Melanoma targeting with alpha-melanocyte stimulating hormone analogs labeled with fac-[Tc-99m(CO)(3)](+): effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized alpha-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of alpha-MSH (Ac-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of alpha-MSH analogs by comparing the pharmacokinetics profile of the (99)mTc-labeled cyclic peptide beta Ala-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2 with that of the linear analog beta Ala-Nle-Asp-His-DPhe-Arg-Trp-Lys-NH2 in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of beta Ala, and the resulting pz-peptide conjugates were reacted with the fac-[Tc-99m(CO)(3)](+) moiety. The Tc-99m(CO)(3)-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic Tc-99m(CO)(3)-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 degrees C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 +/- 0.83 and 11.31 +/- 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear Tc-99m(CO)(3)-pz-peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle(4),DPhe(7))-alpha MSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively)

In vivo and in vitro studies of [M(η6-pseudoerlotinib)2]+ sandwich complexes (M = Re, 99mTc)

The pursuit of molecular imaging for tumors has led to endeavors focused on targeting epidermal growth factor receptors (EGFR) through monoclonal antibodies or radionuclide-labelled EGF analogs with 99mTc, 111In, or 131I. In this context, various 99mTc-labeled EGFR inhibitors using quinazoline structures have been reported based on the so-called pendant approach and on two types of complexes and labelling strategies: “4 + 1” mixed ligand complexes and fac-tricarbonyl complexes. Apart from this approach, which alters lead structures by linking pharmacophores to chelator frameworks through different connectors, the integrated incorporation of topoisomerase and tyrosine kinase inhibitors into Re and 99mTc complexes has not been explored. Here we present [M(η6-inhibitor)2]+ (M = Re, 99mTc) and [Re(η6-bz)(η6-inhibitor)]+ complexes, where the core structure of an EGFR tyrosine kinase inhibitor binds directly to the metal center. These complexes exhibit potential for tumor imaging: initial biological investigations highlight the influence of one versus two bound inhibitors on the metal center

Tricarbonyl M(I) (M = Re, 99mTc) complexes bearing acridine fluorophores : synthesis, characterization, DNA interaction studies and nuclear targeting

© The Royal Society of Chemistry 2010New pyrazolyl-diamine ligands with acridine derivatives at the 4-position of the pyrazolyl ring were synthesized and characterized (L1 and L2). Coordination towards the fac-[M(CO)3]+ (M = Re, 99mTc) led to complexes fac-[M(CO)3(κ3-L)] (L = L1: M = Re1, Tc1; L = L2: M = Re2, Tc2). The interaction of the novel pyrazolyl-diamine ligands (L1 and L2) and rhenium(I) complexes (Re1 and Re2) with calf thymus DNA (CT-DNA) was investigated by a variety of techniques, namely UV-visible , fluorescence spectroscopy and circular and linear dichroism . Compounds L1 and Re1 have moderate affinity to CT-DNA and bind to DNA by intercalation, while L2 and Re2 have a poor affinity for CT-DNA. Moreover, LD measurements showed that L1 and Re1 act as perfect intercalators . By confocal fluorescence microscopy we found that L1 and Re1 internalize and localize in the nucleus of B16F1 murine melanoma cells . The congener Tc1 complex also targets the cell nucleus exhibiting a time-dependent cellular uptake and a fast and high nuclear internalization (67.2% of activity after 30 min). Plasmid DNA studies have shown that Tc1 converts supercoiled (sc) puc19 DNA to the open circular (oc) form.Teresa Esteves and Sofia Gama thank the FCT for a doctoral and postdoctoral research grants (SFRH/BD/29154/2006 and SFRH/BPD/29564/2006, respectively). COST Action D39 is also acknowledge. The QITMS instrument was acquired with the support of the Programa Nacional de Reequipamento Científico (Contract>REDE/1503/REM/2005-ITN) of Fundação para a Ciência e a Tecnologia and is part of RNEM - Rede Nacional de Espectrometria de Massa

NMR structural analysis of MC1R-targeted rhenium(I) metallopeptides and biological evaluation of 99mTc(I) congeners

The melanocortin receptor 1 (MC1R) is a specific molecular target for detection and therapy of melanoma, as it is overexpressed in human melanomas. The design of novel peptide-based MC1R-specific probes for imaging or targeted radionuclide therapy is being actively pursued. Aiming to visualize melanoma in vivo by single photon emission computed tomography (SPECT) imaging using 99mTc(CO) 3-labeled cyclic melanocyte stimulating hormone analogues, we have designed the novel cyclic peptides c[S-NO 2-C 6H-CO-His-DPhe-Arg-Trp-Cys]-Lys-NH 2 (1) and c[NH-NO 2-C 6H 3-CO-His-DPhe-Arg-Trp-Lys]-Lys-NH 2 (2) and their corresponding conjugates c[S-NO 2-C 6H 3-CO-His-DPhe-Arg-Trp-Cys]-Lys(pz)-NH 2 (L1) and c[NH-NO 2-C 6H 3-CO-His-DPhe-Arg-Trp-Lys]- Lys(pz)-NH 2 (L2), which incorporate a pyrazolyl-diamine chelating unit (pz). Upon reaction with adequate organometallic precursors, we prepared the metalated peptides c[S-NO 2-C 6H 3-CO-His- DPhe-Arg-Trp-Cys]-Lys(pz-M(CO) 3)-NH 2 (M = Re (Re1), Tc (Tc1)) and c[NH-NO 2-C 6H 3-CO-His-DPhe-Arg-Trp- Lys]-Lys(pz-M(CO) 3)-NH 2 (M = Re (Re2), Tc (Tc2)). Competitive binding affinity assays demonstrated that metalation of L1 and L2 did not lead to a significant decrease of binding affinity toward MC1R, as concluded by comparing the IC 50 values of Re1 (IC 50 = 690 ± 250 nM) and Re2 (IC 50 = 176 ± 5 nM) to those of the nonmetalated conjugates L1 (IC 50 = 430 ± 100 nM) and L2 (IC 50 = 179 ± 39 nM). Furthermore, the potency of the alkylamine-bridged peptide derivatives is superior to that of the alkylthioaryl-bridged derivatives. NMR structural analysis performed for 1, L1, and Re1 has shown that the peptide moiety displayed an atypical β-turn conformation in solution. The three-dimensional structural features of the peptide moiety were also conserved upon conjugation to the chelator and, most importantly, after metalation. Despite presenting a significant cell internalization degree and moderate retention in murine melanoma cells, the radiopeptides Tc1 and Tc2 displayed poor tumor-targeting properties in a B16F1 melanoma-bearing mouse model. © 2012 American Chemical Society.Peer Reviewe

NMR Insights into the Structure-Function Relationships in the Binding of Melanocortin Analogues to the MC1R Receptor

Linear and cyclic analogues of the alpha-melanocyte stimulating hormone (alpha-MSH) targeting the human melanocortin receptor 1 (MC1R) are of pharmacological interest for detecting and treating melanoma. The central sequence of alpha-MSH (His-Phe-Arg-Trp) has been identified as being essential for receptor binding. To deepen current knowledge on the molecular basis for alpha-MSH bioactivity, we aimed to understand the effect of cycle size on receptor binding. To that end, we synthesised two macrocyclic isomeric alpha-MSH analogues, c[NH-NO2-C6H3-CO-His-DPhe-Arg-Trp-Lys]-Lys-NH2 (CycN-K6) and c[NH-NO2-C6H3-CO-His-DPhe-Arg-Trp-Lys-Lys]-NH2 (CycN-K7). Their affinities to MC1R receptor were determined by competitive binding assays, and their structures were analysed by H-1 and C-13 NMR. These results were compared to those of the previously reported analogue c[S-NO2-C6H3-CO-His-DPhe-Arg-Trp-Cys]-Lys-NH2 (CycS-C6). The MC1R binding affinity of the 22-membered macrocyclic peptide CycN-K6 (IC50 = 155 +/- 16 nM) is higher than that found for the 25-membered macrocyclic analogue CycN-K7 (IC50 = 495 +/- 101 nM), which, in turn, is higher than that observed for the 19-membered cyclic analogue CycS-C-6 (IC50 = 1770 +/- 480 nM). NMR structural study indicated that macrocycle size leads to changes in the relative dispositions of the side chains, particularly in the packing of the Arg side chain relative to the aromatic rings. In contrast to the other analogues, the 22-membered cycle's side chains are favorably positioned for receptor interaction.Peer reviewe

STRUCTURAL CHARACTERIZATION OF RHENIUM(I) METALLOPEPTIDES TO PROBE MELANOCORTIN-1 RECEPTOR

Peer Reviewe

NMR Structural Analysis of MC1R-Targeted Rhenium(I) Metallopeptides and Biological Evaluation of ^99mTc(I) Congeners

The melanocortin receptor 1 (MC1R) is a specific molecular target for detection and therapy of melanoma, as it is overexpressed in human melanomas. The design of novel peptide-based MC1R-specific probes for imaging or targeted radionuclide therapy is being actively pursued. Aiming to visualize melanoma in vivo by single photon emission computed tomography (SPECT) imaging using ^99mTc­(CO)₃-labeled cyclic melanocyte stimulating hormone analogues, we have designed the novel cyclic peptides c­[S-NO₂-C₆H-CO-His-DPhe-Arg-Trp-Cys]-Lys-NH₂ (<b>1</b>) and c­[NH-NO₂-C₆H₃-CO-His-DPhe-Arg-Trp-Lys]-Lys-NH₂ (<b>2</b>) and their corresponding conjugates c­[S-NO₂-C₆H₃-CO-His-DPhe-Arg-Trp-Cys]-Lys­(pz)-NH₂ (<b>L1</b>) and c­[NH-NO₂-C₆H₃-CO-His-DPhe-Arg-Trp-Lys]-Lys­(pz)-NH₂ (<b>L2</b>), which incorporate a pyrazolyl-diamine chelating unit (pz). Upon reaction with adequate organometallic precursors, we prepared the metalated peptides c­[S-NO₂-C₆H₃-CO-His-DPhe-Arg-Trp-Cys]-Lys­(pz-M­(CO)₃)-NH₂ (M = Re (<b>Re1</b>), Tc (<b>Tc1</b>)) and c­[NH-NO₂-C₆H₃-CO-His-DPhe-Arg-Trp-Lys]-Lys­(pz-M­(CO)₃)-NH₂ (M = Re (<b>Re2</b>), Tc (<b>Tc2</b>)). Competitive binding affinity assays demonstrated that metalation of <b>L1</b> and <b>L2</b> did not lead to a significant decrease of binding affinity toward MC1R, as concluded by comparing the IC₅₀ values of <b>Re1</b> (IC₅₀ = 690 ± 250 nM) and <b>Re2</b> (IC₅₀ = 176 ± 5 nM) to those of the nonmetalated conjugates <b>L1</b> (IC₅₀ = 430 ± 100 nM) and <b>L2</b> (IC₅₀ = 179 ± 39 nM). Furthermore, the potency of the alkylamine-bridged peptide derivatives is superior to that of the alkylthioaryl-bridged derivatives. NMR structural analysis performed for <b>1</b>, <b>L1</b>, and <b>Re1</b> has shown that the peptide moiety displayed an atypical β-turn conformation in solution. The three-dimensional structural features of the peptide moiety were also conserved upon conjugation to the chelator and, most importantly, after metalation. Despite presenting a significant cell internalization degree and moderate retention in murine melanoma cells, the radiopeptides <b>Tc1</b> and <b>Tc2</b> displayed poor tumor-targeting properties in a B16F1 melanoma-bearing mouse model