Synthesis and Characterization of Time-resolved Fluorescence Probes for Evaluation of Competitive Binding to Melanocortin Receptors

• Published in: Bioorganic & medicinal chemistry Vol. 21; no. 17; pp. 5029 - 5038
• Main Authors: Alleti, Ramesh, Vagner, Josef, Dehigaspitiya, Dilani Chathurika, Moberg, Valerie E., Elshan, N.G.R.D., Tafreshi, Narges K., Brabez, Nabila, Weber, Craig S., Lynch, Ronald M., Hruby, Victor J., Gillies, Robert J., Morse, David L., Mash, Eugene A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-09-2013
• Subjects: Avidity; Binding, Competitive; Competition binding assays; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Fluorescent probes; HEK293 Cells; Humans; Melanocortin 4 receptor; Pentetic Acid - chemistry; Peptides - chemical synthesis; Peptides - chemistry; Peptides - metabolism; Protein Binding; Receptor, Cholecystokinin B - chemistry; Receptor, Cholecystokinin B - genetics; Receptor, Cholecystokinin B - metabolism; Receptor, Melanocortin, Type 4 - chemistry; Receptor, Melanocortin, Type 4 - genetics; Receptor, Melanocortin, Type 4 - metabolism; Receptors, Melanocortin - chemistry; Receptors, Melanocortin - metabolism; Saturation binding assays; Solid-Phase Synthesis Techniques; Time-resolved fluorescence; DIEA; hMC4R; Competition binding assays; Fluorescent probes; DMSO; TRF; TLC; TFA; BSA; DMEM; HRMS; NDP-α-MSH; THF; FT-ICR MS; Cl-HOBt; Fmoc-PEGO; IC50; HOBt; Saturation binding assays; Fmoc; DMF; DIC; DTPA; MSH; Time-resolved fluorescence; DCM; Melanocortin 4 receptor; MEM; TEAA; CuAAC; PEGO; ESI MS; TBTA; 1-hydroxybenzotriazole; dichloromethane; 6-chloro-1-hydroxybenzotriazole; copper(I)-catalyzed azide-alkyne cycloaddition; triethylammonium acetate; tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine; 9-fluorenylmethyoxycarbonyl; Fourier transform ion cyclotron resonance mass spectrometry; thin-layer chromatography; N,N-dimethylformamide; diisopropylethylamine; IC; high resolution mass spectroscopy; Dulbecco’s Modified Eagle Medium; electrospray ionization mass spectrometry; diethylenetriaminepentaacetic acid; bovine serum albumin; 1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic acid; human melanocortin 4 receptor; half maximal inhibitory concentration; His-DPhe-Arg-Trp; dimethyl sulfoxide; Ser-Nle-Glu-His-DPhe-Arg-Trp; diisopropyl carbodiimide; tetrahydrofuran; trifluoroacetic acid; Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH; 19-amino-5-oxo-3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid; Minimum Essential Medium
• ISSN: 0968-0896; 1464-3391
• DOI: 10.1016/j.bmc.2013.06.052

Probes for use in time-resolved fluorescence competitive binding assays at melanocortin receptors based on the parental ligands MSH(4), MSH(7), and NDP-α-MSH were prepared by solid phase synthesis methods, purified, and characterized. The saturation binding of these probes was studied using HEK-293 cells engineered to overexpress the human melanocortin 4 receptor (hMC4R) as well as the human cholecystokinin 2 receptor (hCCK2R). The ratios of non-specific binding to total binding approached unity at high concentrations for each probe. At low probe concentrations, receptor-mediated binding and uptake was discernable, and so probe concentrations were kept as low as possible in determining Kd values. The Eu-DTPA-PEGO-MSH(4) probe exhibited low specific binding relative to non-specific binding, even at low nanomolar concentrations, and was deemed unsuitable for use in competition binding assays. The Eu-DTPA-PEGO probes based on MSH(7) and NDP-α-MSH exhibited Kd values of 27±3.9nM and 4.2±0.48nM, respectively, for binding with hMC4R. These probes were employed in competitive binding assays to characterize the interactions of hMC4R with monovalent and divalent MSH(4), MSH(7), and NDP-α-MSH constructs derived from squalene. Results from assays with both probes reflected only statistical enhancements, suggesting improper ligand spacing on the squalene scaffold for the divalent constructs. The Ki values from competitive binding assays that employed the MSH(7)-based probe were generally lower than the Ki values obtained when the probe based on NDP-α-MSH was employed, which is consistent with the greater potency of the latter probe. The probe based on MSH(7) was also competed with monovalent, divalent, and trivalent MSH(4) constructs that previously demonstrated multivalent binding in competitive binding assays against a variant of the probe based on NDP-α-MSH. Results from these assays confirm multivalent binding, but suggest a more modest increase in avidity for these MSH(4) constructs than was previously reported.