Design and synthesis of trivalent ligands targeting opioid,cholecystokinin, and melanocortin receptors for the treatment of pain

It has been known that co-administration of morphine with either cholecystokinin (CCK) receptor or melanocortin (MC) receptor antagonists enhance morphine’s analgesic efficacy by reducing serious side effects such as tolerance and addiction.1, 2, 3, 4 Considering these synergistic effects, we have designed trivalent ligands in which all three different pharmacophores for opioid, CCK, and MC receptors are combined in such a way as to conserve their own topographical pharmacophore structures. These ligands, excluding the cyclic compound, were synthesized by solid phase synthesis using Rink-amide resin under microwave assistance in very high yields. These trivalent ligands bind to their respective receptors well demonstrating that the topographical pharmacophore structures for the three receptors were retained for receptor binding. Ligand 10 was a lead compound to show the best biological activities at all three receptors.     

Comparative analysis of antitumor activity of CD40L, RANKL, and 4-1BBL in vivo following intratumoral administration of viral vectors or transduced dendritic cells

The tumor necrosis factor (TNF) family comprises a group of ligands that regulate cell proliferation, differentiation, activation, maturation and apoptosis through interaction with the corresponding TNF receptor family members. In this study, we have evaluated whether adenovirus-mediated intratumoral gene transfer of CD40L, RANKL, or 4-1BBL elicits an immune response to established murine MC38 and TS/A tumors. Intratumoral administration of the recombinant adenoviral vectors expressing CD40L, RANKL or 4-1BBL 7 days post-tumor cell inoculation resulted in significant inhibition of MC38 tumor growth for all three ligands when compared with control groups treated with either saline or control adenovirus. However, intratumoral injection of Ad-4-1BBL or Ad-CD40L resulted in a significantly stronger inhibition of TS/A tumor progression than did Ad-RANKL treatment. We also demonstrated that intratumoral administration of dendritic cells (DC) transduced with adenoviral vectors encoding the TNF-related ligands resulted in a significant inhibition of MC38 tumor growth as compared with control groups treated with Ad-LacZ-transduced DC or saline-treated DC. In addition, DC overexpressing CD40L secreted considerably more IL-12 and expressed higher levels of the co-stimulatory molecules, CD80, CD86 and CD40, than did DC overexpressing LacZ, 4-1BBL or RANKL. We have also demonstrated that DC/CD40L, DC/4-1BBL, and DC/RANKL survived significantly longer than control DC or DC infected with the LacZ vector. Taken together, these results demonstrate that adenoviral gene transfer of CD40L, RANKL or 4-1BBL elicit a significant antitumor effect in two different tumor models, with CD40L gene transfer inducing the strongest antitumor effect.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

Molecular interactions of nonpeptide agonists and antagonists with the melanocortin-4 receptor

The melanocortin-4 (MC4) receptor is a potential therapeutic target for obesity and cachexia, for which nonpeptide agonists and antagonists are being developed, respectively. The aim of this study was to identify molecular interactions between the MC4 receptor and nonpeptide ligands, and to compare the mechanism of binding between agonist and antagonist ligands. Nonpeptide ligand interaction was affected by mutations that reduce peptide ligand binding (D122A, D126A, S190A, M200A, F261A, and F284A), confirming overlapping binding determinants for peptide and nonpeptide ligands. The common halogenated phenyl group of nonpeptide ligands was a determinant of F261A and F284A mutations' affinity-reducing effect, implying this group interacts with the aromatic side chains of these residues. All affected compounds contain this group, the mutations reduced binding of 2,4-dichloro-substituted compounds more than 4-chloro-substituted-compounds, and F284A mutation eliminated the affinity-enhancing effect of 2-chloro-substitution. F261A and F284A mutations reduced the affinity of antagonists more than agonists, suggesting that the stronger ligand interaction with these residues, the lower the ligand efficacy. Supporting this hypothesis, F261A mutation increased the efficacy of nonpeptide antagonist and partial agonist ligands. D122A and D126A mutations reduced nonpeptide ligand interaction. Removing the ligands' derivatized amide group eliminated the effect of the mutations. Interaction of agonists, which bear a common amine within this group, was strongly reduced by D126A mutation (550-3300-fold), suggesting an electrostatic interaction between the amine and the acidic group of D126. These postulated interactions with aromatic and acidic regions of the MC4 receptor are consistent with a molecular model of the receptor. Furthermore, the strength of interaction with the aromatic pocket, and potentially the acidic pocket, controls the signaling efficacy of the ligand.     

Further structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5

Recently we have demonstrated that replacing His(6) by constrained amino acids(2) in the well-known antagonist SHU-9119 resulted in potent and selective antagonist ligands especially at the hMC3R and hMC5 receptors. With the aim to further explore position 6 in the sequence of SHU-9119 and MT-II, we have designed, synthesized, and pharmacologically characterized a series of peptide analogues of MT-II and SHU-9119 at the human melanocortin receptors subtypes MC3R, MC4R and MC5R. All these peptides were modified at position 6 with constrained amino acids which are commercially available. In this study, we have identified new selective ligands for the hMC4R, and an antagonist for the hMC3/hMC4 receptors. Additionally, we have discovered an interesting new selective antagonist at the hMC3R, Ac-Nle-c[Asp-betaAla-DNal(2')-Arg-Trp-Lys]-NH(2) (2, PG-106) which represents an important tool in further biological investigations of the hMC3R. PG-106 will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.     

Synthesis and structure-activity relationships of novel dipeptides and reduced dipeptides as ligands for melanocortin subtype-4 receptor

A series of benzylic piperazines (e.g., 4 and 5) attached to an 'address element', the dipeptide H-D-Tic-D-p-Cl-Phe-OH, 3 has been identified as ligands for the melanocortin subtype-4 receptor (MC4R). We describe herein the structure-activity relationship (SAR) studies on the N-terminal residue of the 'address element'. Several novel dipeptides and reduced dipeptides with high MC4R binding affinities and selectivity emerged from this SAR study.     

Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes

Hepatic stellate cells (HSC) and glomerular mesangial cells (MC) are tissue-specific pericytes involved in tissue repair, a process that is regulated by members of the chemokine family. In this study, we explored the signal transduction pathways activated by the chemokine receptor CXCR3 in vascular pericytes. In HSC, interaction of CXCR3 with its ligands resulted in increased chemotaxis and activation of the Ras/ERK cascade. Activation of CXCR3 also stimulated Src phosphorylation and kinase activity and increased the activity of phosphatidylinositol 3-kinase and its downstream pathway, Akt. The increase in ERK activity was inhibited by genistein and PP1, but not by wortmannin, indicating that Src activation is necessary for the activation of the Ras/ERK pathway by CXCR3. Inhibition of ERK activation resulted in a decreased chemotactic and mitogenic effect of CXCR3 ligands. In MC, which respond to CXCR3 ligands with increased DNA synthesis, CXCR3 activation resulted in a biphasic stimulation of ERK activation, a pattern similar to the one observed in HSC exposed to platelet-derived growth factor, indicating that this type of response is related to the stimulation of cell proliferation. These data characterize CXCR3 signaling in pericytes and clarify the relevance of downstream pathways in the modulation of different biologic responses.     

A possible mechanism for atherosclerosis induced by polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs), aryl hydrocarbon receptor (AHR) ligands, induce atherogenesis. Liver X receptor (LXR) alpha is known to be involved in the control of cholesterol homeostasis. Thus, the purpose of this study was to investigate the effects of 3-methlycholanthrene (MC), one of the PAHs, on LXRalpha-mediated signal transductions. We found that expression of mRNAs for ATP binding cassette A1, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase was suppressed by treatment of HepG2 cells with MC. A luciferase reporter assay revealed that LXRalpha- and SREBP-1c-mediated transactivations were inhibited by MC via AHR. Based on these lines of evidence, we propose that down-regulation of the LXRalpha-regulated genes by PAHs is one of the causes responsible for atherosclerosis induced by PAHs.     

A concise synthesis of 1,4-dihydro-[1,4]diazepine-5,7-dione,a novel 7-TM receptor ligand core structure with melanocortin receptor agonist activity

Finding small non-peptide molecules for G protein-coupled receptors (GPCR) whose endogenous ligands are peptides, is a very important task for medicinal chemists. Over the years, compounds mimicking peptide structures have been discovered, and scaffolds emulating peptide backbones have been designed. In our work on GPCR ligands, including cholecystokinin receptor-1 (CCKR-1) agonists, we have employed benzodiazepines as a core structure. Looking for ways to reduce molecular weight and possibly improve physical properties of GPCR ligands, we embarked on the search for molecules providing similar scaffolds to the benzodiazepine with lower molecular weight. One of our target core structures was 1,4-dihydro-[1,4]diazepine-5,7-dione. There was not, however, a known synthetic route to such molecules. Here we report the discovery of a simple and concise method for synthesis of 2-[6-(1H-indazol-3-ylmethyl)-5,7-dioxo-4-phenyl-4,5,6,7-tetrahydro-[1,4]diazepin-1-yl]-N-isopropyl-N-phenyl-acetamide as an example of a compound containing the tetrahydrodiazepine-5,7-dione core. Compounds from this series were tested in numerous GPCR assays and demonstrated activity at melanocortin 1 and 4 receptors (MC1R and MC4R). Selected compounds from this series were tested in vivo in Peptide YY (PYY)-induced food intake. Compounds dosed by intracerebroventricular and oral routes reduced PYY-induced food intake and this effect was reversed by the cyclic peptide MC4R antagonist SHU9119.     

Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1β and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.     

Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins

Activated platelets are known to express P-selectin, a lectin-likeadhesion receptor (CD62), through which they bind to sialylLewis X (sLe^x) ligands displayed on the membranes of leukocytes.To determine whether direct platelet-platelet interactions viaP-selectin/sLe^x interactions are also possible, we have examinedthe ganglioside extract of human blood platelets for the presenceof sLe^x ligands. Using the sensitive method of high-performancethin-layer chromatography (HPTLC)-immunostaining with the monoclonalantibody (mAb) CSLEX or with sialidase followed by mAbs MC480or PM81, eight sLe^x bands were demonstrated at R₁ 0.01, 0.03,0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10chloroform-methanol-aqueous 0.02% CaCl₂. The sensitivity ofall eight bands to sialidase or endoglycoceramidase confirmedthat they were gangliosides. Comparison of the HPTLC mobilitiesand densities of platelet bands with those from five other humantissues (granulocytes, monoblasts, kidney, aortic endotheliumand erythrocytes) in three different solvents revealed threemajor bands associated with platelets: 3 (R₁ 0.03), 6 (0.08)and 14 (0.21). Platelet bands were demonstrated not to haveresulted from granulocyte contamination. Partial purificationof platelet sLe^x gangliosides by high-performance liquid chromatographyand their reaction with 14 oligosaccharide-specific mAbs (FH4,FH5, LM112-161, LM-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04,SH34, P001 and MC813-70) revealed that band 6 is a multifucosylatedneolacto ganglioside and band 14 is a branched, disialo neolactofucoganglioside. Platelet band 3 combined the features of bothbands 6 and 14, and reacted differently than granulocyte band3. These partial structures resemble gangliosides associatedwith adhesion in other cell systems. It is concluded that plateletsexpress tissue-specific sLe^x gangliosides (sLe^x ligands). Thus,it is possible that platelet-platelet binding may be mediatedat least partially through P-selectin/sLe^x interactions, especiallyafter platelet activation. gangliosides HPTLC-immunostaining platelets selectin ligands sLe^x     

Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor

The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, d-Phe7]-alphaMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 degrees C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor.     

Detection of regions in the MC1 receptor of importance for the selectivity of the MC1 receptor super-selective MS04/MS05 peptides

We have investigated the ability of our earlier identified MS04-MS05 MSH-peptide analogues to bind to chimeric MC1-MC3 receptors. While the MS04 and MS05 peptides bind with nanomolar and sub-nanomolar affinities to the wild type MC1 receptor, they bind only with micromolar affinities for the wild type MC3 receptor, thus being the hitherto most MC1 receptor selective ligands. Upon exchanging portions involving transmembrane regions TM1, TM2-3, and TM6-7 of the MC1 receptor with corresponding portions of the MC3 receptor both of these peptides showed major losses of affinities. By contrast exchanges involving TM4-5 did not appreciably affect the affinity of either MS04 or MS05. Our data suggest that the binding pocket for the MS04-MS05 MSH-peptides is located between TM1-3 and TM6-7 of the melanocortin receptors.     

The Macrophage Scavenger Receptor A Is Host-Protective in Experimental Meningococcal Septicaemia

Macrophage Scavenger Receptor A (SR-A) is a major non-opsonic receptor for Neisseria meningitidis on mononuclear phagocytes in vitro, and the surface proteins NMB0278, NMB0667, and NMB1220 have been identified as ligands for SR-A. In this study we ascertain the in vivo role of SR-A in the recognition of N. meningitidis MC58 (serogroup B) in a murine model of meningococcal septicaemia. We infected wild-type and SR-A^−/− animals intraperitoneally with N. meningitidis MC58 and monitored their health over a period of 50 hours. We also determined the levels of bacteraemia in the blood and spleen, and measured levels of the pro-inflammatory cytokine interleukin-6 (IL-6). The health of SR-A^−/− animals deteriorated more rapidly, and they showed a 33% reduction in survival compared to wild-type animals. SR-A^−/− animals consistently exhibited higher levels of bacteraemia and increased levels of IL-6, compared to wild-type animals. Subsequently, we constructed a bacterial mutant (MC58-278-1220) lacking two of the SR-A ligands, NMB0278 and NMB1220. Mutation of NMB0667 proved to be lethal. When mice were infected with the mutant bacteria MC58-278-1220, no significant differences could be observed in the health, survival, bacteraemia, and cytokine production between wild-type and SR-A^−/− animals. Overall, mutant bacteria appeared to cause less severe symptoms of septicaemia, and a competitive index assay showed that higher levels of wild-type bacteria were recovered when animals were infected with a 1∶1 ratio of wild-type MC58 and mutant MC58-278-1220 bacteria. These data represent the first report of the protective role of SR-A, a macrophage-restricted, non-opsonic receptor, in meningococcal septicaemia in vivo, and the importance of the recognition of bacterial protein ligands, rather than lipopolysaccharide.     

Computational binding studies of human pp60c-src SH2 domain with a series of nonpeptide, phosphophenyl-containing ligands

Monte Carlo/free energy perturbation (MC/FEP) simulations were performed on a series of nonpeptide ligands of the human pp60c-src SH2 domain in order to calculate relative free energies of binding for each compound and to understand the structural requirements for high affinity binding. The amido compound, exhibiting the highest experimental affinity, takes advantage of an interaction with a previously unobserved structural water.     

Inverse agonist activity of agouti and agouti-related protein

Agouti and agouti-related protein (AgRP) are endogenous antagonists of the melanocortin receptors (MCxR). Previous data showed that recombinant full-length agouti and a synthetic fragment of AgRP, AgRP (83-132), are inverse agonists at the MC1R and MC4R, respectively. This study demonstrates the smaller analogs AgRP (87-120) and ASIP [90-132 (L89Y)], and short peptides Yc[CRFFNAFC]Y and Qc[CRFFRSAC]S are also MC4R inverse agonists. Furthermore, the relative affinity of the series of MC4R ligands for displacement of radiolabeled antagonist 125I-AgRP (86-132) versus radiolabeled agonist 125I-NDP-MSH did not correlate with ligand efficacy, which is more consistent with an induced-fit model than a simple two-state model of MC4R activation. These data shed new light on the determinants and mechanism of inverse agonism at the MC4R.     

Melanocortin-5 receptor: a marker of human sebocyte differentiation

Melanocortin receptors (MC1R-MC5R) and their ligands (melanocyte-stimulating hormone (MSH) and adrenocorticotrophin hormone (ACTH)) have been shown to influence physiological functions of cells and organs, including exocrine glands. Since relatively little is known about MC5R expression and function in the human sebaceous gland, we examined expression of MC5R by immunohistochemistry and RT-PCR in human sebaceous cells in vivo and in vitro. In human skin, MC5R was detected only in differentiating, lipid-laden sebaceous cells but not in basal, undifferentiated sebaceous cells. Similarly, in cultured human sebocytes MC5R was only detectable at the onset of differentiation and in fully differentiated cells displaying prominent lipid granules. The lipid profile of the cultured and differentiated human sebocytes was shown to be human sebum-specific using (14)C-acetate labeling and high performance thin layer chromatography. Our studies suggest that MC5R is a marker of human sebocyte differentiation.     

Variability of metapodials in primates with rudimentary digits: Ateles geoffroyi, Colobus guereza, and Perodicticus potto

A tenet of evolutionary theory is that, within a species, phenotypic variability is inversely related to the intensity of stabilizing selection. A corollary is that a rudimentary or vestigial structure should be highly variable. This relationship between rudimentation and variability, however, may simply be part of a continuum, as several studies have shown that variability and size of a structure are inversely related. This study tests whether the first metacarpal (MC1) in Ateles geoffroyi and Colobus guereza and the second metacarpal (MC2) in Perodicticus potto are highly variable in their lengths relative to their other metapodials. The former two species have rudimentary thumbs, and the latter species has a rudimentary index finger. Fourteen other species of primates are included in the comparison. The results show that MC1 in A. geoffroyi and C. guereza and MC2 in P. potto are the relatively shortest first and second metapodials, respectively, in this sample of primates. However, an intraspecific analysis shows that neither MC1 in A. geoffroyi and C. guereza nor MC2 in P. potto is significantly more variable than the other metapodials. Nevertheless, an interspecific analysis shows that MC1 in A. geoffroyi and C. guereza is relatively the most variable among the first metapodials (i.e., MC1 and first metatarsal) in this study. MC2 in P. potto, however, is of relatively low variability compared with the other primates. These contrasting results are interpreted in terms of the developmental and evolutionary biology of digits.