Inverse agonism at G protein‐coupled receptors: (patho)physiological relevance and implications for drug discovery

The largest family of cell surface receptors involved in signal transduction, G protein coupled receptors (GPCRs), are one of the major targets for current drugs as well as new drug development. Ligands interacting with for e.g. adrenergic, histamine, adenosine, opioid, dopamine or serotonin receptors, constitute a large portion of currently used therapeutics. A common property of GPCRs is that upon activation (agonist binding) they transmit signals across the plasma membrane via an interaction with heterotrimeric G proteins (Stadel et al., 1997). The corresponding activated G protein subsequently interacts with an intracellular effector system, such as adenylate cyclase or phospholipase C, leading to a wide variety of distinct physiological responses. Recent evidence suggests that GPCRs have the potential to be ‘active’ even in the absence of an agonist. This exhibition of spontaneous receptor activity has led to the observation that various ligands, previously considered as antagonists with no intrinsic activity, actually can inhibit this spontaneous activity, appearing to possess ‘negative intrinsic activity’. This phenomenon has been termed inverse agonism and the corresponding ligands are referred to as inverse agonists. Although intrinsic constitutive receptor activity and inverse agonism have unequivocally been demonstrated in vitro, (patho)physiological consequences are far from self-evident. Thus, in this review we should like to focus on the expression of inverse agonism under more ‘physiological conditions’, since it appears timely to address the physiological relevance and consequences of this new concept, both in GPCR research and drug discovery. Traditional receptor theory has postulated on a single, ‘quiescent’ receptor state to which agonists bind inducing a conformational change of the receptor to an activated and ‘functional’ state. This view, initially formed in the early 1950s, was more clearly expressed by Del Castillo replacement of four amino acids of the third intracellular by the corresponding of the led to agonist-independent activation of adenylate by et al. had demonstrated that of in the third intracellular of the by the corresponding of the led to receptors that were coupled to like the of adenylate This evidence that the third intracellular is for G protein binding and activation. Since expression of receptors not always in constitutive activity, studies have been in various systems overexpression of the receptor has been of are the expression of in cells leading to receptor to protein et al., or the overexpression of the receptor in cells et al., 1997). In the latter two cell were and the first cell an increase in basal the was not constitutively receptor is to spontaneous activity and various of GPCRs can constitutive activity upon of the G protein involved may to basal of of with various receptor in cells in basal activity of the receptors et al., 1997). This constitutive activity of the receptors was by the antagonists that they as inverse agonists. Thus, of G protein of the active of the receptor the of receptors that are coupled to the G protein and a more for the of inverse agonism. These even in vivo, since GPCRs of constitutively active et al. the various receptor in was over the ligand as an inverse agonist, The effect of was with that it was a from the effect of was in vivo, was in both and in by an effect that was with a both in and the no effects on the CAM has been in et al., 1997). In this the was not from due to the overexpression with CAM resulting in basal and et al. the effects of of various ligands on in with overexpression to overexpression in the by et al. In inverse agonists such as and or were was not by any ligand in the of This the effects of a neutral and inverse agonists the of receptor in the of inverse agonism. Thus, constitutive receptor activity has been demonstrated for GPCRs some form of We will review of studies that may have a more to in pharmacology and These studies cell systems with of receptor expression and or The issue of inverse agonists will be have inverse agonism and constitutive activity on receptors expressed in cell but more or ‘physiological’ of of such studies are in and The receptor in cells was by et al. This receptor had basal activity, as by an increase in basal Although this basal was further by the agonist histamine, the and were to basal inverse agonism in this on the not basal but was to both the increase and the of basal Hence, as a neutral were for the receptor et al., this receptor as a agonist, basal by to the by et al. (1997) the of the serotonin receptor expressed in cells a receptor of of binding in a membrane was used to between the various ligands was as a agonist binding to the same as serotonin was as an inverse agonist since it basal binding by as a neutral It no effect on basal binding by but was to both and of basal The effect of could not be by a of not only had the membranes been but basal activity had been due to receptor activation by the antagonist should have this activation. The of both of the and receptor was by et al. either the or the receptor, higher basal activity to In both cell a agonist, further basal activity. The basal activity in cells the receptor, thus as an inverse agonist for this receptor et al., 1997). effects opposite to agonists in a of a in activity by an as an inverse agonist in two signal and adenylate cyclase were with a ligand that as an inverse agonist, decreasing both basal activity and binding on cells the receptor et al., et al. reported on inverse agonist for the receptor, which basal binding, in contrast to the agonist Inverse effects were various dopamine receptor subtypes. & reported basal receptor activity of the dopamine and receptor, the receptor to higher intracellular basal to the receptor. were for the corresponding dopamine and were to basal the effect of as inverse was more the receptor, since the basal was et al. that various in NG108-15 cells the dopamine receptor. Since dopamine agonists the and as inverse agonists. a had no effect of its on as a neutral The two of the receptor to as and adenylate in one of is to phospholipase to et al. (1997) constitutive receptor activity of and receptors expressed in cells were constitutively active as by an increase in basal to a the receptor not show an increase in basal spontaneous activity of this receptor was more readily for activation of adenylate of a receptor agonist, the not an The of ligands a further of inverse agonism. A is the receptor, a by et al. (1998). This receptor was expressed in and cells receptor of These receptor are in the same as the receptor in cells that the receptor. It was that basal binding in the of a receptor agonist. the basal binding, thereby as an inverse agonist. It that physiological receptor and expressed in cell the receptor is constitutively A for the physiological relevance of inverse is its in experimental conditions that are as to physiological as to inverse agonism in are in most cases to are genetically as Thus, from potential more or ‘physiological may data from cell the receptor of as for the previously NG108-15 cells δ opioid receptors & Herz or from such as or latter may data of receptors physiological or of such studies in which potential inverse agonism was are in & a in G protein activation by antagonists of the receptor in this membrane by to receptors protein, of both basal and binding by was The of was in this by of the membranes with or and both et al. inverse agonism in the receptor. and both basal as well as an effect that was opposite to that of agonists. were for of the receptors, and in a in cells with the receptor inverse agonism in cells the et al. used two of various ligands on both intracellular of and were The ligands used were thus from agonists to inverse agonists. of intrinsic showing that signalling can be used to and ligands as inverse agonists. in studies were the opposite effects of the of as a agonist, and decreasing as an inverse agonist by decreasing and have been to inverse agonism in such ‘physiological The expressed in cells and expressed in inverse effects of in binding et al., adenylate cyclase activity via the expressed in both and a thus as inverse agonists & A cell from was used in the two et al. (1993) on both and as a to the effects of the antagonists and The were first with to the cells more to changes of of both antagonists led to a in in to a that could be readily in changes in that effects were not due to a potential with agonist but to inverse activity of et al. (1993) a but from two and They by increase basal was the with cells of in a of this basal of had the opposite an increase of A effect of was in the absence of This effect was and with not the of since basal was high to the effects by the agonist and the inverse agonist as an inverse agonist on in cells with from et al., have been used to inverse agonism et al., of were and the effects of antagonists on the of the by or were and not only the increase in by but the to in for was within the concept of inverse it was suggested that both and as inverse decreasing the of the receptor in the active state Moreover, both the increase in in the absence of agonist, a model for inverse agonism in receptor showing inverse agonism under ‘physiological are the receptor and the δ opioid receptor. antagonists basal in cells the receptor, of basal et al., was to behave as an inverse agonist the δ opioid receptor, expressed in NG108-15 in both GTPase activity and binding & & 1997). It may be from the above that even in or cell inverse agonism may be readily This led to studies that show evidence of some form of inverse agonism the concept of a two-state receptor model ligand may be as the affinity of the ligand for the two conformational states with agonists higher affinity for the ‘active’ and inverse agonists for the et al., This affinity may be in binding studies with the of of the receptor state. For in receptor systems it is that in the the G protein from the receptor leading to a affinity state of the receptor for agonists & De Lean et al., et Thus, have been in some GPCR as a to from agonists due to the affinity of ligands for the two receptor states et al., et al., model the two conformational states of a receptor and active and their for of this concept to antagonists that are to between the free and G protein-bound form of the receptor may and even with, the of inverse intrinsic of Although a of on antagonist binding, which is inverse to the of on agonist binding, has been previously suggested et al., et al., we should like to its and that to have of of binding of antagonists have been reported in are in affinity and of receptor antagonists and on and membranes, & et al., were in the of a of antagonists for the receptor state. A increase in binding was with two on membranes et al., and chloride on membranes et al., increase in binding of antagonists has been in of receptor and G Costa & Herz (1989) were more the first to show a in binding) upon in the of the δ opioid receptor antagonist The absence of any of antagonist a potential of the negative intrinsic activity of with its affinity for the form of the receptor. interaction of with GPCRs is to in a of the affinity of receptors et al., It is believed that this effect of is to an in in GPCRs This effect of has been to be with intrinsic activity of ligands & the largest in binding for agonists. in cases inhibit agonist binding, their effect on binding has been from no effect to an increase in binding & & et al., Although no was for this of effect the it is to that the inverse agonist of some may be for the of they Hence, upon of this concept may a new on the effects of The increase in binding of some antagonists in the of such as to the opioid receptor & to the δ opioid receptor et al., to the et al., or to the receptor et al., may be of the inverse of The that has been to as an inverse agonist in a et al., is further support for this one may that from binding of ligands, the effect of on the equilibrium is apparent in the of basal of binding & or GTPase activity et al., Costa et al., in in This is not only to the effect of inverse agonists on of basal receptor but the of constitutively active receptors in Thus, the of in binding may to inverse their absence the ability of the to inverse agonism. In are many of receptor systems a This has been to the of the agonist of the receptor involved that interacts a with its Interestingly, under the new by the of the two-state receptor in equilibrium, it is to constitutive activity of the receptor as the potential of of in the of agonists. an the receptor in as an inverse agonist receptors et al., et al., et al., in contrast to agonists in this that by inhibiting this et al., 1997). The same in with receptor the receptor activity et al., for an to such an effect some receptors be Since was to the absence of agonists in this experimental the could only be to constitutive receptor activity and its only to with inverse agonist is the by receptor antagonists and of spontaneous of in et al., It is to whether or not spontaneous are due to constitutive receptor activity and inverse agonists should be over antagonists in such a It should be in this that the of in and hence the with the receptor. are in a of is et al., The potential of or inverse agonists would be of evidence in favour of the physiological relevance of inverse agonism. Although not without protein is considered an inverse agonist. This protein of amino acids and is by the et al. were the first to that protein binding to its receptor, currently referred to as the receptor, with high affinity in the et al. (1997) this observation and the between protein, and on cells that the receptor. in one of the was by protein cell thus as an agonist. of the by and receptor were was not only to basal of but In contrast to the cell protein had effects opposite to as an inverse agonist. the effects of and protein on receptor were both ligands the receptor over a time and to a The two for protein could be interacting a in the signalling a receptor, or protein may be as an inverse agonist on the receptor. this were the protein would with a of constitutive activation of the receptor. constitutive activity of this receptor was not clearly in either preventing an of inverse agonism. the that protein is an inverse agonist on the receptor be is a form of a from the of the It as an inverse agonist on the receptor et al., basal in which the receptor. could be demonstrated in not as a antagonist preventing from binding to the receptor. the inverse effect of was one the signalling In receptor activation in the of of this the concept that inverse agonism has physiological of by via receptors has not been In is an inverse agonist from an on a receptor of This could the for as inverse agonists. The opposite has been protein is an as an inverse agonist on a receptor. was to inhibit the basal of a GPCR within the of et al., This GPCR with receptors and is referred to as et al., receptors are the of activation of receptors in on which is constitutively as an inverse agonist, decreasing basal by is a receptor agonist, but the ability to basal signalling of et al. various receptor ligands, such as and that and as inverse agonists on like is by the and as an antagonist on Thus, ligands or have been as inverse agonists not always on receptors in the same for or for Since the of the concept of inverse of and new in an agonist has Although it is to to to inverse we are more that such a of not or it is is an of a neutral antagonist would be for a ligand with the same affinity for the active and receptor conformation, the it is to on inverse with various of and affinity for two receptor Thus, we should that is only one of ligands whether they are inverse or for we will to the and It is that both of and may for the two antagonists and inverse especially in it is in drug to clearly that may as either antagonists or inverse agonists. In some cases effects may be by with an in an inverse agonist be more We will some that may have relevance or for in drug discovery. It has been for various receptor that with inverse agonists is with of the receptor The increase in receptor may be with drug and For the and which have been to be inverse receptor et al., of the receptor involved could for effects and of the upon of the In such a with a neutral antagonist with an inverse agonist should be considered a more of by was in studies with receptors over cells versus et al., Moreover, a between the of of the receptor and effects has not been found relevance of receptor has not been reported of upon antagonist has been reported on many In one of the studies the receptor on membranes from of was of drug & over the agonists and receptor to a which can intrinsic activity a receptor but to a of from with is to in effects such as and and These effects be with the increase in receptor Since the to an increase of receptors by it was suggested that of would not to the effect of drug on receptor in studies with experimental For et al. reported of by in and of from such studies to the is and should be with of receptors has been of opioid receptors in in et al., has been used to and the of and et al., effects were in in both and In this but it an This especially was as an change from the active to the receptor conformation. In this to is a of the of the active state of the receptor. with inverse agonists have effects on receptors as The G protein involved or receptors the same signalling as the receptor could be et al. between receptors via proteins. not only as an inverse agonist on the receptor it activation in to or by inhibiting activity. protein upon with which was by and further of the cells to the agonist for the of an protein in which the protein is and thus for receptors, was & Milligan however, not of protein upon with inverse agonists for the expressed in NG108-15 of G protein and were by with inverse agonists. the of on G protein not be a common of inverse agonists. et al. reported on the of the serotonin receptor. In the receptor a expression with the inverse agonist led to an of the receptor to agonists This was as an increase in by the agonist Interestingly, was by via receptors These are to the between receptors by et al. In effects may be a of ligand receptor leading to constitutively active receptors are a in Inverse agonists may be since they would the high basal activity by the antagonists would have no These have been reviewed by we will one The and in the receptor for and have been found in with mutant receptors, expressed in an increase in basal but to a they had apparent binding for the ligands and that were two higher those of the receptor et al., et al. used two mutant receptors to for ligands as inverse agonists. and phenomenon is the of This is a of due to the of to et al. demonstrated that as a the receptor in in These constitutive activity the receptor upon expression in and agonist This may that is a physiological for Inverse agonists may an in the of et al. an a corresponding to the of the for this were from and their effects on were A was used to the of by a agonist, to the increase in by a agonist. of the to the to a as This effect to be by and was of the neutral antagonist not and on the a increase was observed. an inverse agonist, the effect of the decreasing The were in the of the two-state receptor model based on the that the an which is only the active of the receptor would the equilibrium the of the receptor the by the the is no to bind and the receptor and an change in is not observed. on the not change the equilibrium between and in this concept. Since some of the receptors are in the conformation, the can the on the receptor and an increase of It has been reported that the of GPCRs are involved in the of are in These are believed to behave in a to the the the receptor, drug with inverse agonists could be more with neutral Moreover, can be used as a research to inverse agonists from neutral have GPCRs with ligand. to be in high The concept of inverse agonism new in such A in which a of spontaneous activity is could not only but with opposite effect In a without constitutive activity, the latter ligands would not be inverse agonism may thus be more on the of the receptor, one of the can be further to new therapeutics. inverse agonists are in such an the may as for further and the discovery of e.g. more or The physiological relevance of inverse agonism is not only but is a and a in drug it a new in drug research and especially in drug since it is that new for inverse agonists versus those for neutral antagonists be for and The support from the agonism. for drug We in this and Their and of this were to support from the in the interaction between receptors and inverse agonists and