ABSTRACT
Perdeuterated poly(styrene) is introduced as an almost artefact-free and arbitrarily scalable alignment medium for measuring residual dipolar couplings and other anisotropic NMR parameters; the spectral quality achievable in this new medium is demonstrated for HSQC spectra leading to the conformational analysis of staurosporine and homonuclear TOCSY-type experiments.
Subject(s)
Artifacts , Gels/chemistry , Magnetic Resonance Spectroscopy/methods , Polystyrenes/chemistry , Anisotropy , Deuterium/chemistry , Staurosporine/chemistryABSTRACT
PURPOSE: Among gynecologic malignancies, ovarian cancer has the highest mortality due to rapid peritoneal dissemination. Treatment failure particularly arises from failure to eliminate disseminated cells. Our aim was to develop peptidic radioligands targeting tumour cell-associated urokinase receptor (uPAR, CD87) for alpha-emitter therapy for advanced ovarian cancer. METHODS: DOTA-conjugated, uPAR-directed ligands were synthesised on solid-phase. Binding of peptides to human cells expressing uPAR was assayed by flow cytofluorometry or, in case of (213)Bi-labelled peptides, by measuring cell-bound radioactivity. Bio-distribution of the (213)Bi-labelled peptide P-P4D was analysed in nude mice 28 days after intraperitoneal inoculation of OV-MZ-6 ovarian cancer cells in the absence or presence of the plasma expander gelofusine. RESULTS: uPAR-selective ligands were developed based on published high-affinity uPAR-binding peptides. For preparation of N-terminally cross-linked divalent ligands, a novel solid-phase procedure was developed. Specific binding of (213)Bi-labelled peptides to monocytoid U937 and OV-MZ-6 cells was demonstrated using the natural ligand of uPAR, pro-uPA, or a soluble form of uPAR, suPAR, as competitors. The pseudo-symmetrical covalent dimer (213)Bi-P-P4D displayed superior binding to OV-MZ-6 cells in vitro. Accumulation of (213)Bi-P-P4D in tumour tissue was demonstrated by bio-distribution analysis in nude mice bearing intraperitoneal OV-MZ-6-derived tumours. Gelofusine reduced kidney uptake of (213)Bi-P-P4D by half. CONCLUSION: Ovarian cancer cells overexpressing uPAR were specifically targeted in vitro and in vivo by (213)Bi-P-P4D. Kidney uptake of (213)Bi-P-P4D was distinctly reduced using gelofusine. Thus, this radiopeptide may represent a promising option for therapy for disseminated ovarian cancer.
Subject(s)
Alpha Particles/therapeutic use , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/radiotherapy , Peptides/chemistry , Peptides/metabolism , Receptors, Urokinase Plasminogen Activator/metabolism , Animals , Bismuth/chemistry , Cell Line, Tumor , Dimerization , Drug Discovery , Enzyme Inhibitors/chemistry , Female , Gene Expression Regulation, Neoplastic , Heterocyclic Compounds, 1-Ring/chemistry , Humans , Kidney/drug effects , Kidney/metabolism , Ligands , Mice , Neoplasm Metastasis , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Peptides/chemical synthesis , Peptides/pharmacokinetics , Polygeline/pharmacology , Radioisotopes , Receptors, Urokinase Plasminogen Activator/antagonists & inhibitors , Receptors, Urokinase Plasminogen Activator/chemistry , Solubility , Substrate Specificity , Tissue DistributionABSTRACT
Hemophilia A, one of the most severe bleeding disorders, results from an inherited deficiency of factor VIII (FVIII) function. Treatment by injection of FVIII has been a common procedure for decades. Nevertheless, the production and purification of FVIII remains a challenging task. Current procedures using immunoaffinity chromatography are expensive and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified by use of octapeptide ligands, but their low protease-resistance limits their application. We here report the systematic rational and combinatorial optimization procedure that allowed us to transfer the octapeptide ligands into a small peptidomimetic. This compound is the smallest ligand known for separation of such a large protein (330 kDa). It not only binds and purifies FVIII with high efficiency but also is stable, protease-resistant, and cheap to produce in preparative scale. Hence it offers a valuable alternative to antibody-based purification procedures.
Subject(s)
Factor VIII/isolation & purification , Indoleacetic Acids/chemical synthesis , Oligopeptides/chemistry , Amino Acid Substitution , Chromatography, Affinity/methods , Drug Stability , Factor VIII/chemistry , Humans , Indoleacetic Acids/chemistry , Ligands , Molecular Mimicry , Oligopeptides/chemical synthesis , Peptide Hydrolases/chemistry , Polymers , Protein Binding , Recombinant Proteins/chemistry , Serum , StereoisomerismABSTRACT
A convenient synthesis of novel bifunctional poly(amino carboxylate) chelating agents allowing chemoselective attachment to highly functionalized biomolecules is described. Based on the well known chelator 1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid (DOTA), we synthesized novel bifunctional chelating agents bearing additional functional groups by alkylating 1,4,7,10-tetraazacyclododecane (cyclen) with one equivalent of para-functionalized alkyl 2-bromophenyl-acetate and three equivalents of tert-butyl 2-bromoacetate. The resulting compounds, which contain an additional carbonyl or alkyne functionality, allow site specific labeling of appropriately functionalized unprotected biomolecules in a rapid manner via click reactions. This was demonstrated by the attachment of our new DOTA derivatives to the somatostatin analogue Tyr3-octreotate by chemoselective oxime ligation and CuI-catalyzed azide-alkyne cycloaddition. Initial biodistribution studies in mice with the radiometalated compound demonstrated the applicability of the described DOTA conjugation.
Subject(s)
Heterocyclic Compounds, 1-Ring/chemistry , Alkynes/chemistry , Amination , Animals , Azides/chemistry , Carbonic Acid/chemistry , Catalysis , Cell Line, Tumor , Chromatography, High Pressure Liquid , Copper/chemistry , Cyclization , Female , Heterocyclic Compounds, 1-Ring/chemical synthesis , Heterocyclic Compounds, 1-Ring/pharmacokinetics , Heterocyclic Compounds, 1-Ring/therapeutic use , Ketones/chemistry , Mice , Mice, Nude , Molecular Structure , Neoplasms/drug therapy , Neoplasms/pathology , Oximes/chemistry , Oxygen/chemistry , Peptides/chemistry , Rats , Tyrosine/chemistry , Xenograft Model Antitumor AssaysABSTRACT
An efficient access to both condensed and conjugated tyrosine analogues of high enantiomeric purity is described. Novel ring-substituted tyrosines were synthesized by Suzuki cross couplings of appropriately protected l-3-iodotyrosine with a series of activated and deactivated boronic acid derivatives to achieve the target compounds in high yields. d- and l-4-hydroxy-1-naphthylalanines were readily prepared from the corresponding alpha-enamide in two different approaches, by asymmetric hydrogenation as well as by unselective hydrogenation and enzymatic resolution of the racemic mixture.
Subject(s)
Tyrosine/analogs & derivatives , Tyrosine/chemical synthesis , Catalysis , Magnetic Resonance Spectroscopy , Monoiodotyrosine/chemistry , StereoisomerismABSTRACT
While residual dipolar couplings (RDCs) are an established method in high-resolution biomolecular NMR, their use for structure determination of small molecules in organic solvents is limited by the alignment media available. Only recently stretched polystyrene (PS) gels were introduced for the measurement of RDCs on small compounds that allowed urgently needed free scalability of the induced anisotropy. Here, the properties of such stretched PS gels in different organic solvents as well as for different magnetic field strengths and temperatures are studied and practical NMR-spectroscopic aspects are discussed.
