Heidi Tanner

A Combinatorial Peptoid Library for the Identification of Novel MSH and GRP / Bombesin Receptor Ligands

A tripeptoid library was synthesized using 69 different primary amines in initially 69 individual reactions by the mix and split approach. The resulting library consisted of 328,509 (69(3)) single compounds, divided in 69 subpools each containing 4,761 entities. The 69 subpools were tested in two binding assays, one for alpha-MSH (alpha-melanotropin) and one for GRP (gastrin-releasing peptide)/bombesin. The sublibraries with the highest affinity to the MSH receptor (i.e. melanocortin type 1 or MC1 receptor) and, respectively, the GRP-preferring bombesin receptor were identified by an iterative process. Individual tripeptoids with good binding activity were resynthesized, analyzed and their dissociation constants and biological activity determined. The KD of the most potent MC1 receptor ligand was 1.58 mumol/l and that of the GRP-preferring bombesin receptor 3.40 mumol/l. Extension of this latter tripeptoid structure whose KD value increased to 280 nmol/l. A similar increase in activity was not observed with the most potent MSH tripeptoid ligand when extended by one residue, but a compound suitable for radioiodination and lacking the N-terminal amino group had a slightly higher binding activity than the tripeptoids (KD approximately 850 nmol/l). These results demonstrate that testing a peptoid library containing 328,509 single compounds led to the successful identification of new ligands for both the MC1 receptor as well as the GRP-preferring bombesin receptor.

Synthesis and characterization of new radioligands for the mammalian melanin-concentrating hormone (MCH) receptor.

Melanin-concentrating hormone (MCH) is a neuropeptide present in the brain of all vertebrates. For the characterization of MCH receptors, a monoiodinated [Phe13, Tyr19]-MCH radioligand analogue was developed. The high susceptibility of [125I]-[Phe13, Tyr19]-MCH to oxidative damage and its very lipophilic nature made it necessary to develop new MCH radioligands. To increase the stability, native methionines were replaced by non-sulphur containing amino acid residues. In one analogue, the L-enantiomer of the phenylalanine residue at position 13 was substituted by the D-enantiomer, which increased the relative affinity of the ensuing [125I]-[D-Phe13, Tyr19]-MCH about 7-fold. The different analogues were iodinated by an enzymatic reaction and used for binding studies with mouse melanoma cells. [125I]-[Met(O)4,8, Phe13, Tyr19]-MCH and [125I]-[Hse4,8, Phe13, Tyr19]-MCH showed only about 19% of total binding and [125I]-[Ser4,8, Phe13, Tyr19]-MCH displayed about 44% of total binding when compared with [125I]-[Phe13, Tyr19]-MCH. Non-specific binding for all tracers was below 11% of total binding of [125I]-[Phe13, Tyr19]-MCH binding. [125I]-[D-Phe13, Tyr19]-MCH was used for saturation binding studies and revealed a KD of 122.7 +/- 15.3 pmol/l. This radioligand was further characterized by association and dissociation binding studies.

Glycosylated DOTA−α-Melanocyte-Stimulating Hormone Analogues for Melanoma Targeting: Influence of the Site of Glycosylation on in Vivo Biodistribution

alpha-Melanocyte-stimulating hormone (alpha-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which is overexpressed on melanotic and amelanotic melanoma cells. alpha-MSH analogues are potential candidates for specific targeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed and tested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenografts in vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim to increase the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an alpha-MSH octapeptide analogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labeling with (111)In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors. The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using both murine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than the parent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particular C-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. By contrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidney ratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 h AUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.

[D-(p-Benzoylphenylalanine)13, tyrosine19]-melanin-concentrating hormone, a potent analogue for MCH receptor crosslinking

A photoreactive analogue of human melanin‐concentrating hormone was designed, [d‐Bpa13,Tyr19]‐MCH, containing the d‐enantiomer of photolabile p‐benzoylphenylalanine (Bpa) in position 13 and tyrosine for radioiodination in position 19. The linear peptide was synthesized by the continuous‐flow solid‐phase methodology using Fmoc‐strategy and PEG‐PS resins, purified to homogeneity and cyclized by iodine oxidation. Radioiodination of [d‐Bpa13,Tyr19]‐MCH at its Tyr19 residue was carried out enzymatically using solid‐phase bound glucose oxidase/lactoperoxidase, followed by purification on a reversed‐phase mini‐column and HPLC. Saturation binding analysis of [125I]‐[d‐Bpa13,Tyr19]‐MCH with G4F‐7 mouse melanoma cells gave a KD of 2.2±0.2×10−10 mol/l and a Bmax of 1047±50 receptors/cell. Competition binding analysis showed that MCH and rANF(1–28) displace [125I]‐[d‐Bpa13,Tyr19]‐MCH from the MCH binding sites on G4F‐7 cells whereas α‐MSH has no effect. Receptor crosslinking by UV‐irradiation of G4F‐7 cells in the presence of [125I]‐[d‐Bpa13,Tyr19]‐MCH followed by SDS‐polyacrylamide gel electrophoresis and autoradiography yielded a band of 45–50 kDa. Identical crosslinked bands were also detected in B16‐F1 and G4F mouse melanoma cells, in RE and D10 human melanoma cells as well as in COS‐7 cells. Weak staining was found in rat PC12 phaeochromocytoma and Chinese hamster ovary cells. No crosslinking was detected in human MP fibroblasts. These data demonstrate that [125I]‐[d‐Bpa13,Tyr19]‐MCH is a versatile photocrosslinking analogue of MCH suitable to identify MCH receptors in different cells and tissues; the MCH receptor in these cells appears to have the size of a G protein‐coupled receptor, most likely with a varying degree of glycosylation. Copyright

INTERACTION OF MELANIN-CONCENTRATING HORMONE (MCH), NEUROPEPTIDE E-I (NEI), NEUROPEPTIDE G-E (NGE), AND α-MSH WITH MELANOCORTIN AND MCH RECEPTORS ON MOUSE B16 MELANOMA CELLS

Melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH) are known to exhibit mostly functionally antagonistic, but in some cases agonistic activities, e.g., in pigment cells and in the brain. Neuropeptide E-I (NEI) displays functional MCH-antagonist and MSH-agonist activity in different behavioral paradigms; the role of neuropeptide G-E (NGE) is not known. This study addressed the question of possible molecular interactions between α-MSH, MCH and the MCH-precursor-derived peptides NEI and NGE at the level of the pigment cell MCH receptor subtype (MCH-Rpc) and the different melanocortin (MC) receptors. Radioreceptor assays using [125I]MCH, [125I]α-MSH and [125I]NEI as radioligands and bioassays were performed with MC1-R-positive and MC1-R-negative mouse B16 melanoma cells and with COS cells expressing the different MC receptors. The IC50s of α-MSH and NEI or NGE for [125I]MCH displacement from mouse MCH-Rpc were 80-fold and, respectively, > 300-fold higher than that of MCH, and the IC50s for MCH and NEI or NGE for [125I]α-MSH displacement from mouse MC1-R were 50,000-fold and > 200,000-fold higher than that of α-MSH. No high-affinity binding sites for NEI were detected on B16 melanoma cells and there was no significant displacement of [125I]α-MSH by MCH, NEI or NGE with MC3-R, MC4-R and MC5-R expressed in COS cells. At concentrations of 100 nM to 10 μM, however, MCH, NEI and NGE induced cAMP formation and melanin synthesis which could be blocked by agouti protein or inhibitors of adenylate cyclase or protein kinase A. This shows that mammalian MCH-precursor-derived peptides may mimic MSH signalling via MC1-R activation at relatively high, but physiologically still relevant concentrations, as e.g. found in autocrine/paracrine signalling mechanisms.

Dimeric DOTA-α-Melanocyte-Stimulating Hormone Analogs: Synthesis and In Vivo Characteristics of Radiopeptides with High In Vitro Activity

Dimeric analogs of α-melanocyte-stimulating hormone (α-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for α-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled α-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [111In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([111In]DOTA)-amide, and 0.36 for [111In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [111In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled α-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.

Endogenous receptor for melanin-concentrating hormone in human neuroblastoma Kelly cells.

Melanin-concentrating hormone (MCH), a cyclic nonadecapeptide, is predominantly expressed in mammalian neurons located in the zona incerta and lateral hypothalamus. Current interest in MCH relates to its role in the control of feeding behaviour. Two receptors for MCH were recently found: MCH-R(1) and MCH-R(2). We show here by RT-PCR analysis and immunofluorescence studies that the human neuroblastoma cell line Kelly expresses MCH and MCH-R(1) but not MCH-R(2). In competition assays using 125I-labelled MCH an inhibitory concentration 50% (IC(50)) of 76nM was determined for MCH, indicating a high affinity of Kelly cells for MCH. MCH induces mitogen-activated protein kinase (MAPK) phosphorylation in Kelly cells but no increase in the intracellular free Ca(2+) concentration. This suggests that MCH signals via Galpha(i)/Galpha(0) in these cells. The presence and functionality of MCH-R(1) renders this neuronal cell a very useful model for future structure-activity studies in a physiological environment mimicking the human brain for the evaluation of potential appetite-regulating drugs.

MSH radiopeptides for targeting melanoma metastases.

Radiolabeled peptides have become important tools for preclinical cancer research and in nuclear oncology they serve as diagnostic and more recently also as therapeutic agents. Whereas the development of receptor-mediated targeting for therapy has been confined to some radiolabeled antibodies and somatostatin/SRIF analogs, recent research into radiolabeled α-Melanocyte-stimulating hormone (α-MSH) and its receptor MC1R (over-)expressed by melanoma tumor cells has demonstrated that small metastatic melanoma lesions in experimental animals are specifically targeted by MSH radiopeptides. Thus MSH radiopharmaceuticals will eventually open a new avenue for the treatment of melanoma metastases in man, provided that the targeting efficiency can be further enhanced and nonspecific incorporation into nontarget organs, e.g., the kidneys, minimized. Some novel MSH lead compounds containing a glyco moiety, added negatively charged groups or a cyclic structure show very promising in vivo targeting characteristics.

DOTA α‐Melanocyte‐Stimulating Hormone Analogues for Imaging Metastatic Melanoma Lesions

Abstract: Scintigraphic imaging of metastatic melanoma lesions requires highly tumor‐specific radiopharmaceuticals. Because both melanotic and amelanotic melanomas overexpress melanocortin‐1 receptors (MC1R), radiolabeled analogues of α‐melanocyte‐stimulating hormone (α‐MSH) are potential candidates for melanoma diagnosis. Here, we report the in vivo performance of a newly designed octapeptide analogue, [βAla3, Nle4, Asp5, D‐Phe7, Lys10]‐α‐MSH3–10 (MSHOCT), which was conjugated through its N‐terminal amino group to the metal chelator 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) to enable incorporation of radiometals (e.g., indium‐111) into the peptide. DOTA‐MSHOCT displayed high in vitro MC1R affinity (IC50 9.21 nM). In vivo [111In]DOTA‐MSHOCT exhibited a favorable biodistribution profile after injection in B16‐F1 tumorbearing mice. The radiopeptide was rapidly cleared from blood through the kidneys and, most importantly, accumulated preferentially in the melanoma lesions. Lung and liver melanoma metastases could be clearly imaged on tissue section autoradiographs 4 h after injection of [111In]DOTA‐MSHOCT. A comparative study of [111In]DOTA‐MSHOCT with [111In]DOTA‐[Nle4, D‐Phe7]‐α‐MSH ([111In]‐DOTA‐NDP‐MSH) demonstrated the superiority of the DOTA‐MSHOCT peptide, particularly for the amount of radioactivity taken up by nonmalignant organs, including bone, the most radiosensitive tissue. These results demonstrate that [111In]DOTA‐MSHOCT is a promising melanoma imaging agent.

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R1 and MCH‐R2, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R1 and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R1 (SLC‐1), the radioligand was [125I]–[Tyr13]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [125I]–[D‐Phe13, Tyr19]‐MCH served as radioligand. The bioassay used for MCH‐R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R1 and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe13 in [Phe13, Tyr19]‐MCH was well tolerated by both MCH‐R1 and B16 MCH‐R, change of configuration to D‐Phe13 in [D‐Phe13, Tyr19]‐MCH or [D‐Phe13]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R1. By contrast, the D‐Phe13 residue increased the affinity of [D‐Phe13, Tyr19]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R1 in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R1.