Radioactive Peptides Research Articles

Radiovirotherapy at twenty

Radiovirotherapy at twenty

Therapeutic Efficacy of 177Lu-Labeled A20FMDV2 Peptides Targeting ανβ6.

Integrin ανβ6 promotes migration and invasion of cancer cells, and its overexpression often correlates with poor survival. Therefore, targeting ανβ6 with radioactive peptides would be beneficial for cancer imaging and therapy. Previous studies have successfully developed radiotracers based on the peptide A20FMDV2 that showed good binding specificity for ανβ6. However, one concern of these ανβ6 integrin-targeting probes is that their rapid blood clearance and low tumor uptake would preclude them from being used for therapeutic purposes. In this study, albumin binders were used to increase tumor uptake for therapeutic applications while the non-albumin peptide was evaluated as a potential positron emission tomography (PET) imaging agent. All peptides used the DOTA chelator for radiolabeling with either 68Ga for imaging or 177Lu for therapy. PET imaging with [68Ga]Ga-DOTA-(PEG28)2-A20FMDV2 revealed specific tumor uptake in ανβ6-positive tumors. Albumin-binding peptides EB-DOTA-(PEG28)2-A20FMDV2 and IBA-DOTA-(PEG28)2-A20FMDV2 were radiolabeled with 177Lu. Biodistribution studies in normal mice showed longer blood circulation times for the albumin binding peptides compared to the non-albumin peptide. Therapy studies in mice demonstrated that both 177Lu-labeled albumin binding peptides resulted in significant tumor growth inhibition. We believe these are the first studies to demonstrate the therapeutic efficacy of a radiolabeled peptide targeting an ανβ6-positive tumor.

Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP

BackgroundSkeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1. MethodsThe modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([14C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin. ResultsThe study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1. ConclusionsThe desensitization to ATP is due to the modification of His-51 of the Zn2 coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized. General significanceThe progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution.

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model.

PurposeMelanocortin receptor 1 (MC1R) is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium (68Ga) labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography (PET) imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide.ProceduresThe MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95°C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors.ResultsIn biodistribution studies, non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracer preparations. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake.ConclusionsWe demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

Photoinactivation of the Staphylococcus aureus Lactose-Specific EIICB Phosphotransferase Component with p-azidophenyl-β-D-Galactoside and Phosphorylation of the Covalently Bound Substrate

Background: The phosphoenolpyruvate (PEP):lactose phosphotransferase system of Staphylococcus aureus transports and phosphorylates lactose and various phenylgalactosides. Their phosphorylation is catalyzed by the Cys476-phosphorylated EIIB domain of the lactose-specific permease enzyme IICB (EIICB^Lac). Phosphorylation causes the release of galactosides bound to the EIIC domain into the cytoplasm by a mechanism not yet understood. Results: Irradiation of a reaction mixture containing the photoactivatable p-azidophenyl-β-D-galactopyranoside and EIICB^Lac with UV light caused a loss of EIICB^Lac activity. Nevertheless, photoinactivated EIICB^Lac could still be phosphorylated with [³²P]PEP. Proteolysis of photoinactivated [³²P]P-EIICB^Lac with subtilisin provided an 11-kDa radioactive peptide. Only the sequence of its first three amino acids (-H-G-P-, position 245–247) could be determined. They are part of the substrate binding pocket in EIICs of the lactose/cellobiose PTS family. Surprisingly, while acid treatment caused hydrolysis of the phosphoryl group in active [³²P]P∼EIICB^Lac, photoinactivated [³²P]P-EIICB^Lac remained strongly phosphorylated. Conclusion: Phosphorylation of the –OH group at C6 of p-nitrenephenyl-β-D-galactopyranoside covalently bound to EIIC^Lac by the histidyl-phosphorylated [³²P]P∼EIIB^Lac domain is a likely explanation for the observed acid resistance. Placing p-nitrenephenyl-β-D-galactopyranoside into the active site of modelled EIIC^Lac suggested that the nitrene binds to the -NH- group of Ser248, which would explain why no sequence data beyond Pro247could be obtained.

Photoaffinity Labeling of Plant Receptor Kinases.

Defining the ligand-binding activity of receptors is important because the binding of ligands is the initial reaction in secreted ligand-dependent cell-to-cell communication. Photoaffinity labeling is one of the most efficient biochemical techniques for detecting direct ligand-receptor interactions. Here, we describe photoaffinity labeling to visualize the direct interaction between peptide ligands and their receptors by using photoactivatable and radioactive peptide ligand derivatives.

Uptake Assay for Radiolabeled Peptides in Yeast.

We describe an assay for measuring the uptake of radioactive peptides into the yeast Saccharomyces cerevisiae. The methods presented here can be adapted to measure a variety of substrates transported into any bacterial or fungal cell via specific carrier-mediated systems.

Glypican-3-targeting F(ab')2 for 89Zr PET of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an increasingly lethal malignancy for which management is critically dependent on accurate imaging. Glypican-3 (GPC3) is a cell surface receptor overexpressed in most HCCs and provides a unique target for molecular diagnostics. The use of monoclonal antibodies (mAbs) that target GPC3 (αGPC3) in PET imaging has shown promise but comes with inherent limitations associated with mAbs such as long circulation times. This study used (89)Zr-conjugated F(ab')2 fragments directed against GPC3 ((89)Zr-αGPC3-F(ab')2) to evaluate the feasibility of the fragments as a diagnostic immuno-PET imaging probe. Immobilized ficin was used to digest αGPC3, creating αGPC3-F(ab')2 fragments subsequently conjugated to (89)Zr. In vivo biodistribution and PET studies were performed on GPC3-expressing HepG2 and GPC3-nonexpressing RH7777 orthotopic xenografts. Reliable αGPC3-F(ab')2 production via immobilized ficin digestion was verified by high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. (89)Zr-αGPC3-F(ab')2 demonstrated F(ab')2-dependent, antigen-specific cell binding. HepG2 tumor uptake was higher than any other tissue, peaking at 100 ± 21 percentage injected dose per gram (%ID/g) 24 h after injection, a value 33- to 38-fold higher than GPC3-nonexpressing RH7777 tumors. The blood half-life of the (89)Zr-αGPC3-F(ab')2 conjugate was approximately 11 h, compared with approximately 115 h for historic mAb controls. This shorter half-life enabled clear tumor visualization on PET 4 h after administration, with a resultant peak tumor-to-liver contrast ratio of 23.3. Blocking antigen-expressing tumors with an excess of nonradiolabeled αGPC3 resulted in decreased tumor uptake similar to native liver. The kidneys exhibited high tissue uptake, peaking at 24 h with 83 ± 12 %ID/g. HepG2 tumors ranging from 1.5 to 7 mm were clearly visible on PET, whereas larger RH7777 tumors displayed signal lower than background liver tissue. This study demonstrates the feasibility of using (89)Zr-αGPC3-F(ab')2 for intrahepatic tumor localization with small-animal PET. Faster blood clearance and lower background liver uptake enable excellent signal-to-noise ratios at early time points. Increased renal uptake is similar to that as has been seen with clinical radioactive peptide imaging. (89)Zr-αGPC3-F(ab')2 addresses some of the shortcomings of whole-antibody immuno-PET probes. Further optimization is warranted to maximize probe sensitivity and specificity in the process of clinical translation.

Metabolic Activation and Major Protein Target of a 1-Benzyl-3-carboxyazetidine Sphingosine-1-phosphate-1 Receptor Agonist

1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened α,β-unsaturated imine conjugated to the ε-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a β-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.

Kinome profiling.

The use of arrays in genomics has led to a fast and reliable way to screen the transcriptome of an organism. It can be automated and analysis tools have become available and hence the technique has become widely used within the past few years. Signal-transduction routes rely mainly on the phosphorylation status of already available proteins; therefore kinases are central players in signal-transduction routes. The array technology can now also be used for the analysis of the kinome. To enable array analysis, consensus peptides for kinases are spot on a solid support. After incubation with cell lysates and in the presence of radioactive ATP, radioactive peptides can be visualized and the kinases that are active in the cells can be determined. The present paper reviews comprehensively the different kinome array platforms available and results obtained hitherto using such platforms. It will appear that this technology does not disappoint its high expectations and is especially powerful because of its species independence. Nevertheless, improvements are still possible and I shall also sketch future possible directions.

The Vitamin K-dependent Carboxylase Generates γ-Carboxylated Glutamates by Using CO2 to Facilitate Glutamate Deprotonation in a Concerted Mechanism That Drives Catalysis

The γ-glutamyl carboxylase converts Glu to carboxylated Glu (Gla) to activate a large number of vitamin K-dependent proteins with diverse functions, and this broad physiological impact makes it critical to understand the mechanism of carboxylation. Gla formation is thought to occur in two independent steps (i.e. Glu deprotonation to form a carbanion that then reacts with CO(2)), based on previous studies showing unresponsiveness of Glu deprotonation to CO(2). However, our recent studies on the kinetic properties of a variant enzyme (H160A) showing impaired Glu deprotonation prompted a reevaluation of this model. Glu deprotonation monitored by tritium release from the glutamyl γ-carbon was dependent upon CO(2), and a proportional increase in both tritium release and Gla formation occurred over a range of CO(2) concentrations. This discrepancy with the earlier studies using microsomes is probably due to the known accessibility of microsomal carboxylase to water, which reprotonates the carbanion. In contrast, tritium incorporation experiments with purified carboxylase showed very little carbanion reprotonation and consequently revealed the dependence of Glu deprotonation on CO(2). Cyanide stimulated Glu deprotonation and carbanion reprotonation to the same extent in wild type enzyme but not in the H160A variant. Glu deprotonation that depends upon CO(2) but that also occurs when water or cyanide are present strongly suggests a concerted mechanism facilitated by His-160 in which an electrophile accepts the negative charge on the developing carbanion. This revised mechanism provides important insight into how the carboxylase catalyzes the reaction by avoiding the formation of a high energy discrete carbanion.

Solid phase synthesis, radiolabeling and biological evaluation of a 99mTc-labeled αVβ3 tripeptide (RGD) conjugated to DOTA as a tumor imaging agent

Solid phase synthesis of peptides-radiometal chelator can facilitate the creation of radioactive peptide libraries to be utilized in high throughput in in vivo screening of targeted molecular imaging agents. An αVβ3 tripeptide derivative DOTA-NH-Arg-Gly-Asp was synthesized by Fmoc solid phase peptide synthesis and analyzed by spectroscopic techniques. In order to radiolabel this RGD peptide with 99mTc-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was incorporated as a chelator. The DOTA-peptide conjugate binds to 99mTc with high efficiency at ambient temperature. The resulting conjugate is stable under physiological conditions for at least 24 h after radiocomplexation. The receptor binding studies of 99mTc-DOTA- αVβ3-tripeptide in established human tumor cell lines U-87MG and BMG revealed Kd values in the nM and μM range respectively. U-87MG tumors in athymic mice were accumulated in the γ-images and major accumulation of the radiotracer was observed in kidneys followed by liver and lungs. High tumor uptake was shown in the U-87MG tumor bearing athymic mice; tumor to muscle ratios reached 8.13 ± 2.18 and 35.09 ± 4.78 at 1 and 4 h after post injection respectively.

Characterization of HKI-272 Covalent Binding to Human Serum Albumin

The study was initiated as an observation of incomplete extraction recovery of N-(4-(3-chloro-4-(2-pyridinylmethoxy)anilino)-3-cyano-7-ethoxy-6-quinolyl)-4-(dimethylamino)-2-butenamide (HKI-272) from human plasma. The objective of this study was to 1) identify the binding site(s) of HKI-272 to human plasma protein(s); 2) characterize the nature of the binding; and 3) evaluate the potential reversibility of the covalent binding. After incubation of [(14)C]HKI-272 with human plasma, the mixture was directly injected on liquid chromatography/mass spectrometry (LC/MS), and an intact molecular mass of HKI-272 human serum albumin (HSA) adduct was determined to be 66,999 Da, which is 556 Da (molecular mass of HKI-272) larger than the measured molecular mass of HSA (66,443 Da). For peptide mapping, the incubation mixture was separated with SDS-polyacrylamide gel electrophoresis followed by tryptic digestion combined with LC/tandem MS. A radioactive peptide fragment, LDELRDEGKASSAK [amino acid (AA) residue 182-195 of albumin], was confirmed to covalently bind to HKI-272. In addition, after HCl hydrolysis, a radioactive HKI-272-lysine adduct was identified by LC/MS. After combining the results of tryptic digestion and HCl hydrolysis, the AA residue of Lys190 of HSA was confirmed to covalently bind to HKI-272. A standard HKI-272-lysine was synthesized and characterized by NMR. The data showed that the adduct was formed via Michael addition with the epsilon-amine of lysine attacking to the beta-carbon of the amide moiety of HKI-272. Furthermore, reversibility of the covalent binding of HKI-272 to HSA was shown when a gradual release of HKI-272 was observed from protein pellet of HKI-272-treated human plasma after resuspension in phosphate buffer, pH 7.4, at 37 degrees C for 18 h.

Radioiodination of Peptide Hormones and Immunoglobuline Preparations: Comparison of the Chloramine T and lodogen Method*)

Following optimization of the reaction conditions, e.g. concentration of oxidizing agents, reaction time, volume of reaction mixture, and pH, chloramine T and the new iodination reagent, Iodogen, were compared for their effectiveness in radioiodination of insulin, glucagon, human growth hormone (hGH), and rabbit anti-mouse IgG. The radioactive peptide hormones prepared were analyzed for the presence of aggregate and breakdown products by polyacrylamide gel electrophoresis (PAGE) at pH 8.9, the rabbit anti-mouse IgG was tested for the presence of low molecular weight damage products by gel filtration on Sephadex G-50. The results demonstrate that with respect to iodine incorporation, specific activity, and immunological reactivity either method can be used to prepare under carefully controlled conditions a wide range of tracers with high specific activity at minimal oxidation damage. These tracers are shown to be highly suitable in radioimmunoassays after previous purification by PAGE and gel filtration, respectively.

Negative and Positive Regulation of MAPK Phosphatase 3 Controls Platelet-derived Growth Factor-induced Erk Activation

MAPK phosphatases (MKPs) are dual specificity phosphatases that dephosphorylate and thereby inactivate MAPKs. In the present study, we provide evidence that platelet-derived growth factor BB (PDGF-BB) regulates MKP3 (DUSP6), which is considered to be a phosphatase highly selective for Erk. Intriguingly, we observed that Mek is positively regulated by MKP3, whereas Erk itself is negatively regulated. In addition, we found that activation of PDGF receptor alpha or beta leads to a rapid proteasomal degradation of MKP3 in a manner that requires Mek activation; this feed-forward mechanism was found to be essential for efficient Erk phosphorylation. We could also demonstrate that PDGF-BB stimulation induces phosphorylation of MKP3 at Ser-174 and Ser-300; phosphorylation of Ser-174 is involved in PDGF-induced MKP3 degradation, since mutation of this site stabilized MKP3. Moreover, activated Erk induces mkp3 expression, leading to restoration of MKP3 levels after 1-2 h and a concomitant dephosphorylation of Erk in cells with activated PDGFRalpha. Reducing the MKP3 level by small interfering RNA leads to an increased Erk activation and mitogenic response to PDGF-BB. In conclusion, MKP3 is an important regulator of PDGF-induced Erk phosphorylation acting in both a rapid positive feed-forward and a later negative feed-back loop.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-Tyr311 Phosphorylation in Cardiomyocyte Caveolae

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCdelta to caveolae and increase PKCdelta-Tyr(311) phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Characterization of Some Molecular Mechanisms Governing Autoactivation of the Catalytic Domain of the Anaplastic Lymphoma Kinase

NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK.

Multifunctional Antibodies by the Dock-and-Lock Method for Improved Cancer Imaging and Therapy by Pretargeting

The Dock-and-Lock (DNL) method, which makes bioactive molecules with multivalency and multifunctionality, is a new approach to develop targeting molecules for improved cancer imaging and therapy. It involves the use of a pair of distinct protein domains involved in the natural association between cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and A-kinase anchoring proteins (AKAPs). The dimerization and docking domain found in the regulatory subunit of PKA and the anchoring domain (AD) of an interactive AKAP are each attached to a biologic entity, and the resulting derivatives, when combined, readily form a stably tethered complex of a defined composition that fully retains the functions of the individual constituents. The DNL method has generated several trivalent, bispecific, binding proteins, each consisting of 2 identical Fab fragments linked site-specifically to a different Fab fragment. For example, 2 identical Fabs reacting with carcinoembryonic antigen (CEA) are bound to a Fab reacting with a hapten peptide that bears a diagnostic or therapeutic radionuclide. Using a 2-step, pretargeting method that separates the bivalent anti-CEA antibody targeting of tumor from the delivery of the radioactive peptide that is captured by the second Fab of the tri-Fab construct, an improved method of cancer imaging and therapy has been developed and shows very sensitive and specific targeting of CEA-expressing tumors for either diagnostic imaging, such as with immunoSPECT and immunoPET, or radioimmunotherapy. Improved therapeutic efficacy is shown with pretargeting in a pancreatic cancer xenograft model given a tri-Fab to a pancreatic cancer MUC1 and the hapten peptide labeled with (90)Y.

Autophosphorylation of Solanum chacoense cytosolic nucleoside diphosphate kinase on Ser117

NDPK catalyses the interconversion of NTPs and NDPs using a phosphohistidine intermediate as part of its catalytic site. Recombinant Solanum chacoense cytosolic NDPK incubated with [gamma-(32)P]ATP was allowed to autophosphorylate and (32)P-labelled P-Ser was identified in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of (32)P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive peptide allowed the identification of a single and conserved P-Ser residue at position 117. Analysis of site-directed mutants where Ser117 was substituted to Asp indicated that the presence of a negative charge at position 117 dramatically lowered the enzyme's catalytic efficiency. Ser autophosphorylation was markedly reduced with increasing ADP concentrations in the autophosphorylation assay. These findings provide evidence that autophosphorylation of cytosolic NDPK on Ser117 could constitute a regulatory mechanism for this important enzyme and that autophosphorylation of Ser117 is modulated by NDP availability.

Identification of in Vivo Phosphorylation Sites on Human Deoxycytidine Kinase: ROLE OF SER-74 IN THE CONTROL OF ENZYME ACTIVITY

Deoxycytidine kinase (dCK) catalyzes the rate-limiting step of the deoxyribonucleoside salvage pathway in mammalian cells and plays a key role in the activation of numerous nucleoside analogues used in anti-cancer and antiviral chemotherapy. Although compelling evidence indicated that dCK activity might be regulated by phosphorylation/dephosphorylation, direct demonstration was lacking. Here we showed that dCK overexpressed in HEK 293T cells was labeled after incubating the cells with [32P]orthophosphate. Sorbitol, which was reported to decrease dCK activity, also decreased the labeling of dCK. These results indicated that dCK may exist as a phosphoprotein in vivo and that its activity can be correlated with its phosphorylation level. After purification of 32P-labeled dCK, digestion by trypsin, and analysis of the radioactive peptides by tandem mass spectrometry, the following four in vivo phosphorylation sites were identified: Thr-3, Ser-11, Ser-15, and Ser-74, the latter being the major phosphorylation site. Site-directed mutagenesis and use of an anti-phospho-Ser-74 antibody demonstrated that Ser-74 phosphorylation was crucial for dCK activity in HEK 293T cells, whereas phosphorylation of other identified sites did not seem essential. Phosphorylation of Ser-74 was also detected on endogenous dCK in leukemic cells, in which the Ser-74 phosphorylation state was increased by agents that enhanced dCK activity. Our study provided direct evidence that dCK activity can be controlled by phosphorylation in intact cells and highlights the importance of Ser-74 for dCK activity.