Design and characterization of alpha-melanotropin peptide analogs cyclized through rhenium and technetium metal coordination

Abstract

alpha-Melanocyte stimulating hormone (alpha-MSH) analogs, cyclized through site-specific rhenium (Re) and technetium (Tc) metal coordination, were structurally characterized and analyzed for their abilities to bind alpha-MSH receptors present on melanoma cells and in tumor-bearing mice. Results from receptor-binding assays conducted with B16 F1 murine melanoma cells indicated that receptor-binding affinity was reduced to approximately 1% of its original levels after Re incorporation into the cyclic Cys4,10, D-Phe7-alpha-MSH4-13 analog. Structural analysis of the Re-peptide complex showed that the disulfide bond of the original peptide was replaced by thiolate-metal-thiolate cyclization. A comparison of the metal-bound and metal-free structures indicated that metal complexation dramatically altered the structure of the receptor-binding core sequence. Redesign of the metal binding site resulted in a second-generation Re-peptide complex (ReCCMSH) that displayed a receptor-binding affinity of 2.9 nM, 25-fold higher than the initial Re-alpha-MSH analog. Characterization of the second-generation Re-peptide complex indicated that the peptide was still cyclized through Re coordination, but the structure of the receptor-binding sequence was no longer constrained. The corresponding 99mTc- and 188ReCCMSH complexes were synthesized and shown to be stable in phosphate-buffered saline and to challenges from diethylenetriaminepentaacetic acid (DTPA) and free cysteine. In vivo, the 99mTcCCMSH complex exhibited significant tumor uptake and retention and was effective in imaging melanoma in a murine-tumor model system. Cyclization of alpha-MSH analogs via 99mTc and 188Re yields chemically stable and biologically active molecules with potential melanoma-imaging and therapeutic properties.

Figure 1

Model structures of APOMSH (Left), ReMSH (Center), and ReCCMSH (Right) derived from NMR analyses. The site of Re coordination is depicted by the filled circle in ReMSH and ReCCMSH molecules.

Figure 2

Imaging of a melanoma tumor in a mouse injected with ^99mTcCCMSH. The γ-camera image of the melanoma-bearing mouse was acquired 30 min postinjection. The lateral image of the mouse with a 400-mg tumor shows a high degree of radioactivity localized in the tumor (b) with lesser amounts present in the kidneys (a), bladder (c), and tail vein injection site (d). The intensity of the γ-emission is color-coded high-to-low, ranging from white-yellow through orange, with dark red representing lowest values.