Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25 ± 10 Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH₂ was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-_c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH₂ and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)₃ units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.     

TERMINAL PHOSPHATE LABELED NUCLEOTIDES: SYNTHESIS,APPLICATIONS, AND LINKER EFFECT ON INCORPORATION BY DNA POLYMERASES

A number of terminal phosphate-labeled nucleotides with three or more phosphates and with varied length linkers attached between the terminal phosphate and the dye have been synthesized. These nucleotides have been tested as substrates for different DNA and RNA polymerases. We have also explored their utility in DNA sequencing, SNP analysis, nucleic acid amplification, quantitative PCR, and other biochemical assays.     

Solid-Phase Synthesis of Heterobivalent Ligands Targeted to Melanocortin and Cholecystokinin Receptors

Heteromultivalency provides a route to increase binding avidity and to high specificity when compared to monovalent ligands. The enhanced specificity can potentially serve as a unique platform to develop diagnostics and therapeutics. To develop new imaging agents based upon multivalency, we employed heterobivalent constructs of optimized ligands. In this report, we describe synthetic methods we have developed for the preparation of heterobivalent constructs consisting of ligands targeted simultaneously to the melanocortin receptor, hMC4R, and the cholecystokinin receptors, CCK-2R. Modeling data suggest that a linker distance span of 20–50 Å is needed to crosslink these two G-protein coupled receptors (GPCRs). The two ligands were tethered with linkers of varying rigidity and length, and flexible polyethylene glycol based PEGO chain or semi-rigid [poly(Pro-Gly)] linkers were employed for this purpose. The described synthetic strategy provides a modular way to assemble ligands and linkers on solid-phase supports. Examples of heterobivalent ligands are provided to illustrate the increased binding avidity to cells that express the complementary receptors.     

Synthesis, immobilization, and solid-state NMR of new phosphine linkers with long alkyl chains

Monodentate and chelating phosphines with long alkyl chains, incorporating ethoxy- or chlorosilane functions for immobilizations, have been synthesized and fully characterized. The new compounds (EtO)₃Si(CH₂)_xPPh₂, Cl₂Si(CH₂CH₂PPh₂)₂, and (EtO)₂Si[(CH₂)_xPPh₂]₂ (x = 7, 11) could be prepared in high yields from cheap starting materials, and they have been characterized by multinuclear NMR spectroscopy and X-ray crystallography. The phosphines have been immobilized on silica in a well-defined manner, and the modified silicas have been studied by ³¹P and ²⁹Si solid-state NMR of the dry materials and of the suspensions.     

Partial characterization of giant extracellular hemoglobin of Glossoscolex paulistus: a MALDI-TOF-MS study

In this work, MALDI-TOF-MS analysis was performed to obtain information on the molecular mass of the different subunits from the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy-form. Experiments were performed for the whole protein at pH 7.0, for the partially dissociated protein at pH 9.0, and for the fraction obtained from gel filtration in Sephadex G-200, at pH 9.0, corresponding to the isolated monomer d. Besides that, experiments were performed for the whole protein treated with 2-mercaptoethanol in order to monitor the effects of reduction of the disulfide bonds, which are expected to maintain the trimer (abc) in the native molecule. The results are compared to those reported for the homologous hemoglobin of Lumbricus terrestris (HbLt) and some tentative assignments are made for the observed polypeptides. The monomer d is found to exist in, at least, two major forms of identical proportions with masses of 16,355 ± 25 and 16,428 ± 24 Da, respectively. Two minor forms were also observed around 16 kDa for the monomers. Upon disulfide bonds reduction the peak associated to the trimer is absent in the mass spectrum, and new peaks assigned tentatively to the monomers a, b and c on the basis of comparison with Lumbricus terrestris hemoglobin literature data are observed. Their molecular masses were 18,258 ± 30, 16,492 ± 24 and 17,363 ± 17 Da, respectively. Two linker chains for HbGp were also observed at 25,817 ± 50 and 26,761 ± 16 Da, and this result is different from HbLt, where four linker chains were reported in the range 24–32 kDa. Finally, trimers (abc) were observed at 51–52 kDa. This partial characterization, performed for the first time, is an important step in the characterization of subunits of this giant extracellular hemoglobin.