Design, synthesis, and biological evaluation of non-peptidic ligands at the Xenopus laevis skin-melanocortin receptor.

Taking the tripeptide D-Trp-Arg-Leu-NH(2) as a lead for a Xenopus laevis skin-melanocortin (MC) receptor antagonist, thirteen non-peptidic compounds were synthesized and biologically evaluated at Xenopus laevis melanophores. Six competitive antagonists (shown by Schild analysis) and one partial agonist were identified with moderate activity (IC(50): 5-10 microM). Tryptophanamides with aliphatic side chains were inactive whereas basic residues restored activity. Introducing an imidazole residue yielded partial agonist activity (EC50: 32 microM). Interestingly, constraining the inactive S-tryptophan-isoamylamide to a beta-carboline ring yielded an MC receptor antagonist (42). The specificity for MC receptors was tested at various G-protein coupled receptors. In conclusion, the synthesis of non-peptidic MC receptor antagonists is described which may serve as lead compounds for further studies.

Differential effects of mu-opioid receptor ligands on Ca(2+) signaling.

Activation of mu-opioid receptors (MORs) transfected into human embryonic kidney 293 cells, caused a multiphasic increase in cytosolic free Ca(2+) levels (Ca(2+)i). The first Ca(2+)i maximum (peak 1) between 5 and 7 s depended on the presence of extracellular Ca(2+) (Ca(2+)e). The second phase peaking at approximately 15 s (peak 2) was independent of Ca(2+)e and thus represents Ca(2+) release from intracellular stores. A decrease in temperature from 37 to 25 degrees C also caused reduction of peak 1 but not peak 2, suggesting that the two responses arise from mechanistically distinct pathways. A delayed Ca(2+)e-dependent third response phase is thought to represent capacitative Ca(2+)e influx evoked after release of Ca(2+) from internal stores. Agonists and antagonists of two major classes of opioid ligands, oxymorphinans (morphine and naloxone) and oripavines (etorphine and diprenorphine), had differential effects on Ca(2+) currents. Although morphine activated both phases with equal potency, etorphine was 20-fold less potent at stimulating peak 1 over peak 2. Similarly, the antagonists, naloxone and diprenorphine, blocked the Ca(2+) response to each agonist with greatly varying potencies. Specifically, concomitant injection of diprenorphine failed to affect peak 1 (thought to represent rapid Ca(2+)e influx) stimulated by morphine while fully blocking peak 2 (intracellular Ca(2+) release). However, diprenorphine potently inhibited peak 1 as well when added to the cells before morphine, indicating limited or slow access of diprenorphine to these morphine binding sites. The existence of multiple, functionally distinct binding site conformations could account for these findings. In conclusion, different opioid ligands can differentially affect Ca(2+) response patterns resulting from MOR activation.

Functional screening of G protein-coupled receptors by measuring intracellular calcium with a fluorescence microplate reader.

Ligand binding studies reveal information about affinity to G protein-coupled receptors (GPCRs) rather than functional properties. Increase in intracellular Ca(2+) appears to represent a universal second messenger signal for a majority of recombinant GPCRs. Here, we exploit Ca(2+) signaling as a fast and sensitive functional screening method for a number of GPCRs coupled to different G proteins. Ca(2+) fluorescence measurements are performed using Oregon Green 488 BAPTA-1/AM and a microplate reader equipped with an injector. Buffer alone or test compounds dissolved in buffer are injected into a cell suspension, and fluorescence intensity is recorded for 30 s. Each of the GPCRs tested--G(q)-coupled P2Y(2), G(s)-coupled dopamine D1 and D5, G(i)-coupled dopamine D2L, and G(q/11)-coupled muscarinic acetylcholine M1--yielded a significant rise in intracellular free [Ca(2+)] on agonist stimulation. Agonist stimulation was dose dependent, as shown for ATP or UTP stimulation of P2Y(2) receptors (EC(50) = 1 microM), SKF38393 stimulation of hD1 and hD5 (EC(50) = 18.1 nM and 2.7 nM), and quinpirole at hD2L (EC(50) = 6.5 nM). SCH23390 (at hD1 and hD5) and spiperone, haloperidol, and clozapine (at hD2L) competitively antagonized the Ca(2+) response. Furthermore, the Ca(2+) assay served to screen suramin analogs for antagonistic activity at P2Y(2) receptors. Screening at dopamine receptors revealed LE300, a new lead for a dopamine receptor antagonist. Advantages of the assay include fast and simple 96- or 384-well plate format (high-throughput screening), use of a visible light-excitable fluorescent dye, applicability to a majority of GPCRs, and simultaneous analysis of distinct Ca(2+) fluxes.

Monitoring intracellular pH changes in response to osmotic stress and membrane transport activity using 5-chloromethylfluorescein.

Intracellular free H+ concentration (pHi) responds to numerous extracellular stimuli. The use of fluorescent indicator dyes to measure pHi is strongly influenced by the ability of target cells to retain activated dye within the cytoplasmic compartment. Here, 3 pH-sensitive indicator dye - acetoxymethyl (AM) esters of SNARF-1 and BCECF, and the thiol-reactive 5-chloromethyfluorescein (CMFDA) - were examined for monitoring pHi. The stability of pH measurements was strongly affected by temperature, cell type, indicator dye, and use of transport inhibitors to prevent dye export. Cellular retention of CMFDA, which forms covalent complexes, was sufficient to permit monitoring of transient pHi changes over extended time periods in a multi-well plate assay format. In human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells, increasing osmotic pressure caused a significant rise in pHi. In contrast, activation of native or transfected beta-adrenergic, cholinergic, and d and m opioid receptors did not measurably affect pHi in HEK293 cells. Decreases in pHi were observed in CHO cells expressing the human H+/peptide transporter PEPT1 upon addition of dipeptide substrates. The use of CMFDA in multi-well formats should facilitate study of osmotic and transport activity and screening for drugs that affect pHi.