Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors

Abstract

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.

Keywords: 1-(9H-fluoren-9-yl)-3,19-dioxo-2,8,11,14,21-pentaoxa-4,18-diazatricosan-23-oic acid; 1-hydroxybenzotriazole; 19-amino-5-oxo-3,10,13,16-tetraoxa-6-azanonadecan-1-oic acid; 6-chloro-1-hydroxybenzotriazole; 9-fluorenylmethyoxycarbonyl; Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH(2); BSA; Cl-HOBt; Competition binding assays; CuAAC; DCM; DIC; DIEA; DMEM; DMF; DMSO; DTPA; Dulbecco’s Modified Eagle Medium; ESI MS; FT-ICR MS; Fluorescent probes; Fmoc; Fmoc-PEGO; Fourier transform ion cyclotron resonance mass spectrometry; HOBt; HRMS; His-DPhe-Arg-Trp; IC(50); MEM; MSH(4); MSH(7); Melanocortin 4 receptor; Minimum Essential Medium; N,N-dimethylformamide; NDP-α-MSH; PEGO; Saturation binding assays; Ser-Nle-Glu-His-DPhe-Arg-Trp; TBTA; TEAA; TFA; THF; TLC; TRF; Time-resolved fluorescence; bovine serum albumin; copper(I)-catalyzed azide-alkyne cycloaddition; dichloromethane; diethylenetriaminepentaacetic acid; diisopropyl carbodiimide; diisopropylethylamine; dimethyl sulfoxide; electrospray ionization mass spectrometry; hMC4R; half maximal inhibitory concentration; high resolution mass spectroscopy; human melanocortin 4 receptor; tetrahydrofuran; thin-layer chromatography; time-resolved fluorescence; triethylammonium acetate; trifluoroacetic acid; tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine.

Figure 1

Top: A portion of the DEPT 135 ¹³C NMR spectrum of 8 in CD₃OD. Bottom: A portion of the ¹³C NMR spectrum of 8 in CD₃OD. The lines near 73 ppm are due to tertiary carbons bearing OH groups and the lines between 76–79 ppm are due to secondary carbons bearing OH groups. The spectral complexity occurs because 8 is a mixture of regioisomers and stereoisomers.

Figure 2

Saturation binding curves for Eu-DTPA-PEGO probes 2–4. Total binding (●). Non-specific binding (■). Specific binding (▲). The graphs in column 3 are truncated expansions of the graphs in column 2 and include a specific binding curve.

Scheme 1

Synthesis of monovalent constructs 12a–15a and divalent constructs 12b–15b.^{a a}Compounds 8, 9, and 10 were obtained as complex mixtures of stereoisomers and regioisomers. Compounds 12a–15a and 12b–15b, derived from 9 and 10, respectively, are likewise assumed to be the related mixtures of stereoisomers and regioisomers.