used to describe a in various conformations, ability to alter receptor activity, theoretical class of agonists that produce receptor activation and production of multiple active receptor states. Response of lower magnitude than that emanating from spontaneous emanates from the hydrolysis of GTP by the G-protein resulting receptor constitutive activity. These kinds of ligands were from activation by R*. As stated above, some receptors can given the name protean agonists after Proteus, the Greek God spontaneously interact with G-proteins to initiate GTPase who could change shape and appearance at will. This class of activity, in the absence of agonist ligands [Figure 2]. This ligands shows positive agonism in some GPCR systems and constitutive activity can be suppressed in suitable in vitro inverse agonism in others.[4] experimental settings by inverse agonists either partially Evidence of Constitutive Activity of GPCRs (partial inverse agonists) or fully (full inverse agonists), Constitutive activity of GPCRs is demonstrated by resulting in action exactly opposite to agonists. Methods to measuring cyclic AMP (cAMP) levels, GTPase activity or by detect inverse agonism are based on the determination of inositol phosphate (IP3) production in in vitro overexpressed ligand affinity at Ri and R* with binding experiments, on the receptor models [Box 1]. The constitutional activity was first modulation of G-protein activity (GTP binding and hydrolysis) demonstrated at -opioid receptors, which paved the ground and change in effectors activity. The receptor agonists (ligands for many further studies, all pointing to the existence of that bind and activate surface receptors) and direct G-protein constitutive receptor activity in (BDZ) receptors

494 Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 Khilnani and Khilnani: Therapeutic implications of inverse agonism

-ARs in transgenic mice. An increased basal cAMP (about Box 1: 2 5-fold higher for 2 than for 1 receptors) output was recorded Transfection: An in-vitro method to study receptor function and the rise was proportional to the receptor density, thus demonstrating constitutive activity for both the subtypes. The process of introduction of nucleic acid material chemically or by Chidiac et al.[10] studied agonist-independent properties of viral vectors in eukaryotic cells is called as transfection. This gene the human -ARs using the baculovirus expression system in transfer technology is a powerful tool to produce knock-out mice with 2 Chinese hamster fibroblasts expressing -ARs. The amount defi cient receptor-related gene function. It is possible to introduce 2 of cAMP production correlated with the activity of  -ARs. gene transfection of  -ARs into murine cardiac myocytes to study 2 2 The increase in the production of cAMP in the absence of receptor function in health and disease. Similarly, human adenosine catecholamines and its reversal by beta-blockers reflected receptor-related cDNA is transfected in Chinese Hamster ovarian spontaneous -ARs activity. In the model for enhanced cell lines (CHO cell lines). In this way adenosine receptors are 2 beta-adrenergic signaling (transgenic mice with heart-specific expressed and can be easily studied by in vitro binding techniques. overexpression of the human -ARs), the -ARs displayed One of the major advantages of stably transfected cell lines is that 1 1 constitutive activity as evidenced by a higher spontaneous a known, constant quantity of the transfected receptor is expressed beating rate of isolated right atria of -transgenic mice for many generations. 1 (which overexpress -ARs) as compared to that of wild-type The use of stably transfected cell lines enables a receptor to be 1 mice (which do not express -ARs).[12] Zhou et al.[13] showed studied in the absence of other related receptor subtypes. The 1 -AR spontaneous activity using the highly novel system of molecular determinants of ligand binding, signal transduction 1 adenovirus-mediated gene transfer of -AR subtypes into pathways of a particular receptor, the nature of interactions between  isolated ventricular myocytes obtained from hearts of / -AR a transfected and overexpressed receptor and endogenous/ 1 2 double knockout mice. exogenous ligands can be found out with this technology. The A -blocker does not simply “block” the receptor, but activity of gene product, such as GTPase, characterize interactions  further inactivates receptor activity beyond its baseline value, between a GPCR and different G-proteins can be assayed. depending on its degree of inverse agonist activity. Antagonists

(at both 1-ARs and 2-ARs), such as metoprolol, carvedilol, and GPCRs.[5] The constitutive activation of G-protein-coupled propranolol, nebivolol, and bisoprolol, have shown inverse receptors (GPCRs) is now well recognized and more than 80% agonist activity.[14,15] The following rank order of “inverse of the classical GPCR antagonists have been found to be inverse efficacy” is reported; timolol ≥ propranolol > alprenolol ≥ agonists.[2,5] The constitutive activity may be present in native > labetalol > dichloroisoproterenol (DCI). The or mutated strains (constitutively activated mutants). Such same rank order was observed using membranes prepared receptors may be underactive and lead to inherited diseases, from Chinese hamster fibroblasts expressing 2-ARs. The such as diabetes insipidus, male pseudohermaphroditism, effect of timolol was partly blocked by labetalol and DCI, in congenital hypothyroidism, retinitis pigmentosa, and congenital an apparently competitive manner. The intracellular cAMP night blindness. The mutation may also render the receptors content of serum-free medium in which cells were cultured overactive and this may also lead to diseases, such as Leydig for assessment of receptor activity was also increased by [6] the expression of -ARs, and that increase was reversed cell tumor and familial hypocalciuria. The constitutive 2 activation may also be seen in genetic variations in receptors by timolol and propranolol. The notable observation was (polymorphisms) that alter coupling efficiency. This is typified that DCI, labetalol, and pindolol produced increase in cAMP concentration in the normal cells. However, when the cell by allelic variants of human beta-adrenergic receptors (-ARs). membranes were prepared from Sf-9 cells (these cells are The Ile164Thr allele of the human  -AR or the Arg389Gly allele 2 devoid of GTP), the system became constitutively active due of the human  –ARs show spontaneous activity, which is not 1 to removal of GTP and, under these circumstances, and these seen in naturally stable -ARs.[7] The spontaneous activity so ligands produced the depression of the basal cAMP levels. obtained may result in ligand-induced opening of L-type Ca2+ These two observations show that these beta-blockers are channels. Binding of “inverse agonists” on these constitutively actually inverse agonists.[10-12] active receptors reduces their activity. Inverse agonists Other -blockers, such as bisoprolol, bucindolol and may initiate signal transduction cascade activity different metoprolol, and propranolol, were also shown to have inverse from GTPase activity and cAMP production. Antagonists to agonist activity.[16,17] Studies show that propranolol reduces [cysteine–leukotriene receptor (CysLT) receptors] montelukast basal cAMP accumulation (via  -mediated inverse agonism) and zafirlukast are actually inverse agonists because these 2 but stimulates  -mediated mitogen-activated protein antagonists decrease the basal IP production in cells expressing 2 3 kinase (MAP) activity.[18] MAP activation is known to occur [8] CysLT receptors. with cytokines, growth factors, and adrenergic overactivity. Inverse Agonistic Activity on Various Receptors and Therefore, propranolol can simultaneously act as an inverse its Signifi cance agonist through a Gs-coupled mechanism (reduced cAMP) while stimulating an alternative G-protein-independent mechanism -adrenoceptors [18,19] (MAP kinase activation). In overexpressed human 2 Spontaneous activity is demonstrated in 1-ARs and 2-ARs receptors, bucindolol has a low inverse agonist profile that by several scientists in a variety of models.[9-11] Engelhardt correlates with its minimal reduction in Holter-monitored [9] et al. studied constitutive activity of the human 1-ARs and lowest 24-h heart rate—a clinical measure of -AR inverse

Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 495 Khilnani and Khilnani: Therapeutic implications of inverse agonism agonism.[20] Carvedilol and metoprolol exert different inverse Whereas, phentolamine and idazoxan are reported to be without agonist activity at -ARs. Metoprolol is a strong inverse agonist inverse agonism, that is, are neutral antagonists.[28] This study that reduces basal -AR activity to a higher degree than the shows that some classical 2A-antagonists are actually inverse weak inverse agonist carvedilol. Thus, carvedilol and metoprolol agonists. Medetomidine has 2 enantiomers. Dexmedetomidine have different negative inotropic effects at equivalent levels of behaves as an agonist (it causes Ca++ mobilization and -AR occupation. This is indicated by experiments on isolated depression of forskolin-stimulated cAMP production), whereas muscle strip preparations from human ventricular myocardium. levomedetomidine, a weak agonist, reduces intracellular While at 100% -AR occupation, force of contraction is reduced Ca++ levels and further increases forskolin-stimulated cAMP by 27% with carvedilol and by 89% by metoprolol.[19,20] production and therefore, it can be classified as an inverse This information can be useful in selecting a particular agonist.[25] -blocking agent in cardiovascular disease. -blockers have 5 It has been shown that chronic elevation of second ancillary properties, such as partial agonism, cardioselectivity, messengers produced by constitutive G-protein activity and membrane stabilizing effect, lipophilicity/hydrophility, can lead to cell transformation. The 1-ARs have been and vasodilating actions.[18] To these, the property of inverse shown to be agonist-independent proto-oncogenes.[26,29] agonist effects can be added that may determine the utility of GTPase-inhibiting mutations constitutively activate alpha -blockers’ in cardiovascular disorders. subunits of signal-transducing guanine nucleotide-binding In spontaneously hypertensive rats with cardiac hypertrophy, proteins (G-proteins). Constitutive GPCR activity leading to binding characteristics of -blockers on 1-ARs change in a way chronic elevation of cell metabolism may also have a role in that the inverse agonist activity of metoprolol is reduced.[21] In promoting the growth of tumors.[28,29] Overexpression of specific congestive heart failure(CHF), 1-ARs are reduced in number GPCRs in tumor cells have been reported to have proliferative [22,23] and 2 activity is overexpressed and inverse agonists may properties. Also there is evidence to show that inhibition of be excellent drugs to treat this condition. Inverse agonists these cellular effects can inhibit tumor growth, and therefore enhance baseline atrial tension more than neutral antagonists. -AR inverse agonists may play role as potential antitumor Inverse agonist activity is of importance for -AR regulation agents.[30] Rauwolscine (an inverse agonist) treatment reduced also. In chronic heart failure, -ARs are downregulated due mitotic index and enhanced apoptosis in mouse mammary to chronic sympathetic activation. Inactivation of -ARs by tumor cell lines.[31] inverse agonists inhibits phosphorylation of receptors, and thus Benzodiazepine receptors desensitization and downregulation. Strong inverse agonist, BDZ–GABA receptor complex is a pentameric structure such as metoprolol, but not the weak inverse agonist carvedilol, having 3 different subunits , , and  in different proportions; leads to upregulation of ventricular -AR density.[21,24] the usual subunits being 122. The BDZ–GABA receptor Therefore, different pharmacologic profiles of carvedilol complex is the site of action for several central nervous system and metoprolol lead to different effects on hemodynamics, depressants, such as general anesthetics, benzodiazepines, especially in situations of -adrenergic stimulation. In this , non-BDZ , and antiepileptics. Depending respect, at a submaximal level of stimulation (low-grade on the affinity and efficacy, these agents act as partial/full exercise), carvedilol may have advantages compared with inverse agonists or antagonists. BDZ inverse agonists are metoprolol because cardiac output is increased to a similar the compounds that produce paradoxical anxiety and have degree with a lesser increase of heart rate. This signifies a more proconvulsive effects and can be partial or full inverse economical mechanism to respond to exercise. In contrast, at agonists. RO15-4513 is partial inverse agonist at recombinant a maximal exercise level, metoprolol is advantageous because 1, 2, 3, 5GABAA receptors and produces severe anxiety maximum heart rate and oxygen consumption are improved rather than the sedative effect of the BDZs.[32] Synthetic BDZ by metoprolol to a higher degree than by carvedilol. This may ligand, FG-7142, induces sweating, nausea, palpitations, explain metoprolol-induced improvement in submaximal and tightness in the chest, restlessness, tremors, and feelings of maximal exercise tolerance, whereas carvedilol improves only worry; all markers of increased anxiety. Interestingly, it also [18,19,24] submaximal exercise tolerance in clinical studies. Thus antagonizes several behavioral and neurochemical effects of the role of inverse agonism in cardiovascular hemodynamics ethanol. Similarly, RO19-4603 an inverse agonist, antagonizes facilitates future selection of a -blocker in CHF. effects of ethanol on locomotor behavior and suppresses

2A-Adrenergic receptors (2A-ARs) ethanol intake in selectively bred alcohol-preferring (P) [32] Constitutive activity in 1- and 2-ARs have been shown rats. These exciting discoveries are paving a way for the recently.[25] Isolation of receptors in stable Chinese hamster development of newer analogs for the treatment of chronic ovary cell lines expressing the porcine 2A-ARs in its wild-type alcoholism. Other examples of BDZ-inverse agonists are and constitutively activated mutant (CAM)T373K forms have DMCM hydrochloride (4-Ethyl-6,7-dimethoxy-9H-pyrido[3,4-b] enabled researchers to study inverse agonism at 2A-adrenergic indole-3-carboxylic acid methyl ester hydrochloride) and butyl [25] receptors. Activation of both Gi and Gs was enhanced for b-carboline-3-carboxylate -CCB, both having anxiogenic and CAM receptors. cAMP production was suppressed in cells proconvulsant activity.[32] with CAM 2A-ARs and this suppression was reversed by The 1 subunits are involved in sedation and amnesia, 2 some 2-adrenergic antagonists (indicating that these have subunits subserve anxiolytic and effects and [33] inverse agonist action). The order of inverse efficacy is shown 5 subunits are involved in memory impairment. Therefore, to be rauwolscine > yohimbine > RX821002 > MK912.[25-27] compounds that have different efficacies at the various subtypes would show dissociation of anxiolytic action from

496 Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 Khilnani and Khilnani: Therapeutic implications of inverse agonism sedation. Attempts have been made to dissociate anxiolytic (N, N-diallyl-Tyr-Aib-Aib-Phe-Leu). These developments are effects from sedative, dependence, and proconvulsive actions. used to understand the complex effects of opiates on behavior. Neuroactivity of a number of flavonoids (which act as partial The basal (constitutional) activity at  receptors may play a agonists) is mediated by modulation of GABAA receptor role in opioid dependence. Agonists cause opioid receptor function via BDZ sites. In whole-cell studies internalization and downregulation and treatment with inverse on neuroblastoma IMR-32 cells expressing native GABAA agonists would result in upregulation of opioid receptors. receptors, 6,2-dihydroxyflavone (DHF), and FG-7142 decreased In a study, the intrinsic effects of ligands were tested GABA-induced currents, which could be blocked by flumazenil, a by measuring GTPS binding to cell membranes and cAMP BDZ site antagonist.[34] In mouse behavioral models, DHF elicited levels in intact cells. Some ligands, such as , were significant anxiety-like effects in the elevated plus-maze test and identified as inverse agonists (suppressing basal signal activity). enhanced cognitive performance in the passive avoidance test, Naloxone and were neutral antagonists (not affecting suggesting that they have inverse agonist activity. However, no basal signaling) in untreated cells, but had inverse agonistic proconvulsant effect was shown by DHF. In electrophysiological effects in -pretreated mice. In contrast, 6- and studies on subtypes of recombinant GABAA receptors expressed 6-naltrexol and 6-naloxol, and 6-naltrexamine were neutral in HEK 293T cells (human embryonic kidney cells easily express antagonists regardless of morphine pretreatment. In acute and receptor activity), DHF decreased GABA-induced currents chronic mouse models of morphine-dependence, 6-naltrexol in some pentmeric isoforms of GABA-receptors, such as caused significantly reduced withdrawal jumping compared

132, 232, or 532, but not in 332 and normal 122 to naloxone and naltrexone, at doses effective in blocking receptors. The results demonstrated DHF as a partial inverse morphine antinociception.[38] This supports the hypothesis that agonist-like modulator of GABAA receptors with selectivity in naloxone-induced withdrawal symptoms result at least in part receptor subtypes. The DHF subtype-selectivity suggested that from suppression of basal signaling activity of  receptors in 3-containing subtypes could be a mediator of the convulsive morphine-dependent animals. [34] activities of GABAA receptors. Another implication is Histaminergic receptors (H1;H2;H3) development of anxioselective compounds, which have low All the 3 histaminergic receptors exhibit constitutional potential of withdrawal syndrome after prolonged use. Abrupt activity. Transient expression of the wild-type human histamine termination of the treatment of humans with BDZs leads to H1 receptor in SV40-(simian virus infected) immortalized African a rapid onset of discontinuation syndrome characterized by green monkey kidney cells reveals an agonist-independent anxiety, muscle spasms, and occasionally convulsions. In a elevation of the basal levels of the second messenger inositol study, mice withdrawn from chronic treatment with alprazolam trisphospate, suggestive of constitutional activity. Several showed anxiety, muscle rigidity, and seizures between days 1 histamine H receptor antagonists ( and loratidine) and 28 after termination of the treatment. Replacement of 1 reduced this constitutive activity. Inverse alprazolam with a -carboline selective agonist, abecarnil for agonism, that is, stabilization of an inactive conformation of the 7 days, prevented the occurrence of the signs of dependence.[35] human histamine H1 receptor, may therefore be a key component Opioid receptors of the antiallergic of clinically used Constitutive activity at  receptors is shown by various . The older antihistaminic, , was also workers in different in vitro models and chronic exposure to found to reduce basal constitutional activity of H1 receptors. The morphine enhances this constitutional activation at  receptors. effect of mepyramine on ATP-induced signaling was specifically

It is hypothesized that this constitutional activation is important neutralized by Gall overexpression, indicating that mepyramine in the development of by morphine.[28] is able to reduce G-protein availability.[39] Furthermore,

Naloxone has been shown to act as an inverse agonist at the the cardiotoxic and anti-inflammatory effects of certain H1  receptors, stimulating cAMP levels and inhibiting GTPS antihistaminics, such as inhibition of ICAM-1 (intercellular binding in morphine-pretreated, but not untreated, tissues.[36] adhesion molecule-1) expression and the reducing effects of Opioid dependence can be viewed as an overexpression of the bradykinin are attributed to the inverse agonist action.[40] Since active receptors and withdrawal as an abrupt change from the all H1-antihistamines examined to date have shown inverse [36,37] active to the inactive state. agonists, it is suggested that the term “H1-receptor antagonists” [41,42] Gi/o-coupled -opioid receptors were the first GPCRs to be be replaced by “H1-antihistamines.” [1] described as having constitutive activity. Ligand-independent Constitutional activity is also demonstrated at H2 35 activity of  receptors is measured by [ S]GTPS binding assay receptors. Many H2 receptor antagonists, such as , (An assay in which G-protein activation is assessed with [35S] , tiotidine, and , described previously as guanosine-5-O-(3-thio)triphosphate (GTPS) binding). Such pure H2 antagonists, actually behave as inverse agonists and ligand-independent activity of this receptor was shown in diminish basal cAMP levels. By using transfected Chinese mouse neuroblastoma-rat glioma hybrid cells (NG108-15 hamster ovarian (CHO) cells expressing different densities of cells), which endogenously express the murine -opioid wild-type H2 receptors or uncoupled H2 (Leu124Ala) receptors, [37] receptor. The constitutional activity of these  receptors can considerable agonist-independent H2 receptor activity was be selectively manipulated by inverse agonists. Treatment with found.[43] Ranitidine and cimetidine acted as inverse agonists inverse agonists upregulates the surface  receptors. There are (both induced H2 receptor upregulation), whereas burimamide two types of inverse agonists acting on  receptors. Alkaloid was shown to be a neutral antagonist. The inverse agonism inverse agonists (RTI-5989-1, RTI-5989-23, RTI-5989-25) is specific at H2 receptors because no change in receptor are more potent than the peptide inverse agonist ICI-174864 density was observed after H1 or H3 antagonist treatment or

Indian Journal of Pharmacology | October 2011 | Vol 43 | Issue 5 497 Khilnani and Khilnani: Therapeutic implications of inverse agonism after incubation with the structural analog of cimetidine, VUF antidyskinetic activity against MPTP (1-methyl-4 phenyl-1,2,3,6, [44] 8299, which has no H2 antagonistic effects. The displayed tetrahydropyridine) primate model of extrapyramidal disorder, inverse agonism of H2 antagonists offer a plausible explanation suggesting that this compound may be an efficacious for the observed development of tolerance after prolonged antipsychotic and antidyskinesia agent.[50] Pimavanserin [45] clinical use. (ACP-103), a highly selective 5-HT2A inverse agonist, which Similarly, constitutive activity is also demonstrated in 2 attenuates psychosis in patients with Parkinson’s disease with isoforms of H3 receptors in rodent brain. H3 receptors control psychosis and is devoid of worsening of motor and nonmotor [44,46] [51] histaminergic neuron activity in vivo. , a H3 inverse side effects. In addition, APD125, a selective serotonin agonist/antagonist is shown to modulate striatal 5-HT2A receptor inverse agonist, significantly improves sleep activity by this mechanism.[46] Other studies have shown that maintenance in primary .[52] intraperitoneal injections of an imidazole-based receptors (CB ;CB ) H receptor inverse agonist, enhances histamine release in the 1 2 3 Many cannabinoid effects are now known to be mediated by brain and potentiates cocaine-induced locomotion hyperactivity CB receptors that are present in the central nervous system as [47] 1 in mice by this action. well as in certain neuronal and nonneuronal peripheral tissues 5-Hydroxytryptamine receptors (5HT , 5HT , 5HT 5HT ) 1 2 6, 7 or by CB2 receptors that are found mainly in cells of the immune The heterogeneity of 5-HT receptors is well known. system. Both these receptors are GPCRs and thus exhibit These receptors play diverse roles. Receptor Selection and significant constitutional activity. SR141716A is a selective Amplification Technology (RSAT) is used to study of the 5-HT 2 CB1 receptor inverse agonist and SR144528 has selective CB2 subclass of serotonin receptors. RSAT is a phenotypic assay of [53] receptor inverse agonist activity. AM630 is a CB2-selective receptor function that involves the heterologous expression of inverse agonist and has a weak at CB1 receptors. receptors in mammalian fibroblasts. Using this technology, it is [53] L759633 and L759656 are both potent CB2-selective agonists. demonstrated that native 5-HT2A receptors possess significant There are endogenous ligands derived from membrane [48-50] constitutive, or agonist-independent, receptor activity. The [54] lipids, which bind with CB1 and CB2 receptors. Hemopressin activating mutations in 5-HT2A receptors may be causative is such a peptide ligand that acts as an antagonist as well as or predisposing to neuropsychiatric disease, or modulate inverse agonist. This agent acts as an antinociceptive in different [48] response to treatment with antipsychotics. The creation in vivo models of pain. This has opened new avenues for newer [54] of a transgenic mouse model that exhibits increased 5-HT2A analgesic agents. The central cannabinoid (CB1) receptors play receptor activity may provide an excellent model of preclinical a role in controlling food consumption and are therefore targets evaluation of antipsychotic drugs having 5-HT receptor [55] 2A for evaluation of antiobesity medications. Rimonaband, an activity. Many compounds used in psychiatry are found to be antagonist at CB receptors and taranaband, a highly selective [48] 1 potent 5-HT2A inverse agonists. Thus 5-HT2A receptor inverse inverse agonist at CB1 receptors were developed for their use in agonism may be the molecular mechanism of antipsychotic obesity. Unfortunately both these agents have been withdrawn efficacy in humans. 5-HT system also plays a role in learning due to serious psychiatric manifestations. AM251, an inverse [49] and memory. Meneses reported that post-training injection agonist at CB receptors, has and anorectic action of SB-224289 (5-HT receptor inverse agonist) facilitated 1 1B in mice. The tail-suspension test (TST) and forced-swim test learning and this effect was partially reversed by GR127935 (FST) are used as tests sensitive to antidepressant compounds. (a 5-HT / ), but not by MDL100907 1B 1D AM251 significantly reduced immobility at 10 mg/kg in the (a 5-HT receptor antagonist) or (a 5-HT 2A 1D/2A/7 TST and at 1 and 10 mg/kg in the FST. In addition, it reduced receptor antagonist) at low doses. Hence, 5-HT receptor 1B fasting-induced hyperphagia in mice.[55] These observations inverse agonists or antagonists could represent drugs for the suggest that it has significant antidepressant and anorectic treatment of learning and memory dysfunctions.[49] In order action. CB receptors have immunoregulatory role. An inverse to determine the possible relationship between antipsychotic 2 agonist at CB receptors, Sch414319 is a potent modulator of drug properties and inverse agonist activity at 5HT and 5HT 2 6 7 immune cell mobility in vivo.[56] receptors, constitutively activated forms of these receptors were created by site-specific mutagenesis. Typical and atypical Angiotensin receptors (AT1) antipsychotic drugs were assayed for their potencies as Constitutional activation is demonstrated at AT1 receptors. inverse agonists at these mutated receptors.[49] The inverse Losartan, candesartan, and olmesartan have been shown to agonist mechanism-of-action of the possess inverse agonist effects in assays evaluating c-fos gene expression and phosphorylated extracellular signal-regulated drugs showed greater inverse agonism at the 5HT7 receptors as compared to typical agents.[50] protein kinases.[57] Mechanical stretching of cardiac cells

An important implication of inverse agonism at serotonin activates the AT1 receptors without the involvement of receptors is development of compounds which have angiotensin II and that is inhibited by the inverse agonistic antipsychotic and antiparkinsonian activities. Selective activity of candesartan and olmesartan. It is conceptually novel that AT receptor directly mediates mechanical stress-induced 5HT2A/ receptor inverse agonists have therapeutic potential 1 for neurodegenerative diseases. These demonstrate in vivo cellular responses, and inverse-agonist activity of AT1-receptor efficacy in models of psychosis and dyskinesias. This includes blockers (ARBs), such as candesartan, emerges as an important activity in reversing MK-801 (dizocilpine, also known as MK-801, pharmacological parameter that determines its efficacy in is a noncompetitive antagonist of the N-methyl-D-aspartate preventing organ damage (stretch-induced hypertrophy) in receptors)-induced locomotor dysfunction and also has cardiovascular diseases.[58]

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- (-AR) -blockers are presently contraindicated for asthmatics,

2-Adrenergic agonists are sheet anchor in therapy of but might actually be able to prevent serious attacks when bronchial asthma. Short-acting agents, such as salbutamol used judiciously on long-term basis.[63] Bond and colleagues and terbuline, are used by inhalation to terminate an acute proposed that duration of therapy might be a key determinant attack. Long-acting 2-adrenergic agonists (LABAs) are also of whether -adrenoceptor ligands have a positive or negative given by inhalation to prevent recurrent bronchospasm. There effect. They suggested that beta-blockers, which have been have been concerns about increased risk of asthma-related shown to be harmful to asthmatics in the short-term by deaths with the use of LABAs.[59] Specific label warnings have heightening airway constriction, might actually have beneficial been recommended by the Food and Drug Administration effects when administered on long-term basis. To investigate recently indicating judicious use of these agents. In addition, this hypothesis, airway sensitivity to methacholine, which elicits chronic administration of 2-adrenergic agonists has been airway narrowing, is commonly used as provocative test. It was demonstrated to lead to reduced responsiveness resulting monitored in mouse models of asthma after chronic (daily dosing from desensitization, sequestration, and downregulation of over 28 days) and acute (single dose 15 min before methacholine) [60] receptors. This consequence may be an observed loss of administration of a range of 2-ligands; salbutamol (a partial responsiveness seen in asthmatic patients on long-acting 2-agonist), alprenolol (a -antagonist with partial 2-agonist

-agonists leading to tolerance or . Furthermore, activity), carvedilol and nadolol (both 1/2-antagonists there is also an increased hyper-responsiveness of the with inverse agonist activity at the 2-adrenoceptor). Acute pulmonary airways in response to provocative allergens. One administration of salbutamol decreased airway responsiveness of the reasons for this is the proinflammatory activity of LABAs. to methacholine compared with nontreated asthmatic mice, The proinflammatory role of LABAs is attributed to activation whereas chronic dosing had no effect. Similar results were of inflammatory cells, such as type-2 T cells which exhibit observed for alprenolol, indicating that it was behaving as a -agonist. Carvedilol and nadolol, on the other hand, 2AR-agonist-promoted proinflammatory properties and/or 2 to activation of alternative signaling pathways independent although acutely detrimental, significantly reduced airway of cAMP activation.[60] This proinflammatory activity is also sensitivity after chronic administration. The authors then used [61] radioligand-binding assays to examine -adrenoceptor density attributed to the constitutive activity of 2ARs. Nguyen et al.  in mouse lungs. They found the density of -adrenoceptors proposed that 2ARs activation, resulting from constitutive 2AR  activity, endogenous catecholamines, or inhaled -agonists, to be significantly lower in asthmatic mice compared with has a positive effect on airway inflammation that generates controls. They also reported that the capacity to reduce airway intermediates, which cause airway smooth muscle contraction responsiveness in asthmatic mice correlated with a ligand’s and promotes airway mucus secretion. ability to increase -adrenoceptor density. Salbutamol and Inverse agonistic activity is demonstrated in some alprenolol increased -adrenoceptor density in the lungs after beta-receptor antagonists, such as nadolol.[10] It is hypothesized an acute dose but had no effect after chronic administration, that inverse agonism may reduce airway inflammation. whereas carvedilol and nadolol had no acute effect on receptor density, but significantly increased -adrenoceptor levels with 2AR inverse agonists by inhibiting 2AR activity may reduce allergic inflammation and nullify or modestly inhibit the chronic dosing. In view of these findings the -adrenoceptor blockers with inverse agonist activity are attractive long-term bronchoprotective/relaxing effect of 2AR activity in bronchial smooth musculature.[61-63] therapeutic options in chronic airway obstruction. There are Chronic administration of cardioselective  inverse agonists theoretical advantages in the use. There will not be tachyphylaxis do not change pulmonary function in patients with congective on long-term use and improvement in signal mechanism will heart failure (CHF) and chronic obstructive pulmonary disease counteract the inflammation underlying the airway narrowing. At [62] present it remains to be known as to how and when a -blocker (COPD) or asthma. Forced expiratory volume (FEV1), a standard measure of pulmonary function, was unchanged in patients be started in asthma.[63,65] treated with cardioselective  inverse agonists. Therefore, Dopaminergic receptors (D1, D2, D3, D4) chronic administration of cardioselective  inverse agonists are Constitutional activation is demonstrated in dopaminergic safe in patients with CHF and pulmonary airway disease. Chronic receptors. The antipsychotic drugs are conventionally thought to administration of an inverse agonist has the effect of upregulating act as antagonists at D2 receptors but recently these the population of active -adrenergic receptors.[62-64] drugs have been shown to possess inverse agonist properties Nguyen et al. have recently studied the effects of at this receptor.[66] Interestingly, several compounds previously beta-blockers—ICI 118,551 and nadolol (full inverse agonists classified as D2 receptor antagonists behaved as inverse agonists at -ARs)—on airway inflammation in rats and showed 35 2 at the D3 receptor also, that is, they inhibited the basal [ S] GTPS that chronic administration of beta-blockers decreased binding in a dose-dependent fashion.[66] was full airway hyper-responsiveness in a murine model of asthma. inverse agonist but clozapine was a partial inverse agonist.[66] In NG Administration of beta-blockers in a murine model of 108-15 cells but not in wild-type cells, haloperidol, , allergic asthma resulted in a reduction in total cell counts, and various other antipsychotics inhibited basal [3H] thymidine eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-1 incorporation in a concentration-dependent manner. In contrast, in bronchoalveolar lavage, and attenuated epithelial mucin other dopamine antagonists, such as nafadotride, a D3-preferring content and morphologic changes. These results indicate antagonist, was without effect.[67] The concentration–response that in a murine model of asthma, chronic administration of curve of haloperidol was shifted to the right in the presence of beta-blockers reduce inflammation and mucous production.[61] nafadotride. These data indicate that some dopamine antagonists

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