Order of amino acids in C-terminal cysteine-containing peptide-based chelators influences cellular processing and biodistribution of <Superscript>99m</Superscript>Tc-labeled recombinant Affibody molecules

Affibody molecules constitute a novel class of molecular display selected affinity proteins based on non-immunoglobulin scaffold. Preclinical investigations and pilot clinical data have demonstrated that Affibody molecules provide high contrast imaging of tumor-associated molecular targets shortly after injection. The use of cysteine-containing peptide-based chelators at the C-terminus of recombinant Affibody molecules enabled site-specific labeling with the radionuclide ^99mTc. Earlier studies have demonstrated that position, composition and the order of amino acids in peptide-based chelators influence labeling stability, cellular processing and biodistribution of Affibody molecules. To investigate the influence of the amino acid order, a series of anti-HER2 Affibody molecules, containing GSGC, GEGC and GKGC chelators have been prepared and characterized. The affinity to HER2, cellular processing of ^99mTc-labeled Affibody molecules and their biodistribution were investigated. These properties were compared with that of the previously studied ^99mTc-labeled Affibody molecules containing GGSC, GGEC and GGKC chelators. All variants displayed picomolar affinities to HER2. The substitution of a single amino acid in the chelator had an appreciable influence on the cellular processing of ^99mTc. The biodistribution of all ^99mTc-labeled Affibody molecules was in general comparable, with the main difference in uptake and retention of radioactivity in excretory organs. The hepatic accumulation of radioactivity was higher for the lysine-containing chelators and the renal retention of ^99mTc was significantly affected by the amino acid composition of chelators. The order of amino acids influenced renal uptake of some conjugates at 1 h after injection, but the difference decreased at later time points. Such information can be helpful for the development of other scaffold protein-based imaging and therapeutic radiolabeled conjugates.     

Nucleotides Part LXXX: Synthesis of 3′-O Fluorescence Labeled Thymidine Derivatives and Their 5′-O-Triphosphates

A new labeling technique attaching a fluorescent pteridine derivative (3, 5) via a linker onto the 3′-OH group of 5′-O-dimethoxytritylthymidine (7) was developed to lead to the conjugates 8 and 11. After detritylation to give 9 and 12, the final conversion into the corresponding 5′-triphosphates (13, 14), which were isolated as sodium salts, was performed by known methods.     

A Universal Approach to Prepare Reagents for DNA-Assisted Protein Analysis

The quality of DNA-labeled affinity probes is critical in DNA-assisted protein analyses, such as proximity ligation and extension assays, immuno-PCR, and immuno-rolling circle amplification reactions. Efficient, high-performance methods are therefore required for isolation of pure conjugates from reactions where DNA strands have been coupled to antibodies or recombinant affinity reagents. Here we describe a universal, scalable approach for preparing high-quality oligonucleotide-protein conjugates by sequentially removing any unconjugated affinity reagents and remaining free oligonucleotides from conjugation reactions. We applied the approach to generate high-quality probes using either antibodies or recombinant affinity reagents. The purified high-grade probes were used in proximity ligation assays in solution and in situ, demonstrating both augmented assay sensitivity and improved signal-to-noise ratios.     

Probing the MvaI Methyltransferase Region that Interacts with DNA: Affinity Labeling with the Dialdehyde-Containing DNA Duplexes

Abstract

Affinity labeling of methyltransferase MvaI by DNA duplexes containing oxidized 2′-O-β-D-ribofuranosylcytidine or 1-(β-D-galactopyranosyl)thymine residues was performed. Partial chemical hydrolysis of the covalently bound methylase in the conjugates with the dialdehyde-containing DNA allowed us to determine the amino acid region in the C terminus of methylase MvaI that interacts with DNA.     

Structure-Activity Relationships of 2′,5′-Oligoadenylate Analogue Modifications of Prostate-Specific Membrane Antigen (PSMA) Antagonists

Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer.     

Preparation, 99mTc-labeling, and in vitro characterization of HYNIC and N3S modified RC-160 and [Tyr3]octreotide.

The synthesis of conjugates of two somatostatin analogues, RC-160 and [Tyr3]octreotide with different bifunctional chelators for labeling with Tc-99m, is described. Conjugates with hydrazinonicotinamide (HYNIC) and two N3S compounds (benzoyl MAG3 and a N3S adipate derivative) were prepared on a small scale with high purity allowing evaluation of different bifunctional chelators on the same peptide without extensive peptide synthesis. High in vitro stability and retained binding affinity was found for all conjugates except for the N3S adipate. Peptide conjugates could be labeled at high specific activities (>1 Ci/micromol) with 99mTc, and different coligands were explored for the HYNIC conjugates. The resulting radiolabeled complexes were highly stable and showed binding affinity to somatostatin receptors in the nanomolar range. Varying labeling yield, stability, lipophilicity, and isomerism were found for different coligands used for labeling HYNIC conjugates, with lower lipophilicity, higher stability, and fewer coordination isomers for EDDA and tricine/nicotinic acid as ternary coligand compared to tricine. In particular, HYNIC complexes showed promising results for further in vivo evaluation.     

Design of Angiotensin II Derivatives Suitable for Indirect Affinity Techniques: Potential Applications to Receptor Studies

Abstract

The design of angiotensin II (A II)-derived probes suitable for indirect affinity techniques is presented. Biotin or dinitrophenyl moieties have been added at the N-terminus of A II, through aminohexanoic acid as spacer arm, to generate (6-biotinylamido)-hexanoyl-All (Bio-Ahx-All) and dinitrophenyl- aminohexanoyl-All (Dnp-Ahx-All). Monoiodinated and highly labeled radioiodinated forms of these probes have been prepared. The two bifunctional ligands displayed high affinities for rat liver A II receptors (K_d values in the nanomolar range) and their secondary acceptors: streptavidin and monoclonal anti-Dnp antibodies respectively. Bio-Ahx-All and Dnp-Ahx-All behaved as agonists on several All-sensitive systems. Based on these structural assessments, the parent photoactivable azido probe: Bio-Ahx-(Ala¹,Phe(4N₃)⁸)A II. A II was synthesized and proved to possess similar biological properties than the non-azido compound. The hepatic A II receptor could be covalently labeled by the radioiodinated probe, with a particularly high yield (15-20%); SDS-polyacrylamide gel electrophoresis of solubilized complexes revealed specific labeling of a 65 Kdaltons binding unit, in agreement with previous data obtained with other azido All-derived compounds. The potential applications of these probes are: i) receptor purification by combination of its photoaffinity labeling and adsorption of biotin-tagged solubilized hormone-receptor complexes on avidin gels. ii) cell labeling and sorting. iii) histochemical receptor visualization.     

A New Type of Fluorescence Labeling of Nucleosides,Nucleotides and Oligonucleotides

Abstract

Fluorescein has been coupled to the amino groups of the common nucleo-sides via a carbamoyl spacer to form a new type of conjugates. The corresponding phos-phoramidites have been prepared with Npe and Npeoc protecting groups for application in oligonucleotide synthesis. Hybridizations have been studied in dependence of the fluores-cing label as well as fluorescence quantum yields and fluorescence anisotropy effects.     

Identification of a high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane of suspension-cultured rice cells by affinity labeling

A high-affinity binding protein for the N-acetylchito-oligosaccharide elicitor of phytoalexin biosynthesis was identified by photoaffinity labeling and affinity cross-linking in the plasma membrane of suspension-cultured rice cells. Both a [¹²⁵I]-labeled photolabile 2-(4-azidophenyl)ethylamino conjugate ([¹²⁵I]-GN₈-AzPEA) and a [¹²⁵I]-labeled 2- (4-aminophenyl)ethylamino conjugate ([¹²⁵]-GN₈-APEA) of N-acetylchito-octaose were synthesized. The two conjugates were separately incubated with the plasma membrane prepared by aqueous two-phase partitioning, and covalently cross-linked to the elicitor binding site by irradiation with UV light or treatment with the cross-linking agent glutaraldehyde, respectively. Autoradiography of the SDS-PAGE gel of the solubilized membrane proteins revealed the labeling of a single 75 kDa band in both cases. The incorporation of the radiolabeled ligands into the 75 kDa protein showed a saturable mode of binding, with half-maximal incorporation at 45 and 52 nM for photoaffinity labeling and affinity cross-linking, respectively. The labeling of the 75 kDa protein was inhibited by N-acetylchito-oligasaccharides in a size-dependent manner, and N-acetylchito-octaose (GlcNAc)₈ showed a half-maximal inhibition at concentrations of the order of 10 nM. However, neither chito-octaose (GlcN)₈, cellopentaose nor α-1,4 linked N-acetylgalactosamine octamer (GalNAc)₈ at concentrations as high as 25 μM inhibited the labeling of the 75 kDa protein. These results are in good agreement with the sensitivity and the specificity of the ‘high-affinity binding site’ previously identified by binding assays, as well as with the activities of these oligosaccharides in the induction of phytoalexin biosynthesis and other cellular responses. These results suggest that the 75 kDa protein identified by the affinity labeling represents a functional receptor for this elicitor.     

New Pyrene Derivatives for Fluorescent Labeling of Oligonucleotides

Abstract

A series of pyrene-containing reagents have been synthesized and used for the fluorescent labeling of oligonucleotides.     

Uptake of Oligonucleotides by Keratinocytes

Abstract

Oligonucleotides (ODNs) conjugated to rhodamin (Rh) and 4-[(N-2-chloroethyl-N-methyl)amino] benzylamine were used to investigate ODNs transport into keratinocytes. Affinity labeling of two proteins, 63 and 35 kDa, and the inhibition of the affinity labeling and ODNs uptake by the cells in the presence of nucleic acids, polyanions and trypsin suggest, that the proteins are involved in transport of nucleic acids in keratinocytes.     

Selective Attachment of Oligonucleotides to Interleukin-1β and Targeted Delivery to Cells

Abstract

Conjugates between oligodeoxyribonucleotides and an interleukin-1β mutant protein have been constructed using a heterobifunctional cross-linker. These protein-DNA conjugates had conserved binding activity to the interleukin-1 receptor. The oligonucleotide hybridization properties were unchanged.     

Synthesis and preliminary in vitro evaluation of DOTA-Tenatumomab conjugates for theranostic applications in tenascin expressing tumors

Tenatumomab is an anti-tenascin murine monoclonal antibody previously used in clinical trials for delivering radionuclides to tumors by both pre-targeting (biotinylated Tenatumomab within PAGRIT) and direct ¹³¹Iodine labeling approaches. Here we present the synthesis and in vitro characterization of three Tenatumomab conjugates to bifunctional chelating agents (NHS-DOTA, NCS-DOTA and NCS-DTPA). Results indicate ST8198AA1 (Tenatumomab-DOTAMA, derived by conjugation of NHS-DOTA), as the most promising candidate in terms of conjugation rate and yield, stability, antigen immunoreactivity and affinity. Labeling efficiency of the different chelators was investigated with a panel of cold metals indicating DOTAMA as the best chelator. Labeling of Tenatumomab-DOTAMA was then optimized with several metals and stability performed confirms suitability of this conjugate for further development. ST8198AA1 represents an improvement of the previous antibody forms because the labeling with radionuclides like ¹⁷⁷Lu or ⁶⁴Cu would allow theranostic applications in patients bearing tenascin expressing tumors.     

Potential for tumor therapy with iodine-125 labeled immunoglobulins

Because of the high intranuclear toxicity of Auger-electron emitters, the use of radioiodinated (¹²³I, ¹²⁵I) 5-iodo-2′-deoxyuridine (IUdR)-antibody conjugates for cancer therapy has been examined. The results have demonstrated that all the conditions necessary for labeling DNA in vivo are present: uptake of the radiolabeled immunoglobulin by target cells, its subsequent internalization, the degradation of the IUdR-protein conjugate by lysosomal enzymes, and the incorporation of the radionucleoside into DNA.     

Cleavage of Leishmania Mini-exon Sequence by Oligonucleotides Conjugated to a Dimidazole Construction

Abstract

RNA sequences derived from the Leishmania amazonensis mini-exon and pre-mini-exon sequences have been targeted with complementary oligonucleotides bearing a diimidazole construction mimicking active center of ribonuclease A. The conjugates were shown to cleave the target RNAs at specific positions.     

5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure

Oligonucleotide conjugates bearing two pyrene residues attached to 5′-phosphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (λ_max 382 nm) and a broad emission peak with λ_max 476 nm, which indicates the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5′-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5′-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.     

Inactivation of trypsin-like proteases by active-site-directed sulfonylation: Ability of the departing group to confer selectivity

The p-nitrophenyl ester of p-amidinophenylmethanesulfonic acid had been found to inactivate thrombin by affinity labeling but did not have this action on other proteases of similar specificity such as trypsin, plasmin, or plasma kallikrein [Wong, S.-C., and Shaw, E., Arch. Biochem. Biophys. 161, 536 (1974)]. The ortho- and meta-nitrophenyl esters of this sulfonic acid have now been synthesized and shown to be less selective. In addition to thrombin, trypsin and plasma kallikrein are also inactivated. The ortho isomer is more effective than the meta. Plasmin is unaffected by all three esters. The results are interpreted to reflect geometrical differences in the first departing group subsite of these homologous active centers and to provide an additional structural basis for achieving selectivity of affinity labeling.     

Aldehyde Functions in Synthetic Oligonucleotides

Abstract

new nucleoside analogs with a masked aldehyde function have been synthesized and incorporated in oligodeoxyribonucléotides. After unmasking the aldehyde function, the oligomers were easily coupled to biotin derivatives by reductive amination. Reversed phase HPLC proved to be an unvaluable tool to characterize and purify these conjugates.     

Combinatorial Library of Artificial Ribonucleases

Abstract

Conjugates of lanthanide complexes and oligonucleotides have been shown to bind and to cleave complementary RNA. Modified oligonucleotide building blocks are required to ensure stability of the oligonucleotide against cellular nucleases. For an efficient cleavage, an accessible site on the structured mRNA is required. For the identification of active oligoribonucleotide-lanthanide complex conjugates, we have used a combinatorial library of conjugate sequences.     

Dansyl labeled proteins: Determination of extinction coefficient and number of bound residues with radioactive dansyl chloride

A method is described for preparing fluorescent conjugates of proteins labeled by reaction with methyl-C¹⁴-dansyl chloride and for determining their radioactivity by scintillation counting. The extinction coefficient of the bound dye varies in conjugates of different proteins and is considerably lower than the value generally employed to calculate degree of labeling. For assays of dansyl groups using absorption spectroscopy, a value of ? = 3.4 × 10³M^?1 cm^?1 will probably be approximately correct. However, the radioactive dye technique allows much more accurate determinations of degree of labeling, as well as of the extinction coefficient, which may be of interest in itself.