Alpha-substituted phosphonate analogues of lysophosphatidic acid (LPA) selectively inhibit production and action of LPA

Isoform-selective agonists and antagonists of the lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and biochemical resistance to chemotherapy- and radiotherapy-induced apoptosis. LPA and its analogues are also feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, also known as autotaxin), a central regulator of invasion and metastasis. For cancer therapy, the ideal therapeutic profile would be a metabolically stabilized pan-LPA receptor antagonist that also inhibits lysoPLD. Herein we describe the synthesis of a series of novel alpha-substituted methylene phosphonate analogues of LPA. Each of these analogues contains a hydrolysis-resistant phosphonate mimic of the labile monophosphate of natural LPA. The pharmacological properties of these phosphono-LPA analogues were characterized in terms of LPA receptor subtype-specific agonist and antagonist activity using Ca(2+) mobilization assays in RH7777 and CHO cells expressing the individual LPA GPCRs. In particular, the methylene phosphonate LPA analogue is a selective LPA(2) agonist, whereas the corresponding alpha-hydroxymethylene phosphonate is a selective LPA(3) agonist. Most importantly, the alpha-bromomethylene and alpha-chloromethylene phosphonates show pan-LPA receptor subtype antagonist activity. The alpha-bromomethylene phosphonates are the first reported antagonists for the LPA(4) GPCR. Each of the alpha-substituted methylene phosphonates inhibits lysoPLD, with the unsubstituted methylene phosphonate showing the most potent inhibition. Finally, unlike many LPA analogues, none of these compounds activate the intracellular LPA receptor PPARgamma.     

Alkoxymethylenephosphonate analogues of (Lyso) phosphatidic acid stimulate signaling networks coupled to the LPA2 receptor

An efficient stereocontrolled synthesis afforded alkoxymethylenephosphonate (MP) analogues of lysophosphatidic acid (LPA) and phosphatidic acid (PA). The pharmacological properties of MP-LPA and MP-PA analogues were characterized for LPA receptor subtype-specific agonist and antagonist activity using Ca(2+)-mobilization assays in RH7777 cells expressing the individual LPA(1)-LPA(3) receptors and CHO cells expressing LPA(4). In addition, activation of a PPARgamma reporter gene construct expressed in CV-1 cells was assessed. These metabolically stabilized LPA analogues exhibited an unexpected pattern of partial agonist/antagonist activity for the LPA G-protein-coupled receptor family and the intracellular LPA receptor PPARgamma. Analogues were compared with 18:1 LPA for activation of downstream signaling in HT-29 colon cancer cells, which exclusively express LPA(2), and both SKOV3 and OVCAR3 ovarian cancer cells, which express LPA(1), LPA(2), and LPA(3). Unexpectedly, reverse phase protein arrays showed that four MP-LPA and MP-PA analogues selectively activated downstream signaling in HT-29 cells with greater potency than LPA. In particular, the oleoyl MP-LPA analogue strongly promoted phosphorylation and activation of AKT, MEK, and pS6 in HT-29 cells in a concentration-dependent manner. In contrast, the four MP-LPA and MP-PA analogues were equipotent with LPA for pathway activation in the SKOV3 and OVCAR3 cells. Taken together, these results suggest that the MP analogues may selectively activate signaling via the LPA(2) receptor subtype, while simultaneously suppressing signaling through the LPA(1) and LPA(3) subtypes.     

G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.     

Receptor-Mediated Vascular Smooth Muscle Migration Induced by LPA Involves p38 Mitogen-Activated Protein Kinase Pathway Activation

Lysophosphatidic acid (LPA), a naturally occurring glycerophospholipid, can evoke various biological responses, including cell migration, proliferation and survival, via activation of G protein-coupled receptors (GPCRs). However, the role of LPA receptors and details of LPA signaling in migration are largely unexplored. In this study we detect the expression of LPA1 and LPA3 receptors in rat aortic smooth muscle cells (RASMCs). LPA stimulated RASMCs migration in a dose-dependent manner and induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell migration was significantly inhibited by specific LPA1/LPA3-receptor antagonist Dioctylglycerol pyrophosphate (8:0) (DGPP8.0) at higher concentration. Migration of cells toward LPA was partially, but significantly, reduced in the presence of SB-203580, a p38 MAPK inhibitor, but not PD98059, an ERK inhibitor. In addition, pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 MAPK, ERK phosphorylation and RASMCs migration. These data suggest that LPA-induced migration is mediated through the Gi-protein-coupled LPA1 receptor involving activation of a PTX-sensitive Gi / p38MAPK pathway.     

Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ‐15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid‐phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4 , in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide‐based opioid analgesics.     

Synthesis and biological activity of metabolically stabilized cyclopentyl trisphosphate analogues of D-myo-Ins(1,4,5)P3

We describe the synthesis of four novel metabolically stabilized analogues of Ins(1,4,5)P(3) based on the known cyclopentane pentaol tris(phosphate) 2: tris(phosphorothioate) 3, tris(methylenephosphate) 4, tris(sulfonamide) 5, and tris(sulfate) 6. Of these analogues, only the tris(phosphorothioate) 3 and parent tris(phosphate) 2 bound to the type I InsP(3)R construct. In addition, both the tris(phosphorothioate) 3 and parent tris(phosphate) 2 elicited calcium release in MDA MB-435 breast cancer cells. The Ins(1,4,5)P(3) agonist activities of these two compounds can be rationalized on the basis of computational docking of the ligands to the binding domain of the type I InsP(3)R.     

The Role of Formyl Peptide Receptors in Permanent and Low-Grade Inflammation: Helicobacter pylori Infection as a Model

Formyl peptide receptors (FPRs) are cell surface pattern recognition receptors (PRRs), belonging to the chemoattractant G protein-coupled receptors (GPCRs) family. They play a key role in the innate immune system, regulating both the initiation and the resolution of the inflammatory response. FPRs were originally identified as receptors with high binding affinity for bacteria or mitochondria N-formylated peptides. However, they can also bind a variety of structurally different ligands. Among FPRs, formyl peptide receptor-like 1 (FPRL1) is the most versatile, recognizing N-formyl peptides, non-formylated peptides, and synthetic molecules. In addition, according to the ligand nature, FPRL1 can mediate either pro- or anti-inflammatory responses. Hp(2-20), a Helicobacter pylori-derived, non-formylated peptide, is a potent FPRL1 agonist, participating in Helicobacter pylori-induced gastric inflammation, thus contributing to the related site or not-site specific diseases. The aim of this review is to provide insights into the role of FPRs in H. pylori-associated chronic inflammation, which suggests this receptor as potential target to mitigate both microbial and sterile inflammatory diseases.     

Asymmetric synthesis and receptor pharmacology of the group II mGlu receptor ligand (1S,2R,3R,5R,6S)-2-amino-3-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid-HYDIA

The asymmetric synthesis and receptor pharmacology of (1S,2R,3R,5R,6S)-2-amino-3-Hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (+)-9 (HYDIA) and a few of its O-alkylated derivatives are described. The key step of the synthesis utilizes Sharpless' asymmetric dihydroxylation (AD-beta) for the kinetic resolution of a bicyclic racemic precursor olefin. In contrast to the bicyclic glutamate analogue LY354740, which is a potent and selective agonist for the group II metabotropic glutamate receptors (mGluRs), these new conformationally restricted and also hydroxylated or alkoxylated glutamate analogues are potent and selective antagonists for the group II mGluRs.     

Synthesis, receptor binding, and CNS pharmacological studies of new thyrotropin-releasing hormone (TRH) analogues

As part of our search for selective and CNS‐active thyrotropin‐releasing hormone (TRH) analogues, we synthesized a set of 44 new analogues in which His and pGlu residues were modified or replaced. The analogues were evaluated as agonists at TRH‐R1 and TRH‐R2 in cells in vitro, and in vivo in mice for analeptic and anticonvulsant activities. Several analogues bound to TRH‐R1 and TRH‐R2 with good to moderate affinities, and are full agonists at both receptor subtypes. Specifically, analogue 21 a (R=CH₃) exhibited binding affinities (K_i values) of 0.17 μM for TRH‐R1 and 0.016 μM for TRH‐R2; it is 10‐fold less potent than TRH in binding to TRH‐R1 and equipotent with TRH in binding to TRH‐R2. Compound 21 a , the most selective agonist, activated TRH‐R2 with a potency (EC₅₀ value) of 0.0021 μM , but activated TRH‐R1 at EC₅₀=0.05 μM , and exhibited 24‐fold selectivity for TRH‐R2 over TRH‐R1. The newly synthesized TRH analogues were also evaluated in vivo to assess their potencies in antagonism of barbiturate‐induced sleeping time, and several analogues displayed potent analeptic activity. Specifically, analogues 21 a , b and 22 a , b decreased sleeping time by nearly 50 % more than TRH. These analogues also displayed potent anticonvulsant activity and provided significant protection against PTZ‐induced seizures, but failed to provide any protection in MES‐induced seizures at 10 μmol kg^?1. The results of this study provide evidence that TRH analogues that show selectivity for TRH‐R2 over TRH‐R1 possess potent CNS activity.     

Stable Binding of Full-Length Chemerin Is Driven by Negative Charges in the CMKLR1 N Terminus

The adipokine chemerin is the endogenous ligand of the chemokine-like receptor 1 (CMKLR1), a member of the family of G protein-coupled receptors (GPCRs). This protein ligand plays an important role in obesity and inflammatory processes. Stable receptor–ligand interactions are highly relevant for its different physiological effects such as the migration of immune cells towards sites of inflammation. Here, we demonstrate that negative charges in the CMKLR1 N terminus are involved in the formation of strong contacts with a specific positively charged patch at the surface of full-length chemerin, which is absent in the short nonapeptide agonist chemerin-9, thus explaining its reduced affinity. Using receptor chimera of G protein-coupled receptor 1 (GPR1) and CMKLR1, we were able to identify the residues of this interaction and its relevance for stable full-length chemerin binding. This could help to develop more potent ligands for the treatment of inflammation-related diseases.     

State-Targeting Stabilization of Adenosine A2A Receptor by Fusing a Custom-Made De Novo Designed α-Helical Protein

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A_2A receptor (A_2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A_2AR through straight helical connections without any kinks or intervening loops. The chimeric A_2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.     

5‐Stabilized Phosphatidylinositol 3,4,5‐Trisphosphate Analogues Bind Grp1 PH,Inhibit Phosphoinositide Phosphatases,and Block Neutrophil Migration

Metabolically stabilized analogues of PtdIns(3,4,5)P₃ have shown long‐lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5‐phosphatase‐resistant analogues of PtdIns(3,4,5)P₃, the 5‐methylene phosphonate (MP) and 5‐phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P₃ analogues in four contexts. First, the relative binding affinities of the 3‐MP, 3‐PT, 5‐MP, 5‐PT, and 3,4,5‐PT₃ analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P₃. Third, exogenously delivered analogues severely impair complement factor C5a‐mediated polarization and migration of murine neutrophils. Finally, the new analogues show long‐lived agonist activity in mimicking insulin action in sodium transport in A6 cells.     

Heme oxygenase inhibition by α-(1H-imidazol-1-yl)-ω-phenylalkanes: effect of introduction of heteroatoms in the alkyl linker

Several α-(1H-imidazol-1-yl)-ω-phenylalkanes were synthesized and evaluated as novel inhibitors of heme oxygenase (HO). These compounds were found to be potent and selective for the stress-induced isozyme HO-1, showing mostly weak activity toward the constitutive isozyme HO-2. The introduction of an oxygen atom in the alkyl linker produced analogues with decreased potency toward HO-1, whereas the presence of a sulfur atom in the linker gave rise to analogues with greater potency toward HO-1 than the carbon-containing analogues. The most potent compounds studied contained a five-atom linker between the imidazolyl and phenyl moieties, whereas the most HO-1-selective compounds contained a four-atom linker between these groups. The compounds with a five-atom linker containing a heteroatom (O or S) were found to be the most potent inhibitors of HO-2; 1-(N-benzylamino)-3-(1H-imidazol-1-yl)propane dihydrochloride, with a nitrogen atom in the linker, was found to be inactive.     

Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca²⁺ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we discuss the main mechanisms of GPCR-mediated cell-surface receptors transactivation and the pathways involved in intracellular responses induced by GPCR agonists. These studies may suggest the design of novel strategies for therapeutic interventions.     

Distribution of endogenous farnesyl pyrophosphate and four species of lysophosphatidic Acid in rodent brain

Lysophosphatidic acid (LPA) is the umbrella term for lipid signaling molecules that share structural homology and activate the family of LPA receptors. Farnesyl Pyrophosphate (FPP) is commonly known as an intermediate in the synthesis of steroid hormones; however, its function as a signaling lipid is beginning to be explored. FPP was recently shown to an activator of the G-protein coupled receptor 92 (also known as LPA5) of the calcium channel TRPV(3). The LPA receptors (including GPR92) are associated with the signal transduction of noxious stimuli, however, very little is known about the distribution of their signaling ligands (LPAs and FPP) in the brain. Here, using HPLC/MS/MS, we developed extraction and analytical methods for measuring levels of FPP and 4 species of LPA (palmitoyl, stearoyl, oleoyl and arachidonoyl-sn-glycerol-3 phosphate) in rodent brain. Relative distributions of each of the five compounds was significantly different across the brain suggesting divergent functionality for each as signaling molecules based on where and how much of each is being produced. Brainstem, midbrain, and thalamus contained the highest levels measured for each compound, though none in the same ratios while relatively small amounts were produced in cortex and cerebellum. These data provide a framework for investigations into functional relationships of these lipid ligands in specific brain areas, many of which are associated with the perception of pain.     

Benzyl and naphthalene methylphosphonic acid inhibitors of autotaxin with anti-invasive and anti-metastatic activity

Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) by lysophospholipase D activity, which leads to generation of the growth‐factor‐like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy‐resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Herein we report the synthesis and pharmacological characterization of ATX inhibitors based on the 4‐tetradecanoylaminobenzylphosphonic acid scaffold, which was previously found to lack sufficient stability in cellular systems. The new 4‐substituted benzylphosphonic acid and 6‐substituted naphthalen‐2‐ylmethylphosphonic acid analogues block ATX activity with K_i values in the low micromolar to nanomolar range against FS3, LPC, and nucleotide substrates through a mixed‐mode inhibition mechanism. None of the compounds tested inhibit the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor (LPAR) subtypes. Analogues 22 and 30 b , the two most potent ATX inhibitors, inhibit the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro in a dose‐dependent manner. The average terminal half‐life for compound 22 is 10±5.4 h and it causes a long‐lasting decrease in plasma LPA levels. Compounds 22 and 30 b significantly decrease lung metastasis of B16‐F10 syngeneic mouse melanoma in a post‐inoculation treatment paradigm. The 4‐substituted benzylphosphonic acids and 6‐substituted naphthalen‐2‐ylmethylphosphonic acids described herein represent new lead compounds that effectively inhibit the ATX–LPA–LPAR axis both in vitro and in vivo.     

Mapping the Melatonin Receptor. 8. Selective MT2 Agonists Derived from 5,6-Dihydroindolo[2,1-a]isoquinolines and Related Systems

A series of substituted indolo[2,1-a]isoquinolines and indolo[1,2-a]benzoxazines have been prepared, as melatonin analogues, to investigate the nature of the binding site of the melatonin receptor. Agonist and antagonist potency of all the analogues was measured using the [35S]GTPγS binding assay protocol. The binding affinity of the analogues were measured by competition binding studies against the human MT1 (hMT1) and MT2 (hMT2) receptors stably transfected in Chinese Hamster Ovarian (CHO) cells, using 2-[¹²⁵I]-iodomelatonin, as a ligand. N-Acetyl 2-(10-methoxy-5,6-dihydroindolo[2,1-a]isoquinolin-12-yl)propyl-1-amine (12 a) binds strongly to both the hMT1 and hMT2 receptors, and shows a preference for the hMT2, as does its propanamido counterpart 12 b . The introduction of two methyl groups into their side chain, analogues 15 a and 1 5 b , leads to antagonism, in the case of the former, and drastically diminishes its hMT1 binding; an analogous profile is seen for 15 b , which, however, is a partial agonist. Introduction of chlorine or methoxy groups into ring 4 gives compounds, that are weakly binding, with a preference for MT2. Substitution of oxygen for carbon at position 5 gives the indolo[1,2-c]benzoxazines 33 , 36 a and b , that bind strongly to the human receptors, 33 , 36 b being potent agonists at the melatonin receptors, but do not discriminate between hMT1 and hMT2.     

Improving potency, selectivity, and water solubility of adenosine A1 receptor antagonists: xanthines modified at position 3 and related pyrimido[1,2,3-cd]purinediones

The structure-activity relationships of xanthine derivatives related to the adenosine A(1) receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 1,3-dipropyl-8-(3-noradamantyl)xanthine (KW3902) were investigated by focusing on variations of the 3-substituent. Aromatic residues were well tolerated by the A(1) receptor in that position. A moderate effect of stereochemistry was found for the 3-(1-phenylethyl)-substituted analogue of DPCPX (S>R) at A(1) and A(3) receptors, whereas the opposite stereoselectivity was observed at the A(2) receptor subtypes. A 3-hydroxypropyl substituent was found to be optimal for high A(1) affinity and selectivity. The most potent compound of the present series was 1-butyl-3-(3-hydroxypropyl)-8-(3-noradamantyl)xanthine (10 c), which exhibits a K(i) value of 0.124 nM at rat, and 0.7 nM at human adenosine A(1) receptors, combined with high selectivity (>200-fold) versus the other receptor subtypes. The similarly potent 8-cyclopentyl-3-(3-hydroxypropyl)-1-propylxanthine was converted into a water-soluble phosphate prodrug, which may become a useful pharmacological tool for in vivo studies. 8-Alkyl-2-(3-noradamantyl)pyrimido[1,2,3-cd]purine-8,10-diones, which can be envisaged as xanthine analogues with a fixed 3-propyl substituent, were identified as a new class of potent, selective adenosine A(1) receptor antagonists. For example, compound 14 (8-butyl-substituted) exhibits a K(i) value of 13.8 nM at human A(1) receptors. A selection of the most potent compounds was investigated in [(35)S]GTPgammaS binding assays and showed inverse agonistic activity. Their efficacy was generally lower than that of the full inverse agonist DPCPX, and depended on subtle structural changes. Some of the new compounds belong to the most potent and selective A(1) antagonists described to date.