strategic-screening-characterization-visual-gpcr-mini-g-protein

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice ...

Paul Scherrer Institute

The rapid degradation of neurotensin (NT) limits its clinical use in cancer imaging and therapy. Thus, a new NT(8--13) pseudopeptide, NT-VIII, was synthesized. Some changes were introduced in the sequence of NT(8--13) to stabilize the molecule against enzymatic degradation: Arg(8) was N-methylated, and Lys and Tle replaced Arg(9) and Ile(12), respectively. Finally, (NalphaHis)Ac was coupled to the N-terminus for (99m)Tc(CO)(3) labeling. This peptide was characterized both in vitro and in vivo.

Preclinical evaluation of a new, stabilized neurotensin(8--13) pseudopeptide radiolabeled with (99m)tc.

Neurotensin (NT) receptors in pancreatic and other neuroendocrine tumors are promising targets for imaging and therapeutic purposes. Here, we report on the effect of distinct changes in the peptide chain on catabolism in vitro for five radiolabeled [99mTc] neurotensin analogues having high affinity for neurotensin receptors. Substitution of NT(1-7) by (NalphaHis)Ac--the Tc-binding moiety--combined with a reduced bond 8-9 (CH2NH), N-methylation of peptide bonds or replacement of Ile(12) by tertiary leucin (Tle) led to peptide stabilization of various degrees. Biodistribution studies in nude mice bearing HT29 xenografts showed higher tumor uptake with more stable peptides, yielding high tumor to blood ratios of up to 70.

Stabilization of neurotensin analogues: effect on peptide catabolism, biodistribution and tumor binding.

Overexpression of neurotensin (NT) receptors in exocrine pancreatic cancer and other neuroendocrine cancers make them interesting targets for tumor imaging and therapy. Modifications at the cleavage bonds 8-9 and 11-12 led to the synthesis of NT-XII, NT-XIII and NT-XVIII, three new stabilized analogues. (NalphaHis)Ac was coupled to the N-terminus for labeling with [(99m)Tc]-tricarbonyl.

Double-stabilized neurotensin analogues as potential radiopharmaceuticals for NTR-positive tumors.

Bombesin, a neuropeptide with potential for breast and prostate tumor targeting, is rapidly metabolized in vivo, and as a result, uptake in tumor xenografts in mice is poor. An improvement can be expected from the introduction of nonnatural amino acids and spacers. Leu13 was replaced by cyclohexylalanine and Met14 by norleucine. Two spacers, -betaAla-betaAla- and 3,6-dioxa-8-aminooctanoic acid, were inserted between the receptor-binding amino acid sequence (7-14) of bombesin (BBS) and the retroN(alpha)-carboxymethyl histidine chelator used for labeling with the [99mTc](CO)3 core and the rhenium (Re) congener.

Chemical and biological characterization of new Re(CO)3/[99mTc](CO)3 bombesin analogues.

Neurotensin (NT) and its high affinity receptor (NTR1) are involved in several neoplastic processes. Thus, NT-based radiopharmaceuticals are potential tracers for targeted diagnosis and therapy of NTR-positive tumours. A new analogue based on NT(8-13), NT-XIX, with the three enzymatic cleavage sites stabilised, was synthesised and tested.

A stable neurotensin-based radiopharmaceutical for targeted imaging and therapy of neurotensin receptor-positive tumours.

We assessed the suitability of the radiolanthanide (155)Tb (t1/2=5.32 days, Eγ=87 keV (32%), 105keV (25%)) in combination with variable tumor targeted biomolecules using preclinical SPECT imaging.

Future prospects for SPECT imaging using the radiolanthanide terbium-155 - production and preclinical evaluation in tumor-bearing mice.

Over the last decades, great strides were made in the development of novel implants for the treatment of bone defects. The increasing versatility and complexity of these implant designs request for concurrent advances in means to assess in vivo the course of induced bone formation in preclinical models. Since its discovery, micro-computed tomography (micro-CT) has excelled as powerful high-resolution technique for non-invasive assessment of newly formed bone tissue. However, micro-CT fails to provide spatiotemporal information on biological processes ongoing during bone regeneration. Conversely, due to the versatile applicability and cost-effectiveness, single photon emission computed tomography (SPECT) would be an ideal technique for assessing such biological processes with high sensitivity and for nuclear imaging comparably high resolution (<1 mm). Herein, we employ modular designed poly(ethylene glycol)-based hydrogels that release bone morphogenetic protein to guide the healing of critical sized calvarial bone defects. By combined in vivo longitudinal multi-pinhole SPECT and micro-CT evaluations we determine the spatiotemporal course of bone formation and remodeling within this synthetic hydrogel implant. End point evaluations by high resolution micro-CT and histological evaluation confirm the value of this approach to follow and optimize bone-inducing biomaterials. 

Longitudinal in vivo evaluation of bone regeneration by combined measurement of multi-pinhole SPECT and micro-CT for tissue engineering.

Fibroblast growth factor-2 (FGF-2) is a potent modulator of cell growth and regulation, with improper FGF-2 signaling being involved in impaired responses to injury or even cancer. Therefore, the exploitation of FGF-2 as a therapeutic drives the prerequisite for effective insight into drug disposition kinetics. In this article, we present an In-radiolabeled FGF-2 derivative for noninvasive imaging in small animals deploying single photon emission tomography (SPECT). In-FGF-2 is equally well suitable for in vitro and ex vivo investigations as I-FGF-2. Furthermore, In-FGF-2 permits the performance of in vivo imaging, for example for the analysis of FGF-2 containing pharmaceutical formulations in developmental or preclinical stages. In-FGF-2 had affinity for the low-molecular-weight heparin enoxaparin identical to that of unlabeled FGF-2 (K: 0.6 ± 0.07 μM and 0.33 ± 0.03 μM, respectively) as assessed by isothermal titration calorimetry. The binding of In-FGF-2 to heparan sulfate proteoglycans (HPSGs) and the biological activity were comparable to those of unlabeled FGF-2, with EC values of 12 ± 2 pM and 25 ± 6 pM, respectively. In vivo biodistribution in healthy nude mice indicated a predominant accumulation of In-FGF-2 in filtering organs and minor uptake in the retina and the salivary and pituitary glands, which was confirmed by SPECT imaging. Therefore, In-FGF-2 is a valid tracer for future noninvasive animal imaging of FGF-2 in pharmaceutical development.

Radiolabeled In-FGF-2 Is Suitable for In Vitro/Ex Vivo Evaluations and In Vivo Imaging.

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.

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

 Cu (T  = 12.7 h) is an important radionuclide for diagnostic purposes and used for positron emission tomography (PET). A previous method utilized at Paul Scherrer Institute (PSI) proved to be unreliable and, while a method using anion exchange chromatography is a popular choice worldwide, it was felt a different approach was required to obtain a robust chemical separation method.

Alain Blanc

Center for Radiopharmaceutical Sciences

Paul Scherrer Institute

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

Alain Blanc

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Paul Scherrer Institute

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

Center for Radiopharmaceutical Sciences