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

Ramesh Alleti, Josef Vagner, Dilani Chathurika Dehigaspitiya, Valerie E. Moberg, N. G R D Elshan, Narges K. Tafreshi, Nabila Brabez, Craig S. Weber, Ron Lynch, Victor J Hruby, Robert J. Gillies, David L. Morse, Eugene A Mash

Research output: Contribution to journalArticle

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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 K d 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.

Original languageEnglish (US)
Pages (from-to)5029-5038
Number of pages10
JournalBioorganic and Medicinal Chemistry
Volume21
Issue number17
DOIs
StatePublished - Sep 1 2013

Fingerprint

Melanocortin Receptors
Melanocyte-Stimulating Hormones
Competitive Binding
Fluorescence
Assays
Squalene
Pentetic Acid
Cholecystokinin B Receptor
Ligands
Solid-Phase Synthesis Techniques
HEK293 Cells

Keywords

  • Competition binding assays
  • Fluorescent probes
  • Melanocortin 4 receptor
  • Saturation binding assays
  • Time-resolved fluorescence

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Drug Discovery
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology
  • Clinical Biochemistry
  • Biochemistry

Cite this

Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors. / Alleti, Ramesh; Vagner, Josef; Dehigaspitiya, Dilani Chathurika; Moberg, Valerie E.; Elshan, N. G R D; Tafreshi, Narges K.; Brabez, Nabila; Weber, Craig S.; Lynch, Ron; Hruby, Victor J; Gillies, Robert J.; Morse, David L.; Mash, Eugene A.

In: Bioorganic and Medicinal Chemistry, Vol. 21, No. 17, 01.09.2013, p. 5029-5038.

Research output: Contribution to journalArticle

Alleti, Ramesh ; Vagner, Josef ; Dehigaspitiya, Dilani Chathurika ; Moberg, Valerie E. ; Elshan, N. G R D ; Tafreshi, Narges K. ; Brabez, Nabila ; Weber, Craig S. ; Lynch, Ron ; Hruby, Victor J ; Gillies, Robert J. ; Morse, David L. ; Mash, Eugene A. / Synthesis and characterization of time-resolved fluorescence probes for evaluation of competitive binding to melanocortin receptors. In: Bioorganic and Medicinal Chemistry. 2013 ; Vol. 21, No. 17. pp. 5029-5038.
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AU - Elshan, N. G R D

AU - Tafreshi, Narges K.

AU - Brabez, Nabila

AU - Weber, Craig S.

AU - Lynch, Ron

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N2 - 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 K d 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.

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