Minying Cai

Multiple N-Methylation of MT-II Backbone Amide Bonds Leads to Melanocortin Receptor Subtype hMC1R Selectivity: Pharmacological and Conformational Studies

Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but nonselective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4, and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation that resembles other alpha-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.

Sex-specific mediation of opioid-induced hyperalgesia by the melanocortin-1 receptor.

Background:N-Methyl-d-aspartate receptor antagonists reverse hyperalgesia during morphine infusion in male mice only. Because the melanocortin-1 receptor can act as a female-specific counterpart to N-methyl-d-aspartate receptors in &kgr;-opioid analgesic mechanisms, the authors assessed the contribution of melanocortin-1 receptors to the sex-specific mechanisms underlying morphine hyperalgesia. Methods:The tail-withdrawal test was used to compare the nociceptive responses of male and female C57BL/6J (B6) mice with those of C57BL/6J-Mc1re/e (e/e) mice, spontaneous mutants of the B6 background lacking functional melanocortin-1 receptors, during continuous morphine infusion (1.6 and 40.0 mgkg−1 · 24 h−1). Separate groups of hyperalgesic B6 and outbred CD-1 mice were injected with MK-801 or MSG606, selective N-methyl-d-aspartate and melanocortin-1 receptor antagonists, respectively. Results:Morphine infusion (40.0 mg · kg−1 · 24 h−1) reduced baseline withdrawal latencies by 45–55% in B6 mice of both sexes, indicating hyperalgesia; this increased nociception was manifest in male e/e mice only. Although MK-801 reversed hyperalgesia in male mice only, increasing latencies by 72%, MSG606 increased latencies by approximately 60% exclusively in females. A lower morphine infusion dose (1.6 mg · kg−1 · 24 h−1) reduced baseline withdrawal latencies by 45–52% in B6 and e/e mice of both sexes, which was reversed by MK-801, but not MSG606, in both male and female B6 mice. Conclusions:The data indicate the sex-specific mediation of high-dose morphine-induced hyperalgesia by N-methyl-d-aspartate and melanocortin-1 receptors in male and female mice, respectively, suggesting a broader relevance of this known sexual dimorphism. The data further indicate that the neural substrates contributing to hyperalgesia are morphine dose-dependent.

Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression

Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with ICV infusion of the specific MC3-R agonist, [D-Trp(8)]-gamma-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that ICV infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.

Design of Peptide and Peptidomimetic Ligands with Novel Pharmacological Activity Profiles

Peptide hormones and neurotransmitters are of central importance in most aspects of intercellular communication and are involved in virtually all degenerative diseases. In this review, we discuss physicochemical approaches to the design of novel peptide and peptidomimetic agonists, antagonists, inverse agonists, and related compounds that have unique biological activity profiles, reduced toxic side effects, and, if desired, the ability to cross the blood-brain barrier. Designing ligands for specific biological and medical needs is emphasized, as is the close collaboration of chemists and biologists to maximize the chances for success. Special emphasis is placed on the use of conformational (ϕ-ψ space) and topographical (χ space) considerations in design.

Contribution of the Conserved Amino Acids of the Melanocortin-4 Receptor in d-[Nle4,Phe7]-α-Melanocyte-stimulating Hormone Binding and Signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for α-melanocortin-stimulating hormone (α-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for α-MSH binding and signaling. α-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and α-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter α-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for α-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu100 in TM2, Asp122, Asp126 in TM3 and Trp258, Phe261, His264 in TM6 are involved in α-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for α-MSH binding and signaling.

Design and Microwave-Assisted Synthesis of Novel Macrocyclic Peptides Active at Melanocortin Receptors: Discovery of Potent and Selective hMC5R Receptor Antagonists

Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9(PG-20N) is a selective and competitive hMC5R antagonist, with IC 50 of 130 +/- 11 nM, and a pA 2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC 50 values of 38 +/- 3 nM and 58 +/- 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.

Design and synthesis of novel χ2-constrained phenylalanine, naphthylalanine, and tryptophan analogues and their use in biologically active melanotropin peptides

A series of novel hydrophobic, bulky χ2-constrained phenylalanine, naphthylalanine, and tryptophan derivatives was designed and synthesized. The key steps involved asymmetric hydrogenations of α-enamides using Burks DuPHOS-based Rh(I) catalysts to give high enantiomerically pure α-amino acid derivatives. The subsequent Suzuki cross couplings of the amino acid analogues with boronic acid derivatives afforded these aromatic substituted amino acids in high yields and with high enantioselectivity. The incorporation of these novel χ2-constrained amino acids into peptides and peptidomimetics provides fruitful information in the development of peptide and peptidomimetic ligands of melanotropins and an understanding of the interactions between ligands and receptors/acceptors.

Design of novel melanotropin agonists and antagonists with high potency and selectivity for human melanocortin receptors

alpha-MSH and gamma-MSH are the natural endogenous hormones for the human melanocortin-1, 3, 4 and 5 receptors (hMC1R, hMC3R, hMC4R and hMC5R). These and more potent, stable and prolonged acting analogues such as NDP-alpha-MSH, MT-II and SHU-9119 are not very receptor selective. To develop potent and selective agonist and antagonist ligands for the melanocortin receptors we have used state-of-the-art biophysical studies, computational chemistry, and design of conformational and topographical constraints with novel templates.

Cell signaling and trafficking of human melanocortin receptors in real time using two-photon fluorescence and confocal laser microscopy: differentiation of agonists and antagonists.

Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G‐protein‐coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine‐labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two‐photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists‐bound melanocortin receptors, on the other hand, did not recruit β‐arrestins, and remained in the cell membrane even after long‐term (30 min) treatment. Agonist‐mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin‐dependent endocytosis, but not to caveolae inhibitors. In summary, agonist‐mediated internalization of all subtypes of melanocortin receptors are dependent upon β‐arrestin‐mediated clathrin‐coated pits, whereas, β‐arrestin‐2 conjugated green fluorescence protein (β‐arrestin‐2‐GFP) recruitment is not dependent on protein kinase A activation. Real time two‐photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.

New Paradigms and Tools in Drug Design for Pain and Addiction

New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.