Oligoproline-Based Mono- and Bivalent Minigastrin and Octreotide Analogues: Synthesis, in Vitro, and in Vivo Characterization

Medullary thyroid cancer (MTC) and small cell lung cancer (SCLC) are both neuroendocrine tumours (NETs) with only very limited therapy options. The overexpression of peptide binding G-protein coupled receptors on the surface of cancer cells can be used for selective tumour targeting with radioactively labelled peptides for imaging and therapy. However, peptidic radioligands for the so-called peptide receptor radionuclide therapy (PRRT) often have limitations in terms of stability, specificity, bioavailability, and tumour tissue retention. The heterogeneous expression profile of the target receptors is another important factor that can be limiting for PRRT, since target receptor expression levels can fall below the threshold of detection depending on the state of the disease. With this thesis several strategies to address these limitations were investigated. We explored the advantages of multivalent ligands with more than one recognition motif for the simultaneous targeting of either two receptors of the same type (homodivalent ligands) or two different types of receptors (heterodivalent ligands). The cholecystokinin receptor subtype 2 (CCK2R) and the somatostatin receptor subtype 2 (SSTR2) were chosen as target receptors, since they are both overexpressed on the cells of several types of NETs including MTC and SCLC. Minigastrin analogues to target the CCK2R and octreotide analogues to target the SSTR2 were conjugated over a rigid oligoproline backbone, which ensures well defined distances between the recognition motifs, providing additional information about a distance-dependence of the biological performance. We successfully developed homodivalent ligands to target the CCK2R with higher in vitro cellular uptake compared to the non-conjugated reference compound, demonstrating the benefit of multivalency. We further could show a distance-dependence in the in vitro performance of the homodivalent octreotide based ligands that target the SSTR2. The heterogeneity of NETs was successfully addressed by our heterodivalent ligand with good CCK2R mediated and SSTR2 mediated cellular uptake into tumour cells, which therefore is less dependent on the expression level of one individual target receptor. The search for optimized recognition motifs for multivalent ligands led to the investigation and optimization of novel monovalent minigastrin- and octreotide-type ligands. Minigastrin analogues with different linkers gave important insights into the structure activity relationship between gastrins and the CCK2R (especially regarding the role of the pentaglutamic acid sequence in gastrin), whereas modifications of octreotides led to the development of a highly potent [Tyr3]octreotide analogue. In addition, the attachement of a human serum album binding moiety to monovalent minigastrin analogues for significantly improved pharmacokinetics was investigated

Concise Asymmetric Synthesis and Pharmacological Characterization of All Stereoisomers of Glutamate Transporter Inhibitor TFB-TBOA and Synthesis of EAAT Photoaffinity Probes

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-β-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

Elucidating the structure–activity relationship of the pentaglutamic acid sequence of minigastrin with cholecystokinin receptor subtype 2

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 β -glutamic acid residues were evaluated and compared to the lead compound PP-F11N (DOTA-[d-Glu ,Nle ]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 < (β -Glu) < (d-Glu) . 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

Dual, Site-Specific Modification of Antibodies by Using Solid-Phase Immobilized Microbial Transglutaminase

ISSN:1439-4227ISSN:1439-763

Distance-Dependent Cellular Uptake of Oligoproline-Based Homobivalent Ligands Targeting GPCRs—An Experimental and Computational Analysis

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. © 2020 American Chemical Society.ISSN:1043-1802ISSN:1520-481