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±3.9 nM and 4.2±0.48 nM, 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.

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

“…Hence, the desired metal is usually introduced after cleavage of the peptide from the resin and purification, utilizing an excess of a metal halide (1.2–3 eq). In our hands, purification using the commonly employed technique of reversed-phase chromatography [3, 6] did not achieve adequate removal of unchelated metal ion contaminants. Recent literature describes the removal of excess metal ions from DTPA and DOTA chelates by means of “passive” removal techniques, such as size-exclusion chromatography and dialysis [7,8].…”

“…Given the low yield (9%) previously reported [3] for the synthesis of Eu-DTPA-PEGO-MSH(7)-NH 2 , an approach leading to a higher synthetic efficiency was devised, utilizing a different resin for solid-phase peptide synthesis (see Scheme 1). The metal-free peptide precursor DTPA-PEGO-MSH(7)-NH 2 was obtained in 64% yield following HPLC purification, and was characterized by mass spectrometry.…”

“…DTPA-PEGO-MSH(7)-NH 2 was synthesized manually via an N α -Fmoc solid-phase peptide synthesis strategy (Scheme 1) using a modified version of the previously published procedure [3]. Instead of Tentagel S resin (0.24 mmol/g loading) used in the previous work, Rink amide AM resin (200–400 mesh, 0.68 mmol/g loading) was used.…”

“…The reaction mixture was stirred at rt overnight. Salts were removed using a Sep-Pak ® C 18 reverse-phase (500 mg) column as previously reported [3, 6]. The product-containing fractions were concentrated and lyophilized to obtain the product Eu-DTPA-PEGO-MSH(7)-NH 2 as a fluffy white solid (9.1 mg) in 76% yield.…”

“…Due to their unique properties [5], luminescent lanthanide chelates are versatile nonradiolabeled alternatives to organic fluorophores, particularly in applications where background autofluorescence is a significant problem [6]. For example, the europium diethylenetriaminepentaacetic acid (DTPA 2 ) chelated probe Eu-DTPA-PEGO-MSH(7)-NH 2 and similar compounds have been routinely used to conduct in vitro binding experiments, where the time-resolved fluoresence from the lanthanide provides the readout in the assay [3]. However, the presence of unchelated Eu ions in formulations of probes such as Eu-DTPA-PEGO-MSH(7)-NH 2 can result in high background fluoresence and can lead to poor results in binding assays by overshadowing specific binding of the probe to targeted receptors (see Figure 1).…”