RESUMEN
Tumor targeting with bivalent radiolabeled ligands for GPCRs is an attractive means for cancer imaging and therapy. Here, we studied and compared the distance dependence of homobivalent ligands for the human gastrin-releasing peptide receptor (hGRP-R) and the somatostatin receptor subtype II (hSstR2a). Oligoprolines were utilized as molecular scaffolds to enable distances of 10, 20, or 30 Å between two identical, agonistic recognition motifs. In vitro internalization assays revealed that ligands with a distance of 20 Å between the recognition motifs exhibit the highest cellular uptake in both ligand series. Structural modeling and molecular dynamics simulations support an optimal distance of 20 Å for accommodating ligand binding to both binding sites of a GPCR dimer. Translation of these findings to the significantly higher complexity in vivo proved difficult and showed only for the hGRP-R increased tumor uptake of the bivalent ligand.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/farmacocinética , Prolina/análogos & derivados , Prolina/farmacocinética , Receptores de Bombesina/agonistas , Receptores de Somatostatina/agonistas , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Ligandos , Ratones SCID , Simulación de Dinámica Molecular , Neoplasias/metabolismo , Oligopéptidos/farmacología , Prolina/farmacología , Receptores de Bombesina/metabolismo , Receptores de Somatostatina/metabolismoRESUMEN
Derivatized minigastrin analogues make up a promising class of candidates for targeting cholecystokinin receptor subtype 2 (CCK2R), which is overexpressed on cancer cells of various neuroendocrine tumors. The pentaglutamic acid sequence of minigastrin influences its biological properties. In particular, it plays a crucial role in the kidney reuptake mechanism. However, the importance of the binding affinity and interaction of this region with the receptor on a molecular level remains unclear. To elucidate its structure-activity relationship with CCK2R, we replaced this sequence with various linkers differing in their amount of anionic charge, structural characteristics, and flexibility. Specifically, a flexible aliphatic linker, a linker with only three d-Glu residues, and a structured linker with four adjacent ß3-glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu1-6,Nle11]gastrin-13). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the minigastrin derivatives, which allowed radiolabeling with Lutetium-177. The levels of In vitro internalization into MZ-CRC1 cells and in vivo tumor uptake as well as human blood plasma stability increased in the following order: aliphatic linker < three d-Glu < (ß3-Glu)4 < (d-Glu)6. The in vitro and in vivo behavior was therefore significantly improved with anionic charges. Computational modeling of a CCK2 receptor-ligand complex revealed ionic interactions between cationic residues (Arg and His) of the receptor and anionic residues of the ligand in the linker.
Asunto(s)
Gastrinas/química , Gastrinas/farmacología , Receptor de Colecistoquinina B/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular Tumoral , Estabilidad de Medicamentos , Gastrinas/farmacocinética , Humanos , Ratones , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Distribución TisularRESUMEN
Microbial transglutaminase (MTG) was stably solid-phase immobilized on glass microbeads by using a second-generation dendronized polymer. Immobilized MTG enabled the efficient generation of site-specifically conjugated proteins, including antibody fragments, as well as whole antibodies through distinct glutamines and, unprecedentedly, also through lysines with various bifunctional substrates with defined stoichiometries. With this method, we generated dual, site-specifically modified antibodies comprising a fluorescent probe and a metal chelator for radiolabeling-a strategy anticipated to design antibodies for imaging and simultaneous therapy. Furthermore, we provide evidence that immobilized MTG features higher siteselectivity than soluble MTG.
Asunto(s)
Anticuerpos/metabolismo , Enzimas Inmovilizadas/metabolismo , Streptomyces/enzimología , Transglutaminasas/metabolismo , Especificidad por SustratoRESUMEN
Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-ß-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.