Putative endogenous ligands for the benzodiazepine receptor.

The recent discovery of pharmacologically relevant, high affinity, stereospecific binding sites for the benzodiazepines in the central nervous system (CNS) has rekindled investigations concerning the mechanism of action of these drugs. It has become increasingly clear that elucidation of benzodiazepine action will provide new and important insights into the neurochemical substances of seizure activity, centrally mediated muscle relaxation and anxiety, three major actions of this class of drugs.The existence of a functional receptor for the benzodiazepines, compounds not present $\text{in vivo}$, suggests that endogenous substances exist that serve as natural substrates for this receptor. Furthermore, the characterization of endogenous benzodiazepine receptor ligands affords an opportunity to determine the neurochemical mechanisms underlying the pharmacologic and behavioral effects manifested by the benzodiazepines.Using receptor binding methodology to assay tissue extracts for [³H] diazepam binding inhibitory activity, putative endogenous ligands for the benzodiazepine receptor have been isolated and identified as the purine nucleosides. Compounds such as inosine and hypoxanthine exhibit competitive inhibition of [³H] diazepam binding. The low affinity purinergic inhibition of diazepam binding is consistent with their $\text{in vivo}$ concentrations. Distinct structure-activity relationships exist for the purines with subtle structural alterations having marked effects on diazepam binding inhibitory potency. The methylxanthine stimulants, caffeine, theophylline, and theobromine, also competitively inhibit diazepam binding, suggesting that some of their actions may be mediated by the benzodiazepine receptor.The purines also have “benzodiazepine-like” pharmacologic properties, since they have been shown to antagonize pentylenetetrazol induced seizures in mice in a dose dependent manner. Neurophysiologic studies have also shown that iontophoresis of inosine on cultured mouse primary neurons produce neurotransmitter like effects. Furthermore, these effects are similar to those observed with flurazepam, a finding that provides additional evidence for the “benzodiazepine-like” properties of the purines.The preliminary studies outlined below indicate that the purines are good candidates as putative endogenous ligands for the benzodiazepine receptor and provide a foundation for future studies that concern the homeostatic mediation of seizure activity and anxiety.     

Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4

Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor 1 (OGR1) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR1 (51%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF10A cells. These effects are insensitive to treatment with BN52021, WEB-2170, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = 159 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not sphingosine 1-phosphate (S1P), PAF, psychosine (Psy), glucosyl-beta1'1-sphingosine (Glu-Sph), galactosyl-beta1'1-ceramide (Gal-Cer), or lactosyl-beta1'1-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a receptor with high affinity to SPC and low affinity to LPC, and that multiple cellular functions can be transduced via this receptor.     

Thyroid receptor ligands. Part 4: 4'-amido bioisosteric ligands selective for the thyroid hormone receptor beta

Based on the examination of the X-ray crystallographic structures of the LBD of TRalpha and TRbeta in complex with KB-141 (2), a number of novel 4'-hydroxy bioisosteric thyromimetics were prepared. Optimal affinity and beta-selectivity (33 times), was found with a medium-sized alkyl-substituted amido group; iso-butyl (12c). It can be concluded that bioisosteric replacements of the 4'-hydroxy position represent a new promising class of TRbeta-selective synthetic thyromimetics.     

Polyamines are potent ligands for the capsaicin receptor TRPV1

Polyamines are important endogenous regulators of ion channels and are known to modulate inflammation and nociception. Here we investigated effects of polyamines on the capsaicin receptor TRPV1, a major ion channel expressed in nociceptive sensory afferents. Extracellular spermine, spermidine, and putrescine directly activated TRPV1 in a charge-dependent manner, both in heterologous expression systems and sensory neurons. The threshold for activation by spermine was approximately 500 microm at room temperature. At lower concentrations, spermine enhanced capsaicin-evoked currents with an EC50 of approximately 5 microm. Further, polyamines freely permeated TRPV1 (estimated relative permeabilities compared with Na+ were between 3 and 16), and spermine reduced the single channel conductance from 96 to 49 pS. Experiments with TRPV1 mutants identified extracellular acidic residues critical for polyamine regulation. Neutralization of aspartate 646 (D646N) abolished direct activation by spermine, whereas neutralization of this same aspartate (D646N) or glutamate 648 (E648A) inhibited spermine-induced sensitization. These data show that polyamines, by virtue of their cationic charge, can regulate the activity of TRPV1. Extracellular polyamines are present in considerable concentrations in the gastrointestinal tract and at synapses, and these levels increase during inflammation and cancer. Therefore, polyamine regulation of TRPV1 in these tissues may be relevant to a variety of physiological and pathophysiological states.     

Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances

The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with 3H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.     

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A). Chinese hamster ovary cells stably transfected to express the SR-A bound and internalized 4-fold more human platelet-derived sAPP than control cells. The binding and internalization of sAPP were inhibited by the SR-A antagonist fucoidin. In addition, sAPP competed as effectively as fucoidin for SR-A-mediated cell association and degradation of (125)I-AcLDL. To determine if the KPI domain is required for the binding of sAPP to the SR-A, APP751 and APP695 were expressed in Chinese hamster ovary cells, and sAPP751 and sAPP695 purified from the medium were tested for their binding to the SR-A. sAPP751 and sAPP695 were equally effective in competing for the cell association of (125)I-AcLDL by SR-A-expressing cells, demonstrating that the KPI domain is not essential for binding. We also found that sAPP751 is present in extracts of atherosclerotic lesions and that sAPP competes for the SR-A-mediated cell association of oxidized low density lipoprotein. Deletion mutagenesis indicated that a negatively charged region of APP (residues 191-264) contributes to binding to the SR-A. These results suggest that the SR-A contributes to the clearance of sAPP and that sAPP competes for the cell association of other SR-A ligands.     

Leukotriene B4 receptor and the function of its helix 8

More than 30 lipid ligands, which express their biological activities through cognate G-protein-coupled receptors (GPCRs), have been reported. Among them, leukotriene B(4) (LTB(4)) is a potent lipid mediator involved in host defense, inflammation, and the immune responses. Two GPCRs for LTB(4) (BLT1 and BLT2) have been cloned and analyzed. Recent studies using genetically engineered mice suggest that BLT1 plays an important role in several inflammatory diseases including ischemic reperfusion tissue injury, atherosclerosis, and bronchial asthma. BLT1 is also a good tool to study the molecular mechanism of GPCR activation and inactivation in vitro. In this brief review, we focus on the biological and biochemical properties of BLT1 with special attention to the putative helix 8 of the receptor.     

Photoaffinity labeling of the Ah receptor

A series of halodibenzo-p-dioxins with the photolabile aryl azide functional group were synthesized and screened as potential photoaffinity labels for the Ah receptor, and 2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin was selected for radiosynthesis with 125I (specific activity 2176 Ci/mmol, equilibrium dissociation constant, KD = 0.76 nM). Following incubation of this 125I-labeled photoaffinity ligand with the protamine sulfate-precipitated fraction of C57BL/6J mouse liver cytosol, and irradiation with long wavelength ultraviolet light, the radiolabeled macromolecules were precipitated with acetone and analyzed by denaturing gel electrophoresis and autoradiography. Among the labeled products, two peptides with apparent molecular masses of 95,000 and 70,000 daltons had the following properties: 1) they were selectively labeled at low ligand concentrations; 2) they were labeled in approximately a 1:1 ratio; 3) co-incubation with receptor agonists inhibited the photoaffinity labeling of both peptides to a similar extent, and structure activity relationship for inhibition of labeling by these agonists corresponded to that for their binding affinity to the Ah receptor; 4) upon nondenaturing chromatographic separation of photoaffinity labeled cytosol on high performance liquid chromatography size exclusion and anion exchange columns, the 95- and 70-kDa peptides coelute; 5) the migration of these peptides upon denaturing electrophoresis is the same in the presence or absence of a thiol reducing agent; and 6) proteolysis of the 95- and 70-kDa peptides produces a similar pattern of cleavage peptides. The simplest structure of the Ah receptor in mouse liver cytosol, appears to be a dimer composed of two noncovalently linked subunits of apparent molecular masses of 95 and 70 kDa, which have homologous structure and similar ligand binding sites, but other possibilities are discussed.     

Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR

The orphan nuclear receptor SXR coordinately regulates drug clearance in response to a wide variety of xenobiotic compounds. This signaling system protects the body from exposure to toxic compounds; however, it can also pose a severe barrier to drug therapy. We now demonstrate that the human immunodeficiency virus (HIV) protease inhibitor ritonavir binds SXR and activates its target genes. This represents an example of a commonly used therapeutic agent that effectively activates SXR. We also show that other protease inhibitors are weaker (saquinavir) or unable to activate SXR (nelfinavir, indinavir) thus defining analogs that fail to induce SXR-regulated clearance pathways. Interestingly, HIV protease inhibitors are distinct from previously known SXR ligands in that they are peptide mimetic compounds. This expands the ligand specificity of SXR to include this unique chemical class whose pharmaceutical significance is expanding. Finally, we show that SXR ligands activate expression of multiple resistance protein 2, a critical regulator of bile flow and biliary drug excretion. These findings have important implications for the role of SXR in regulating drug clearance and hepatic disorders associated with impaired bile flow.     

Triarylethylene bisphenols with a novel cycle are ligands for the estrogen receptor

We have prepared a series of triarylethylene and triarylethane systems, analogues of the selective antiestrogen tamoxifen, in which the alkyl substituent is tethered to the distal, rather than the proximal aryl ring by a 5-, 6-, or 7-membered carbocycle. This unusual cyclic structure rigidifies the ligand and adds bulk in a manner that is different from the more typical cyclization to the proximal aryl ring, as in the antiestrogen nafoxidine. These new systems were prepared efficiently by the addition of a benzylic sodium reagent, generated from the corresponding chloride by treatment with sodium naphthalenide, to a doubly protected 4,4'-dihydroxybenzophenone, followed by dehydration and deprotection. In all cases, formation of the exocyclic alkene was preferred. Two of the corresponding alkanes could be obtained by catalytic hydrogenation. All of these compounds have relatively high binding affinity for the estrogen receptor, and some of them demonstrate a significant level of affinity selectivity for the estrogen receptor alpha subtype. Accommodation of these newly rigidified cyclic triarylethylene systems into the ligand-binding pocket of the estrogen receptor can be visualized by molecular modeling.     

Dysfunctional mitochondria contain endogenous high-affinity human Toll-like receptor 4 (TLR4) ligands and induce TLR4-mediated inflammatory reactions

Mitochondria, known to share many common features with prokaryotic cells, accumulate several endogenous ligands of the pattern-recognition Toll-like receptor 4 (TLR4), such as the heat shock proteins (Hsp) 70 and 60. TLR4 specifically recognises and responds to LPS of Gram-negative bacteria and participates in both autoimmune reactions and tissue regeneration due to its ability to recognise endogenous ligands. In the present study we show that mitochondria extracts obtained from hydrogen peroxide-dysfunctionalised cells induce a pro-inflammatory response in human THP-1 myeloid leukaemia cells. This inflammatory response was similar to that caused by LPS and much stronger than that induced by the extracts of normal mitochondria. Such reactions include activation of stress-adaptation hypoxia-inducible factor 1 alpha (HIF-1α) and expression/release of the pro-inflammatory cytokines IL-6 and TNF-α. Pre-treatment of THP-1 myeloid macrophages with TLR4-neutralising antibody before exposure to mitochondria extracts or LPS attenuated the inflammatory responses. Signalling pathways recruited by TLR4 in response to LPS and mitochondria-derived ligands were found to be the same. An in vitro ELISA-based TLR4-ligand binding assay, in which the ligand-binding domain of human TLR4 was immobilised, showed that mitochondria extracts contain endogenous TLR4 ligands. These results were verified in surface plasmon resonance experiments in which the affinity of the ligands derived from dysfunctional mitochondria was comparable with that of LPS and was much higher than that observed for normal mitochondria.     

Intracellular location of the Ah receptor

The subcellular distribution of the Ah receptor from the mouse hepatoma line, Hepa-1, was investigated following cytochalasin B treatment and cell enucleation. Probing the resultant cytoplast and nucleoplast fractions with radiolabelled tetrachlorodibenzo-p-dioxin (TCDD) revealed the presence of a specifically bound peak of receptor only in the cytoplast fraction. However, the quantity of receptor recovered in these experiments was only 10–12% of the expected value. We therefore undertook an investigation to determine the fate of the Ah receptor in the presence of cytochalasin B. Incubation of Hepa-1 cells with this compound resulted in a rapid loss or inactivation of cytosolic binding activity with a concomitant decrease in the amount of receptor partitioned into the nucleus at all time periods examined. Control experiments indicated that cytochalasin B did not compete with TCDD for binding to the Ah receptor and furthermore, that its mechanism of action could not be attributed to a non-specific effect on all cytosolic proteins. The results obtained are discussed in relation to the proposed models for induction by the estrogen and glucocorticoid binding receptors.     

FLRT proteins are endogenous latrophilin ligands and regulate excitatory synapse development

Latrophilins (LPHNs) are a small family of G protein-coupled receptors known to mediate the massive synaptic exocytosis caused by the black widow spider venom α-latrotoxin, but their endogenous ligands and function remain unclear. Mutations in LPHN3 are strongly associated with attention deficit hyperactivity disorder, suggesting a role for latrophilins in human cognitive function. Using affinity chromatography and mass spectrometry, we identify the FLRT family of leucine-rich repeat transmembrane proteins as endogenous postsynaptic ligands for latrophilins. We demonstrate that the FLRT3 and LPHN3 ectodomains interact with high affinity in trans and that interference with this interaction using soluble recombinant LPHN3, LPHN3 shRNA, or FLRT3 shRNA reduces excitatory synapse density in cultured neurons. In addition, reducing FLRT3 levels with shRNA in vivo decreases afferent input strength and dendritic spine number in dentate granule cells. These observations indicate that LPHN3 and its ligand FLRT3 play an important role in glutamatergic synapse development.     

Heparan sulfate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma

RPTPsigma is a cell adhesion molecule-like receptor protein tyrosine phosphatase involved in nervous system development. Its avian orthologue, known as cPTPsigma or CRYPalpha, promotes intraretinal axon growth and controls the morphology of growth cones. The molecular mechanisms underlying the functions of cPTPsigma are still to be determined, since neither its physiological ligand(s) nor its substrates have been described. Nevertheless, a major class of ligand(s) is present in the retinal basal lamina and glial endfeet, the potent native growth substrate for retinal axons. We demonstrate here that cPTPsigma is a heparin-binding protein and that its basal lamina ligands include the heparan sulfate proteoglycans (HSPGs) agrin and collagen XVIII. These molecules interact with high affinity with cPTPsigma in vitro, and this binding is totally dependent upon their heparan sulfate chains. Using molecular modelling and site-directed mutagenesis, a binding site for heparin and heparan sulfate was identified in the first immunoglobulin-like domain of cPTPsigma. HSPGs are therefore a novel class of heterotypic ligand for cPTPsigma, suggesting that cPTPsigma signaling in axons and growth cones is directly responsive to matrix-associated cues.     

Tumor biomarker glycoproteins in the seminal plasma of healthy human males are endogenous ligands for DC-SIGN

DC-SIGN is an immune C-type lectin that is expressed on both immature and mature dendritic cells associated with peripheral and lymphoid tissues in humans. It is a pattern recognition receptor that binds to several pathogens including HIV-1, Ebola virus, Mycobacterium tuberculosis, Candida albicans, Helicobacter pylori, and Schistosoma mansoni. Evidence is now mounting that DC-SIGN also recognizes endogenous glycoproteins, and that such interactions play a major role in maintaining immune homeostasis in humans and mice. Autoantigens (neoantigens) are produced for the first time in the human testes and other organs of the male urogenital tract under androgenic stimulus during puberty. Such antigens trigger autoimmune orchitis if the immune response is not tightly regulated within this system. Endogenous ligands for DC-SIGN could play a role in modulating such responses. Human seminal plasma glycoproteins express a high level of terminal Lewis(x) and Lewis(y) carbohydrate antigens. These epitopes react specifically with the lectin domains of DC-SIGN. However, because the expression of these sequences is necessary but not sufficient for interaction with DC-SIGN, this study was undertaken to determine if any seminal plasma glycoproteins are also endogenous ligands for DC-SIGN. Glycoproteins bearing terminal Lewis(x) and Lewis(y) sequences were initially isolated by lectin affinity chromatography. Protein sequencing established that three tumor biomarker glycoproteins (clusterin, galectin-3 binding glycoprotein, prostatic acid phosphatase) and protein C inhibitor were purified by using this affinity method. The binding of DC-SIGN to these seminal plasma glycoproteins was demonstrated in both Western blot and immunoprecipitation studies. These findings have confirmed that human seminal plasma contains endogenous glycoprotein ligands for DC-SIGN that could play a role in maintaining immune homeostasis both in the male urogenital tract and the vagina after coitus.     

Endogenous bile acids are ligands for the nuclear receptor FXR/BAR.

The major metabolic pathway for elimination of cholesterol is via conversion to bile acids. In addition to this metabolic function, bile acids also act as signaling molecules that negatively regulate their own biosynthesis. However, the precise nature of this signaling pathway has been elusive. We have isolated an endogenous biliary component (chenodeoxycholic acid) that selectively activates the orphan nuclear receptor, FXR. Structure-activity analysis defined a subset of related bile acid ligands that activate FXR and promote coactivator recruitment. Finally, we show that ligand-occupied FXR inhibits transactivation from the oxysterol receptor LXR alpha, a positive regulator of cholesterol degradation. We suggest that FXR (BAR) is the endogenous bile acid sensor and thus an important regulator of cholesterol homeostasis.