Allosteric agonist modulators

This article discusses a model to describe the effects of molecules that bind to a site on the receptor separate from that of the endogenous agonist to actively produce receptor signals (direct agonism). In addition, these molecules also modify the biological responses of the endogenous agonist (either potentiation or antagonism). The effects of such compounds in high-throughput screening assays are described as well as their effects on the dose-response curves to conventional agonists.     

Kappa agonist CovX-Bodies

Small peptidic kappa agonists were covalently linked to the reactive lysine of the CovX antibody to create compounds having potent activity at the kappa receptor with greatly extended half-life when compared to the parent peptide as exemplified by compound 20.     

A novel intestinal-restricted FXR agonist

In this study, a new intestinal-restricted FXR agonist named fexaramine-3 (Fex-3) was developed and investigated both in vitro and in vivo. Fex-3 could selectively activate intestinal FXR and promote the expression of BSEP and SHP while suppressing CYP7A1 which is involved in bile acids syntheses better than the reported intestinal-restricted FXR agonist fexaramine (Fex). We demonstrated that Fex-3 targeted on FXR in ileum and has better selectivity than Fex. And the study of utilizing Fex-3 to reduce obesity was undergoing.     

Spinal analgesia: Comparison of the mu agonist morphine and the kappa agonist ethylketazocine

The sites of analgesic action of the mu agonist morphine and the purported kappa agonist ethylketazocine (EKC) were compared. Using local drug injections and parenteral administration of drugs to spinalized rats, our data support a predominantly spinal site of action for EKC and a major supraspinal action for morphine in antinociceptive tests. This spinal analgesic action of EKC was dose dependent and naloxone reversible indicating opiate receptor involvement. The possibility that EKC activates a spinal kappa receptor population is under further study.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Combination dopamine agonist and prostaglandin agonist treatment of cystic endometrial hyperplasia-pyometra complex in the bitch

Cystic endometrial hyperplasia-pyometra (CEH-P) complex is a progesterone-dependent disease that requires medical treatment in bitches intended for breeding. To test the efficacy and safety of a combined protocol and to assess the effect of age, stage of cycle, previous steroid hormone administration and parity on treatment, 29 bitches diagnosed with CEH-P complex were treated daily with cabergoline 5 microg/kg PO and cloprostenol 1 microg/kg SC for 7-14 days, along with supportive antibiotic and hydration therapies. Before treatment, and on Days 3, 7 and 14, all bitches were evaluated clinically and uterine horn diameter measured during trans-abdominal ultrasonography. Twenty-four of 29 bitches were cured by either Day 7 or 14. Nine bitches had mild digestive side effects. Clinical signs related to pyometra began to improve markedly as early as Day 2 of treatment. Uterine diameters decreased (P < 0.05) by Day 3 of treatment, and continued to gradually decrease, reaching normal size by Day 14. Relapses occurred in 6 of 29 cases. Pregnancy was achieved in one of the two young bitches bred after treatment. No significant relationships were found between success rate and age, stage of the estrous cycle, previous hormone administration or parity. Although no variables affecting treatment results could be identified, this combination of compounds was found to be an efficient and safe for treatment of CEH-P.     

Design of a partial PPARdelta agonist

Structure based ligand design was used in order to design a partial agonist for the PPARdelta receptor. The maximum activation in the transactivation assay was reduced from 87% to 39%. The crystal structure of the ligand binding domain of the PPARdelta receptor in complex with compound 2 was determined in order to understand the structural changes which gave rise to the decrease in maximum activation.     

Minor structural modifications convert a selective PPARalpha agonist into a potent, highly selective PPARdelta agonist

We report the solid-phase synthesis and pharmacological evaluation of a new series of small-molecule agonists of the human peroxisome proliferator-activated receptor delta (PPARdelta) based on a lead structure from our PPARalpha program. Compound 33 showed good pharmacokinetics.     

Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist

The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a "steric doorstop" holding the protein in an open and active conformation.     

The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist

Meta III is formed during the decay of rhodopsin's active receptor state at neutral to alkaline pH by thermal isomerization of the retinal Schiff base C15=N bond, converting the ligand from all-trans 15-anti to all-trans 15-syn. The thereby induced change of ligand geometry switches the receptor to an inactive conformation, such that the decay pathway to Meta III contributes to the deactivation of the signaling state at higher pH values. We have examined the conformation of Meta III over a wider pH range and found that Meta III exists in a pH-dependent conformational equilibrium between this inactive conformation at neutral to alkaline pH and an active conformation similar to that of Meta II, which, however, is assumed at very acidic pH only. The apparent pKa of this transition is around 5.1 and thus several units lower than that of the Meta I/Meta II photoproduct equilibrium with its all-trans 15-anti ligand, but still about 1 unit higher than that of the opsin conformational equilibrium in the absence of ligand. The all-trans-15-syn-retinal chromophore is therefore not an inverse agonist like 11-cis- or 9-cis-retinal, which lock the receptor in an inactive conformation, but a classical partial agonist, which is capable of activating the receptor, yet with an efficiency considerably lower than the full agonist all-trans 15-anti. As the Meta III chromophore differs structurally from this full agonist only in the isomeric state of the C15=N bond, this ligand represents an excellent model system to study principal mechanisms of partial agonism which are helpful to understand the partial agonist behavior of other ligands.     

Quantifying agonist activity at G protein-coupled receptors

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors. Receptors behave as quantal switches that alternate between active and inactive states (Figure 1). The active state interacts with specific G proteins or other signaling partners. In the absence of ligands, the inactive state predominates. The binding of agonist increases the probability that the receptor will switch into the active state because its affinity constant for the active state (K(b)) is much greater than that for the inactive state (K(a)). The summation of the random outputs of all of the receptors in the population yields a constant level of receptor activation in time. The reciprocal of the concentration of agonist eliciting half-maximal receptor activation is equivalent to the observed affinity constant (K(obs)), and the fraction of agonist-receptor complexes in the active state is defined as efficacy (ε) (Figure 2). Methods for analyzing the downstream responses of GPCRs have been developed that enable the estimation of the K(obs) and relative efficacy of an agonist. In this report, we show how to modify this analysis to estimate the agonist K(b) value relative to that of another agonist. For assays that exhibit constitutive activity, we show how to estimate K(b) in absolute units of M(-1). Our method of analyzing agonist concentration-response curves consists of global nonlinear regression using the operational model. We describe a procedure using the software application, Prism (GraphPad Software, Inc., San Diego, CA). The analysis yields an estimate of the product of K(obs) and a parameter proportional to efficacy (τ). The estimate of τK(obs) of one agonist, divided by that of another, is a relative measure of K(b) (RA(i)). For any receptor exhibiting constitutive activity, it is possible to estimate a parameter proportional to the efficacy of the free receptor complex (τ(sys)). In this case, the K(b) value of an agonist is equivalent to τK(obs)/τ(sys). Our method is useful for determining the selectivity of an agonist for receptor subtypes and for quantifying agonist-receptor signaling through different G proteins.     

ICV NPY Y1 receptor agonist but not Y5 agonist induces torpor-like hypothermia in cold-acclimated Siberian hamsters

The reduced metabolism derived from daily torpor enables numerous small mammals, including Siberian hamsters, to survive periods of energetic challenge. Little is known of the neural mechanisms underlying the initiation and expression of torpor. Hypothalamic neuropeptide Y (NPY) contributes to surviving energetic challenges by both increasing food ingestion and reducing metabolic expenditure. Intracerebroventricular injections of NPY in cold-acclimated Siberian hamsters induce torpor-like hypothermia comparable to natural torpor. Multiple NPY receptor subtypes have been identified, and the Y1 receptor and Y5 receptor both contribute to the orexigenic effect of NPY. The purpose of this research was to compare and contrast the effects of Y1 receptor activation by a specific Y1 agonist ([D-Arg25]-NPY) or Y5 receptor activation by a specific Y5 agonist ([D-Trp34]-NPY) on body temperature and subsequent food intake in cold-acclimated Siberian hamsters. Intracerebroventricular injections of Y1 agonist produced torporlike hypothermia closely resembling that induced by intracerebroventricular NPY. The intracerebroventricular Y5 agonist infrequently produced hypothermia reaching criterion for torpor and that failed to resemble either NPY-induced or natural torpor. Combined injections of Y1 and Y5 agonists resulted in hypothermia comparable to Y5 agonist treatments alone, negating the mimicry of NPY treatment seen with Y1 agonist alone. Prior treatment with Y1 agonist or Y5 agonist surprisingly had lingering effects on NPY-induced torpor expression, Y1 agonist enhanced and Y5 agonist inhibited the effect of NPY. The ability of NPY to induce torporlike hypothermia, especially its initiation, most likely involves activation of the NPY Y1 receptor subtype.     

Mapping the agonist binding site of the nicotinic acetylcholine receptor. Orientation requirements for activation by covalent agonist

To characterize the structural requirements for ligand orientation compatible with activation of the Torpedo nicotinic acetylcholine receptor (nAChR), we used Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to tether primary or quaternary amines at defined positions within the agonist binding site of nAChRs containing mutant alpha- or gamma-subunits expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and 2-aminoethylmethanethiosulfonate acted as irreversible antagonists when tethered at alphaY93C, alphaY198C, or gammaE57C, as well as at alphaN94C (2-aminoethylmethanethiosulfonate only). [2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine to binding site Cys, also acted as an irreversible antagonist when tethered at alphaY93C, alphaN94C, or gammaE57C. However, MTSET modification of alphaY198C resulted in prolonged activation of the nAChR not reversible by washing but inhibitable by subsequent exposure to non-competitive antagonists. Modification of alphaY198C (or any of the other positions tested) by [(trimethylammonium)methyl]methanethiosulfonate resulted only in irreversible inhibition, while modification of alphaY198C by [3-(trimethylammonium)propyl]methanethiosulfonate resulted in irreversible activation of nAChR, but at lower efficacy than by MTSET. Thus changing the length of the tethering arm by less than 1 A in either direction markedly effects the ability of the covalent trimethylammonium to activate the nAChR, and agonist activation depends on a very selective orientation of the quaternary ammonium within the agonist binding site.     

A natural history of "agonist"

This paper constructs a brief history of the biochemical term agonist by exploring the multiple meanings of the root ag?n in ancient Greek literature and describing how agonist first appeared in the scientific literature of the 20th century in the context of neurophysiologists' debates about the existence and properties of cellular receptors. While the narrow scientific definition of agonist may appear colorless and dead when compared with the web of allusions spun by the ancient Greek ag?n, the scientific power and creativity of agonist actually resides precisely in its exact, restricted meaning for biomedical researchers.     

8-O-acetylharpagide is not an ecdysteroid agonist

We have reinvestigated the activity of 8-O-acetylharpagide, an iridoid glucoside, as an ecdysteroid agonist. Elbrecht et al. (Insect Biochem. Mol. Biol. 26 (1996) 519) isolated a preparation of this compound from Ajuga reptans L. and ascribed ecdysteroid agonist activity on the basis of the induction of an ecdysteroid-like response in Drosophila melanogaster KcO cells, the displacement of [3H]ponasterone A from the Drosophila receptor and the activation of an ecdysteroid-regulated gene in a transactivation assay. We provide evidence that the agonist activity derives from contaminating ecdysteroids; A. reptans is a species rich in ecdysteroids. Purified 8-O-acetylharpagide is not active in the D. melanogaster B(II) cell bioassay, neither as an agonist nor as an antagonist, nor does it displace [3H]ponasterone A from dipteran or lepidopteran ecdysteroid receptor complexes.