Molecular modeling of melanocortin receptors

Molecular modeling of G-protein coupled receptors (GPCRs) remains a challenge due to the limited availability of structural information for the receptors. Molecular modeling approaches for melanocortin receptors (MCRs) fall into three categories: structure-based, ligand-based, and proteochemometric. Homology modeling combined with the information obtained from site-directed mutagenesis of receptors, recombined chimeric mutations of receptors and the structures of melanocortin type 4 receptor (MC4R) peptide ligands, has provided insights on detailed ligand-receptor interactions. Still, homology models based on the structures of bacteriorhodopsin (bR) or bovine rhodopsin as templates have not reached atomic level accuracy, making them unsuitable for rational drug design. On the other hand, availability of a large number of potent ligands of MCRs, especially those for the therapeutically important MC4R, has fueled ligand-based approaches, including automated pharmacophore query optimization and pharmacophore-based virtual screening. Proteochemometrics, a novel technology for the analysis of intermolecular interactions between ligand and receptor, has also shown great value in obtaining detailed information on molecular recognition and providing guidance to ligand design. In this review, the strengths and limitations of homology modeling, pharmacophore modeling and proteochemometrics modeling of MCRs are evaluated.     

Identifying novel adenosine receptor ligands by simultaneous proteochemometric modeling of rat and human bioactivity data

The four subtypes of adenosine receptors form relevant drug targets in the treatment of, e.g., diabetes and Parkinson's disease. In the present study, we aimed at finding novel small molecule ligands for these receptors using virtual screening approaches based on proteochemometric (PCM) modeling. We combined bioactivity data from all human and rat receptors in order to widen available chemical space. After training and validating a proteochemometric model on this combined data set (Q(2) of 0.73, RMSE of 0.61), we virtually screened a vendor database of 100910 compounds. Of 54 compounds purchased, six novel high affinity adenosine receptor ligands were confirmed experimentally, one of which displayed an affinity of 7 nM on the human adenosine A(1) receptor. We conclude that the combination of rat and human data performs better than human data only. Furthermore, we conclude that proteochemometric modeling is an efficient method to quickly screen for novel bioactive compounds.     

Cytisine derivatives as high affinity nAChR ligands: synthesis and comparative molecular field analysis

A number of new N-substituted cytisine derivatives were prepared and tested, along with similar compounds already described by us and others, as high affinity neuronal acetylcholine receptor ligands. Structure-affinity relationships were discussed in the light of our recently proposed pharmacophore model for nicotinic receptor agonists. The most significant physicochemical interactions modulating the receptor-ligand binding were detected at the three dimensional (3D) level by means of comparative molecular field analysis (CoMFA). The best predictive PLS model was a single-field steric model showing good statistical figures: n = 17, Q2 = 0.717, s(ev) = 0.566, r2 = 0.942, s = 0.275.     

Structure, function and regulation of the melanocortin receptors

Melanocortin receptors belong to the seven-transmembrane (TM) domain proteins that are coupled to G-proteins and signaled through intracellular cyclic adenosine monophosphate. Many structural features conserved in other G-protein coupled receptors (GPCRs) are found in the melanocortin receptors. There are five melanocortin receptor subtypes and each of the melanocortin receptor subtypes has a different pattern of tissue expression and has its own profile regarding the relative potency of different melanocortin peptides. α-, β-, and γ-MSH and ACTH are known endogenous agonist ligands for the melanocortin receptors. Agouti and AgRP are the only known naturally occurring antagonists of the melanocortin receptors. We have examined the molecular basis of all five human melanocortin receptors for different ligand binding affinities and potencies using chimeric and mutated receptors. Our studies indicate that human melanocortin MC₁ receptor, human melanocortin MC₃ receptor, human melanocortin MC₄ receptor and human melanocortin MC₅ receptor utilize orthosteric sites for non selective agonists, α-MSH and NDP-?α-MSH, high affinity binding and utilize allosteric sites for selective agonist or antagonist binding. Furthermore, our results indicate that molecular determinants of human melanocortin MC₂ receptor for ACTH binding and signaling are different from that of other melanocortin receptors. Many studies also indicate that agonists can induce different conformation changes of melanocortin receptors, which then lead to the activation of different signaling pathways, even when the expression level of receptor and the strength of stimulus–response coupling are the same. This finding may provide new information for the design of drugs for targeting melanocortin receptors.     

Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC(2)-selective ligands

A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C(6)-VIP), behaved as a VPAC(2) preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C(6)-VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C(6)-VIP, its analog AcHis(1)-VIP, and the VPAC(2)-selective agonist Ro 25-1553 on the wild-type VPAC(1) and VPAC(2) receptors and on the chimeric receptors exchanging the different domains between both receptors. VIP had a normal affinity and efficacy on the chimeras starting with the amino-terminal VPAC(2) receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC(2) receptors is supported by the amino-terminal extracellular domain, whereas the ability to prefer C(6)-VIP over VIP is supported by the VPAC(2) fifth transmembrane (TM5)-EC(3) receptor domain. These results further support the hypothesis that the central and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular amino-terminal region domain, whereas the amino-terminal VIP amino acids bind to the TM receptor core. VIP had a reduced affinity and efficacy on the N-VPAC(1)/VPAC(2) and on the N-->EC(2)-VPAC(1)/VPAC(2) chimeric receptors. C(6)-VIP behaved as a high-affinity agonist on these constructions. The antagonists [AcHis(1),D-Phe(2),Lys(15),Arg(16), Leu(27)]VIP(3-7)/GRF(8-27) and VIP(5-27) had comparable affinities for the wild-type receptors and for the two latter chimeras, supporting the hypothesis that these chimeras were properly folded but unable to reach the high-agonist-affinity, active receptor conformation in response to VIP binding.     

Incorporation of a bioactive reverse-turn heterocycle into a peptide template using solid-phase synthesis to probe melanocortin receptor selectivity and ligand conformations by 2D 1H NMR

By use of a solid-phase synthetic approach, a bioactive reverse turn heterocycle was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein-coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nanomolar potency. Biophysical structural analysis [2D (1)H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families compared to the reference peptide and result in a novel template for further structure-function studies.     

Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro5, DNal (2')8]-gamma-MSH selectivity

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with DNal (2') in gamma-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, DNal (2')8]-gamma-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, DNal (2')8]-gamma-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, DNal (2')8]-gamma-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, DNal (2')8]-gamma-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, DNal (2')-gamma-MSH selective activity at MC4R.     

Design, synthesis, and biological studies of efficient multivalent melanotropin ligands: tools toward melanoma diagnosis and treatment

To achieve early detection and specific cancer treatment, we propose the use of multivalent interactions in which a series of binding events leads to increased affinity and consequently to selectivity. Using melanotropin (MSH) ligands, our aim is to target melanoma cells which overexpress melanocortin receptors. In this study, we report the design and efficient synthesis of new trivalent ligands bearing MSH ligands. Evaluation of these multimers on a cell model engineered to overexpress melanocortin 4 receptors (MC4R) showed up to a 350-fold increase in binding compared to the monomer, resulting in a trivalent construct with nanomolar affinity starting from a micromolar affinity ligand. Cyclic adenosine monophosphate (cAMP) production was also investigated, leading to more insights into the effects of multivalent compounds on transduction mechanisms.