68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

Background: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment

Biophysical Chemistry

Little is known about structural alterations of proteins within the polymeric films of paints. For the first time, hydrogen‑deuterium exchange mass spectrometry (HDX-MS) was implemented to explore the conformational alterations of proteins resulting from their interaction with inorganic pigments within the early stages of the paint film formation. Intact protein analysis and bottom-up electrospray-ionisation mass spectrometry strategies combined with progressively increasing deuterium incubation times were used to compare the protein structures of the model protein hen egg-white lysozyme (HEWL) extracted from newly dried non-pigmented films and newly dried films made from a freshly made mixture of HEWL with lead white pigment (2PbCO3 Pb(OH)2). The action of other pigments was also investigated, expanding the HDX study with a global approach to paint models of HEWL mixed with zinc white (ZnO), cinnabar (HgS) and red lead (Pb3O4) pigments. The results show structural modifications of HEWL induced by the interaction with the pigment metal ions during the paint formulation after drying and prior to ageing. Both the charge distribution of HEWL proteoforms, its oxidation rate and its deuterium absorption rate, were influenced by the pigment type, providing the first insights into the correlation of pigment type/metal cation to specific chemistries related to protein stability.Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-60 ITN

Affinity ranking of peptide-polyphenol non-covalent assemblies by mass spectrometry approaches

Energy Resolved Mass Spectrometry of peptide-polyphenol complexes was used to characterize the affinity of a selection of 21 polyphenols for peptides from human salivary basic Proline Rich Proteins (PRPb) having variable lengths. The affinity scale determined in the gas phase for a 14 amino acid long peptide is in agreement with results obtained in the liquid phase with the same PRPb probe immobilized on magnetic beads and those of sensory descriptive analysis of polyphenols astringency. (C) 2015 Elsevier Ltd. All rights reserved

Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion

PiP(2) favors an alpha-helical structure of non-recombinant Hsp12 of Saccharomyces cerevisiae

Hsp12 is a small heat shock protein of Saccharomyces cerevisiae upregulated in response to various stresses. Non recombinant Hsp12 has been purified and characterized. Using circular dichroism (CD), Isothermal Titration Calorimetry (ITC) and Differential Scanning Calorimetry (DSC), it has been demonstrated that the native Hsp12 is monomeric and intrinsically disordered (IDP). Hsp12 gains in structure in the presence of specific lipids (PiP(2)). The helical form binds to liposomes models membrane with high affinity, leading to their rigidification. These results suggest that hydrophobic and ionic interactions are involved. Hsp12 is most likely a membrane chaperone expressed during stresses in Saccharomyces cerevisiae

68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2

International audienceBackground: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients.Methods: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature.Results: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5–5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min.Conclusion: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment

A new reliable, transposable and cost-effective assay for absolute quantification of total plasmatic bevacizumab by LC MS/MS in human plasma comparing two internal standard calibration approaches

The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC-MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500 mu g/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies

Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis

Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a beta-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis

A METHOD FOR QUANTIFYING BEVACIZUMAB

The present invention relates to the use of an Internal Standard compound in a method for quantifying Bevacizumab in a sample by mass spectrometry, wherein the said Internal Standard compound is described in the specification

Biochimica et biophysica acta

Cell-penetrating peptides (CPP) are able to efficiently transport cargos across cell membranes without being cytotoxic to cells, thus present a great potential in drug delivery and diagnosis. While the role of cationic residues in CPPs has been well studied, that of Trp is still not clear. Herein 7 peptide analogs of RW9 (RRWWRRWRR, an efficient CPP) were synthesized in which Trp were systematically replaced by Phe residues. Quantification of cellular uptake reveals that substitution of Trp by Phe strongly reduces the internalization of all peptides despite the fact that they strongly accumulate in the cell membrane. Cellular internalization and biophysical studies show that not only the number of Trp residues but also their positioning in the helix and the size of the hydrophobic face they form are important for their internalization efficacy, the highest uptake occurring for the analog with 3 Trp residues. Using CD and ATR-FTIR spectroscopy we observe that all peptides became structured in contact with lipids, mainly in alpha-helix. Intrinsic tryptophan fluorescence studies indicate that all peptides partition in the membrane in about the same manner (Kp~10(5)) and that they are located just below the lipid headgroups (~10A) with slightly different insertion depths for the different analogs. Plasmon Waveguide Resonance studies reveal a direct correlation between the number of Trp residues and the reversibility of the interaction following membrane washing. Thus a more interfacial location of the CPP renders the interaction with the membrane more adjustable and transitory enhancing its internalization ability