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

Linear and cyclic analogues of the α-melanocyte stimulating hormone (α-MSH) targeting the human melanocortin receptor 1 (MC1R) are of pharmacological interest for detecting and treating melanoma. The central sequence of α-MSH (His-Phe-Arg-Trp) has been identified as being essential for receptor binding. To deepen current knowledge on the molecular basis for α-MSH bioactivity, we aimed to understand the effect of cycle size on receptor binding. To that end, we synthesised two macrocyclic isomeric α-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 ¹H and 13C 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-C6 (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.

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.

Evaluation of novel 99mTc(I)-labeled homobivalent α-melanocyte-stimulating hormone analogs for melanocortin-1 receptor targeting.

Aiming to apply the multivalency concept to melanoma imaging, we have assessed the in vivo melanocortin type 1 receptor (MC1R)-targeting properties of (99m)Tc(I)-labeled homobivalent peptide conjugates which contain copies of the α-melanocyte-stimulating hormone (α-MSH) analog [Ac-Nle(4), Asp(5), D-Phe(7), Lys(11)]α-MSH4-11 separated by linkers of different length (L(2) nine atoms and L(3) 14 atoms). The MC1R-binding affinity of L(2) and L(3) is significantly higher than that of the monovalent conjugate L(1). Metallation of these conjugates yielded the complexes fac-[M(CO)(3)(k(3)-L)](+) (M is (99m)Tc/Re; 1/1a, L is L(1); 2/2a, L is L(2); 3/3a, L is L(3)), with IC(50) values in the subnanomolar and nanomolar range. The MC1R-mediated internalization of 2 and 3 is higher than that of 1 in B16F1 melanoma cells. Biodistribution studies in melanoma-bearing mice have shown low nonspecific accumulation with a tumor uptake that correlates with IC(50) values. However, no correlation between tumor uptake and valency was found. Nevertheless, 2 displayed the highest tumor retention, and the best tumor to nontarget organ ratios.

Re and Tc tricarbonyl complexes: from the suppression of NO biosynthesis in macrophages to in vivo targeting of inducible nitric oxide synthase.

The in vivo molecular imaging of nitric oxide synthase (NOS), the enzyme responsible for the catalytic oxidation of l-arginine to citrulline and nitric oxide (NO), by noninvasive modalities could provide valuable insights into NO/NOS-related diseases. Aiming at the design of innovative (99m)Tc(I) complexes for targeting inducible NOS (iNOS) in vivo by SPECT imaging, herein we describe a set of novel (99m)Tc(CO)3 complexes (2-5) and the corresponding rhenium surrogates (2a-5a) containing the NOS inhibitor N(ω)-nitro-l-arginine. The latter is linked through its α-NH2 or α-COOH group and an alkyl spacer of variable length to the metal center. The complexes 2a (propyl spacer) and 3a (hexyl spacer), in which the α-NH2 group of the inhibitor is involved in the conjugation to the metal center, presented remarkable affinity for purified iNOS, being similar to that of the free nonconjugated inhibitor (K(i) = 3-8 µM) in the case of 3a (K(i) = 6 µM). 2a and 3a are the first examples of organometallic complexes that permeate through RAW 264.7 macrophage cell membranes, interacting specifically with the target enzyme, as confirmed by the suppression of NO biosynthesis in LPS-treated macrophages (2a, ca. 30% inhibition; 3a, ca. 50% inhibition). The (99m)Tc(I)-complexes 2 and 3, stable both in vitro and in vivo, also presented the ability to cross cell membranes, as demonstrated by internalization studies in the same cell model. The biodistribution studies in LPS-pretreated mature female C57BL6 mice have shown that 2 presented an overall higher uptake in most tissues of the LPS-treated mice compared to the control group (30 min postinjection). This increase is significant in lung (3.98 ± 0.63 vs to 0.99 ± 0.13%ID/g), which is known to be the organ with the highest iNOS expression after LPS treatment. These results suggest that the higher uptake in that organ may be related to iNOS upregulation.

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

New 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, (99m)Tc) led to complexes fac-[M(CO)(3)(kappa(3)-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.

Thiosemicarbazonate complexes with affinity for amyloid-ß fibers: synthesis, characterization and biological studies.

Aim: Obtain radioimages of amyloid-ß fibers using 99mTc-complexes. Methodology: Tridentate thiosemicarbazone and thiocarbonohydrazone ligands containing fragments (stilbene, azobenzene, benzothiazole or benzoxazole) with affinity for amyloid-ß fibers and its Re(I) complexes have been prepared. The molecular structures of several ligands and complexes were determined by x-ray diffraction. Binding affinity studies toward Aß1-42 fibers were performed for the ligands and Re(I) complexes. The ability of formation of some 99mTc(I) complexes, their biodistribution and in vivo stability have been established. Results & conclusion: Complexes of stilbene and benzothiazole thiosemicarbazonates show similar affinity for amyloid-ß fibers to the free ligand. These 99mTc complexes present a reasonable in vivo stability and a low capability to cross the blood-brain barrier although not sufficient to brain amyloid imaging.

A (99m)Tc(CO)(3)-labeled pyrazolyl-alpha-melanocyte-stimulating hormone analog conjugate for melanoma targeting.

Melanoma primary tumors can be, in most cases, removed surgically, whereas there is no satisfactory treatment for metastatic melanoma, being almost always lethal at this stage. Therefore, early detection of primary melanoma tumors is essential. The finding that melanocortin-1 receptor (MC1R) is overexpressed in isolated melanoma cells and melanoma tissues led to the radiolabeling of several alpha-melanocyte-stimulating hormone (alpha-MSH) analogs for early detection and treatment of melanoma. We have coupled the alpha-MSH analog Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys-NH(2), through the epsilon-amino group of Lys(11), to a pyrazolyl-containing chelator (pz). The resulting pz-alpha-MSH analog reacted with the fac-[(99m)Tc(CO)(3)](+) moiety, giving [Ac-Nle(4),Asp(5),d-Phe(7),Lys(11)(pz-(99m)Tc(CO)(3))]alpha-MSH(4-11) in high yield, high specific activity and high radiochemical purity. This radioconjugate, which presents remarkable stability in vitro, exhibited time- and temperature-dependent internalization (4 h at 37 degrees C; 56.7% maximum internalization) and high cellular retention (only 38% was released from the cell after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake [4.2+/-0.9%ID/g, at 4 h postinjection (p.i.)] was also obtained in melanoma-bearing C57BL6 mice. The in vivo affinity and specificity of the radioconjugate to MC1R were demonstrated by receptor-blocking studies with the potent NDP-MSH agonist (63.5% reduction in tumor uptake at 4 h p.i.).

Chronic neuropeptide Y infusion into the lateral ventricle induces sustained feeding and obesity in mice lacking either Npy1r or Npy5r expression.

Neuropeptide Y (NPY) is a powerful orexigenic neurotransmitter. The NPY Y1 and Y5 receptors have been implicated in mediating the appetite-stimulating activity of NPY. To further investigate the importance of these two receptors in NPY-induced hyperphagia after chronic central administration, we used mice lacking either Npy1r or Npy5r expression. NPY infusion into the lateral ventricle of wild-type mice stimulated food intake and induced obesity over a 7-d period. Fat pad weight as well as plasma insulin, leptin, and corticosterone levels were strongly increased in NPY-treated mice. In addition, NPY infusion resulted in a significant decrease in hypothalamic NPY and proopiomelanocortin expression. Interestingly, the lack of either Npy1r or Npy5r expression in knockout mice did not affect such feeding response to chronic NPY infusion. Moreover, the obesity syndrome that developed in these animals was similar to that in wild-type animals. Taken together, these data strongly suggest biological redundancies between Y1 and Y5 receptor signaling in the NPY-mediated control of food intake.

Influence of the bifunctional chelator on the pharmacokinetic properties of 99mTc(CO)3-labeled cyclic α-melanocyte stimulating hormone analog.

Aiming at the design of specific melanocortin-1 receptor (MC1R) targeted imaging probes, we report on the effect of different azolyl-ring substitution patterns (carboxylate at the 4-position and/or methyl groups at the 3,5 positions) of pyrazolyl-diamine bifunctional chelators (Pz(2)-Pz(4)) on the pharmacokinetic profile of the (99m)Tc(CO)3-labeled lactam bridge-cyclized α-melanocyte stimulating hormone derivative, ßAlaNleCycMSH(hex). Three pyrazolyl-diamine-containing chelators were conjugated to ßAlaNleCycMSHhex, with the resulting peptide conjugates displaying subnanomolar MC1R binding affinity. Biodistribution studies in B16F1 melanoma-bearing mice show that all radiopeptides present a good melanoma uptake. The introduction of a carboxylate group in the azolyl-ring leads to a remarkable reduction of the kidney (>89%) and liver (>91%) accumulation for (99m)Tc(CO)3-Pz(3)-ßAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-ßAlaNleCycMSH(hex) when compared to the radiopeptide (99m)Tc(CO)3-Pz(1)-ßAlaNleCycMSH(hex), where that group is absent. The good tumor uptake and favorable tumor-to-nontarget-organs ratios of (99m)Tc(CO)3-Pz(3)-ßAlaNleCycMSH(hex) and (99m)Tc(CO)3-Pz(4)-ßAlaNleCycMSH(hex) highlights the potential of both compounds as melanoma imaging agents.

99mTc(I)/Re(I) tricarbonyl complexes for in vivo targeting of melanotic melanoma: Synthesis and biological evaluation.

The (99m)Tc (I) tricarbonyl complexes fac-[(99m)Tc(κ(3)-L)(CO)(3)] (Tc1-Tc6) containing N-ethylpyrrolidine and N,N-diethylethylamine groups for melanin binding, were evaluated in vitro and in vivo as radioactive probes for the targeting of melanotic melanoma. Aiming at the modification of their size, topology and lipophilicity, Tc1-Tc6 were obtained based on an S,N,O-donor bifunctional chelator (BFC) derived from cysteamine and on pyridyl- and pyrazolyl-containing N,N,O-donor BFCs. Tc1-Tc6 were chemically identified by HPLC comparison with the Re congeners (Re1-Re6) that were synthesized at the macroscopic level and fully characterized by common analytical techniques. With the exception of Tc5 and Tc6, these (99m)Tc complexes are moderately lipophilic, and bind to melanin with moderate to high affinity (23-87%). The cell uptake of Tc1-Tc6, expressed as a percentage of total activity per million cells, spanned between 0.86 and 21.02% for the melanotic B16-F1 cell line and between 0.49% and 13.58% for the amelanotic A375 cell line. In the B16-F1 cell line, Tc1, Tc3 and Tc4 showed moderate cellular uptake values (>10% at 4 h of incubation). In the amelanotic A375 cell line, only Tc4 has shown a moderate cell uptake (>10% at 4 h of incubation), with all the other compounds displaying a relatively poor uptake, i.e. inferior to 5%. Competition studies with haloperidol have shown that the involvement of sigma receptors in cellular uptake and retention is likely to occur for Tc4. Complex Tc1, stabilized with the S,N,O-donor BFC and containing a N,N-diethylethylamine group, presented the most promising biological profile for in vivo targeting of melanoma, showing a moderate tumor uptake of 2.17% ID/g at 1 h p.i in a B16-F1 melanoma-bearing mouse and rather favorable target/non-target ratios with values as high as 16.9 and 5.2 for tumor/muscle and tumor/blood ratios, respectively.

Radiometallated peptides for molecular imaging and targeted therapy.

In developed countries, cancer is the second leading cause of death, being only surpassed by cardiovascular diseases. To develop tumor-targeted tools to localize and treat cancer at an early stage is a multidisciplinary area fuelled by the convergence of biology, medicine, chemistry, physics and engineering. Chemists, in particular, play a critical role in this effort, as they are continuously challenged to use innovative chemical strategies to develop 'smart drugs'. The in vitro observation that peptide receptors are overexpressed in certain tumors, as compared to endogenous expression levels, has prompted the use of such receptors as targets and the design of radiolabelled peptide-based tools for targeted nuclear molecular imaging and therapy. Such approach has gained increased interest over the last two decades, driven in particular by the success of OctreoScan(®) and by the increasing knowledge concerning overexpression of regulatory peptide receptors in tumor tissues. Selected peptides that target a variety of disease related receptors are in place and have been labeled with different radiometals, using mainly the bifunctional approach. This review begins by summarizing some relevant aspects of the coordination chemistry of the metals studied for labeling peptides. Then, we provide an overview of the chemical strategies explored to improve the biological performance of different families of radiometallated peptides for nuclear molecular imaging and/or targeted radionuclide tumor therapy.

Melanocortin-1 receptor-targeting with radiolabeled cyclic α-melanocyte-stimulating hormone analogs for melanoma imaging.

Melanoma is a type of skin cancer known for its high aggressiveness, early dissemination of metastases, and poor prognosis once metastasized. Thus, early diagnosis of melanoma is a key issue for increasing patient survival. The overexpression of melanocortin-1 receptors (MC1R) in isolated melanoma cells and melanoma tissues led to the radiolabeling of several linear and cyclic MC analogs for melanoma imaging or therapy. Cyclization of α-melanocyte stimulating hormone (α-MSH) peptides has been successfully used to improve binding affinity and in vivo stability of peptides. Herein, we describe the different peptide cyclization strategies recently reported for radiolabeled α-MSH analogs and discuss how such strategies affect MC1R binding affinity, pharmacokinetic profile, and MC1R-melanoma imaging. This review also highlights how the nature of the radiometal and labeling approach influence those properties. Among the cyclized α-MSH peptides reported, (99m)Tc/(111)In-labeled metal-cyclized and lactam bridge-cyclized peptides displayed the highest melanoma and lowest renal uptake values in B16/F1 melanoma-bearing mice and became the most promising tools to be further explored as potential melanoma imaging probes.

Preparation and biological evaluation of cyclopentadienyl-based 99mTc-complexes [(Cp-R)99mTc(CO)3] mimicking benzamides for malignant melanoma targeting.

The biological evaluation of half-sandwich (99m)Tc-complexes that surrogate iodobenzamide with a high affinity for melanin tumor tissue is described. We have synthesized via retro Diels-Alder reaction two models of (99m)Tc complexes which possess the piano stool [Cp(99m)Tc(CO)(3)] motif instead of a phenyl ring as in the original iodobenzamide (123)I-N-(N-benzylpiperidin-4-yl)-2-iodobenzamide (2-IBP) and N-(2-diethylaminoethyl)-4-iodobenzamide (BZA). Diels-Alder products 2a-b (HCp-CONHR)(2) (2a, R=2-diethylaminoethyl; 2b, R=benzylpiperidin-4-yl) were prepared and reacted with fac-[(99m)Tc(H(2)O)(3)(CO)(3))](+) 1 in water to produce the corresponding (99m)Tc complexes [(2a)(99m)Tc(CO)(3))] 4a and [(2b)(99m)Tc(CO)(3))] 4b. The structures of the (99m)Tc complexes on the no-carrier-added level have been confirmed by chromatographic comparison with the corresponding rhenium complexes 3a and 3b, macroscopically characterized by IR, NMR, ESI-MS and X-ray crystallography for 3a [triclinic, P-1, a=7.3518(1) A, b=8.0309(2) A, c=17.5536(3) A, alpha=99.1260(5) degrees, beta=90.4215(14) degree , gamma=117.0187(11) degrees]. The radioconjugate 4b showed good in vitro stability. In murine melanoma B16F1 cells, significant cellular uptake (43.9% of the total applied activity) was attained after 4 h at 37 degrees C with about 50% of the cell-associated radioactivity being internalized in the cells (22% of the applied activity). Furthermore, in melanoma-bearing C57BL6 mice, tumor uptake values of 3.39+/-0.50 %ID g(-1) and 3.21+/-0.26 %ID g(-1) at 1 and 4 h postinjection, respectively, were observed indicating a good retention of 4b in the tumor.

Re and (99m)Tc organometallic complexes containing pendant l-arginine derivatives as potential probes of inducible nitric oxide synthase.

Aiming to design radioactive compounds based on the core "(99m)Tc(CO)(3)" for probing inducible nitric oxide synthase (iNOS) levels in vivo, we have synthesized conjugates containing a pyrazolyl-diamine chelating unit and pendant l-arginine analogues (substrates and inhibitors of NOS). Reaction of the conjugates with fac-[M(CO)(3)](+) (M = Re, (99m)Tc) gave bioorganometallic complexes of the type fac-[M(CO)(3)(k(3)-L)] in good yield. After in vitro testing using the oxyhemoglobin NO capture assay, we concluded that the affinity of the inhibitor-containing conjugates to iNOS seems to be less affected upon metallation with rhenium than the substrate-containing conjugates. The complexes bearing guanidino substituted analogues of l-arginine still present considerable inhibitory action (N(omega)-monomethyl-l-arginine, K(i) = 36 microM; N(omega)-nitro-l-arginine, K(i) = 84 microM), being the first examples of organometallic complexes able to inhibit the iNOS. These results seem to indicate that (99m)Tc(CO)(3)-labeled L-argininine analogues, namely NOS inhibitors, may hold potential for monitoring increased levels of iNOS in vivo.

Melanoma targeting with alpha-melanocyte stimulating hormone analogs labeled with fac-[99mTc(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 99mTc-labeled cyclic peptide betaAla-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH2 with that of the linear analog betaAla-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 betaAla, and the resulting pz-peptide conjugates were reacted with the fac-[99mTc(CO)3]+ moiety. The 99mTc(CO)3-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic 99mTc(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 99mTc(CO)3-pz-peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle4,DPhe7)-alphaMSH 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).