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Caroline J. Springer - One of the best experts on this subject based on the ideXlab platform.
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Detection of the Prodrug-Activating Enzyme Carboxypeptidase G2 Activity with Chemical Exchange Saturation Transfer Magnetic Resonance
Molecular Imaging and Biology, 2014Co-Authors: Yann Jamin, Caroline J. Springer, Thomas R Eykyn, Evon Poon, Simon P RobinsonAbstract:Purpose The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by Carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy. Procedures Z spectra of solutions of the CPG2 substrate, 3,5-difluorobenzoyl- l -glutamate (amide), and glutamate (amine) were acquired at 11.7 T, 37 °C, across different pH (5–8). The ability of CEST-MR to monitor CPG2-mediated release of glutamate was assessed in extracts of CPG2-expressing cancer cells and purified solution of CPG2. Results The addition of CPG2 to a solution containing 3,5-difluorobenzoyl- l -glutamate led to a marked and progressively increasing CEST effect (+3 ppm), concomitant with the time-dependent release of glutamate induced by CPG2. Conclusion CEST-MR allows the detection of CPG2 activity in vitro and supports the translation of CEST-MRI to assess CPG2-based gene therapy in vivo .
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detection of the prodrug activating enzyme Carboxypeptidase G2 activity with chemical exchange saturation transfer magnetic resonance
Molecular Imaging and Biology, 2014Co-Authors: Yann Jamin, Caroline J. Springer, Thomas R Eykyn, Evon Poon, Simon P RobinsonAbstract:Purpose The purpose of this study is to evaluate if the differential exchange rates with bulk water between amine and amide protons can be exploited using chemical exchange saturation transfer magnetic resonance (CEST-MR) to monitor the release of glutamate induced by Carboxypeptidase G2 (CPG2), an enzyme utilized in cancer gene therapy.
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noninvasive detection of Carboxypeptidase G2 activity in vivo
NMR in Biomedicine, 2011Co-Authors: Yann Jamin, Caroline J. Springer, Thomas R Eykyn, Evon Poon, Simon P Robinson, Lynette A Smyth, Martin O Leach, Geoffrey S PayneAbstract:The pseudomonad protein, Carboxypeptidase G2 (CPG2), is a prodrug-activating enzyme utilized in the targeted chemotherapy strategies of antibody-and gene-directed enzyme prodrug therapy (ADEPT and GDEPT). We have developed a noninvasive imaging approach to monitor CPG2 activity in vivo that will facilitate the preclinical and clinical development of CPG2-based ADEPT and GDEPT strategies. Cleavage of the novel reporter probe, 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu), by CPG2, in human colon adenocarcinoma WiDr xenografts engineered to stably express CPG2, was monitored using F-19 MRSI. The high signal-to-noise ratio afforded by the two MR-equivalent F-19 nuclei of 3,5-DFBGlu, and the 1.4 ppm F-19 chemical shift difference on CPG2-mediated cleavage, enabled the dynamics and quantification of the apparent pharmacokinetics of 3,5-DFBGlu and its CPG2-mediated cleavage in the tumor to be evaluated. In addition, the apparent rate of increase of 3,5-difluorobenzoic acid concentration could also provide a biomarker of CPG2 activity levels in tumors of patients undergoing CPG2-based therapies, as well as a biomarker of treatment response. The addition of in vivo reporter probes, such as 3,5-DFBGlu, to the armamentarium of prodrugs cleaved by CPG2 affords new applications for CPG2 as a gene reporter of transgene expression. Copyright (C) 2010 John Wiley & Sons, Ltd.
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hyperpolarized 13 c magnetic resonance detection of Carboxypeptidase G2 activity
Magnetic Resonance in Medicine, 2009Co-Authors: Yann Jamin, Caroline J. Springer, Simon P Robinson, Cristina Gabellieri, Lynette A Smyth, Steven Reynolds, Martin O Leach, Geoffrey S Payne, Thomas R EykynAbstract:Carboxypeptidase G2 (CPG2) is a bacterial enzyme that is currently employed in a range of targeted cancer chemotherapy strategies such as gene-directed enzyme prodrug therapy (GDEPT). Employing dynamic nuclear polarization (DNP) and natural abundance 13 C magnetic resonance spectroscopy (MRS), we observed the CPG2-mediated conversion of a novel hyperpolarized reporter probe 3,5-difluorobenzoyl-L-glutamic acid (3,5-DFBGlu) to 3,5-difluorobenzoic acid (3,5-DFBA) and L-glutamic acid (L-Glu) in vitro. Isotopic labeling of the relevant nuclei with 13 C in 3,5-DFBGlu or related substrates will yield a further factor of 100 increase in the signal-to-noise. We discuss the feasibility of translating these experiments to generate metabolic images of CPG2 activity in vivo. Magn Reson Med 62: 1300 –1304, 2009. © 2009 Wiley-Liss, Inc.
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a novel technique to monitor Carboxypeptidase G2 expression in suicide gene therapy using 19f magnetic resonance spectroscopy
NMR in Biomedicine, 2009Co-Authors: Laura Mancini, Caroline J. Springer, Frank Friedlos, Lawrence Davies, Martin O Leach, Geoffrey S Payne, Maria Falckminiotis, Andrzej S K DzikjuraszAbstract:Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid, a prodrug for the GDEPT enzyme Carboxypeptidase-G2 (CPG2; K-m = 1.71 mu M; k(cat) = 732 s(-1)), was measured with F-19 magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K-m = 3.5 mu M, k(cat) = 747 s(-1)). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well-resolved F-19 MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half-life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent. Copyright (C) 2009 John Wiley & Sons, Ltd.
R G Melton - One of the best experts on this subject based on the ideXlab platform.
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Carboxypeptidase G2 rescue in patients with methotrexate intoxication and renal failure
British Journal of Cancer, 2005Co-Authors: S. Buchen, R G Melton, A Zoubek, Udo Bode, D Ngampolo, C. Hasan, Günter Henze, Gudrun FleischhackAbstract:High-dose methotrexate (HD-MTX) of more than 1 g m−2 body surface area, administered by a prolonged intravenous infusion with leucovorin (LV, D,L-5-formyltetrahydrofolic acid) rescue, has demonstrated clinical activity in malignancies and diseases, such as acute lymphoblastic leukaemia, lymphoma, osteosarcoma, and head and neck cancer. Methotrexate is primarily excreted by the renal route. It may cause acute nephrotoxicity, which is presumed to result from its precipitation or its relatively insoluble metabolites in acidic urine. This nephrotoxicity leads to delayed MTX elimination, ineffective rescue and a marked increase of other nonhaematological and haematological toxicities associated with MTX, such as myelosuppression, oro-gastrointestinal mucositis and dermatitis. In early studies using HD-MTX, severe toxicity occurred in approximately 10% of patients and there was a 6% mortality rate (Ahmad et al, 1978). The introduction of pretreatment hydration, alkalinisation of urine, routine monitoring of serum MTX concentrations and postinfusion guided LV rescue has decreased the incidence of severe and life-threatening toxicity after HD-MTX from 10% to less than 1% (Juergens et al, 1983; Allegra, 1990). However, severe nephrotoxicity of HD-MTX leading to potentially lethal toxicity still occurs. For patients with decreased MTX clearance due to renal dysfunction, therapeutic options are few and of limited efficacy. When serum MTX concentrations are persistently greater than 10–100 μM, high doses of LV are not likely to completely reverse the toxicity of MTX (Goldman and Levy, 1968; Pinedo et al, 1976; Frei et al, 1980; Grem et al, 1991). Thymidine may be used in case of MTX toxicity, but due to its rapid clearance it must be administered in high doses by continuous infusion (Ensminger and Frei, 1977; Howell et al, 1978; Howell et al, 1980; van den Bongard et al, 2001). Neither LV nor thymidine has any effect on the underlying problem of delayed MTX elimination. Peritoneal dialysis is ineffective, most likely due to a combination of factors including the protein binding, the high degree of ionisation and the low lipid solubility of MTX (Ahmad et al, 1978). Haemodialysis and haemoperfusion have variable efficacy and usually produce only transient decreases of plasma MTX concentrations (Gibson et al, 1978; Relling et al, 1988; Saland et al, 2002). Alternative methods to treat persistently elevated MTX levels are therefore needed. The Carboxypeptidase G class of enzymes hydrolyse the C-terminal glutamate residue from folic acid and classical antifolates, such as MTX, rendering them inactive (Levy and Goldman, 1967; Chabner et al, 1972, Goldman, 1975). Carboxypeptidase G1 (CPDG1) was originally extracted from Pseudomonas stutzeri. During the 1970s, CPDG1 was administered successfully to a small number of patients with brain tumours and to one patient with pre-existing renal failure after MTX administration. However, the bacterial source for CPDG1 is no longer available (Bertino et al, 1974; Abelson et al, 1978; Minton et al, 1983). A recombinant form of CPDG2, cloned from Pseudomonas strain RS-16, is now available as an investigational drug (Minton et al, 1983; Sherwood et al, 1985). Carboxypeptidase G2 hydrolyses MTX rapidly to the inactive metabolites DAMPA (4-[[2,4-diamino-6-(pteridinyl)methyl]-methylamino]-benzoic acid) and glutamate. In contrast to CPDG1, CPDG2 has a higher affinity for MTX (Km 8 μM) than LV (Km 120 μM) and 5-methyltetrahydrofolate (Km 35 μM), the primary circulating metabolite of LV. In studies performed in rhesus monkeys, a single CPDG2 dose of 50 units (U) kg−1 lowered serum MTX steady-state concentration from 10 μM to nontoxic levels of <0.05 μM within 30 min (Adamson et al, 1992). In addition, the bolus injection of CPDG2 (a dose of 50 U kg−1 in combination with LV and partial thymidine rescue) resulted in a rapid 95–99% reduction of plasma levels of MTX in patients with renal dysfunction after HD-MTX (Widemann et al, 1995, 1997; Zoubek et al, 1995). This study will demonstrate the feasibility, effectiveness and toxicity of an emergency therapy with CPDG2 in a large European patient collective with acute MTX intoxication and renal failure.
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crystal structure of Carboxypeptidase G2 a bacterial enzyme with applications in cancer therapy
Structure, 1997Co-Authors: Siân Rowsell, R G Melton, Richard A Pauptit, D M Blow, Alec D Tucker, Peter BrickAbstract:Abstract Background: Carboxypeptidase G enzymes hydrolyze the C-terminal glutamate moiety from folic acid and its analogues, such as methotrexate. The enzyme studied here, Carboxypeptidase G 2 (CPG 2 ), is a dimeric zinc-dependent exopeptidase produced by Pseudomonas sp. strain RS-16. CPG 2 has applications in cancer therapy: following its administration as an immunoconjugate, in which CPG 2 is linked to an antibody to a tumour-specific antigen, it can enzymatically convert subsequently administered inactive prodrugs to cytotoxic drugs selectively at the tumour site. CPG 2 has no significant amino acid sequence homology with proteins of known structure. Hence, structure determination of CPG 2 was undertaken to identify active-site residues, which may in turn provide ideas for protein and/or substrate modification with a view to improving its therapeutic usefulness. Results: We have determined the crystal structure of CPG 2 at 2.5 A resolution using multiple isomorphous replacement methods and non-crystallographic symmetry averaging. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site, and a separate smaller domain that forms the dimer interface. The two active sites in the dimer are more than 60 A apart and are presumed to be independent; each contains a symmetric distribution of carboxylate and histidine ligands around two zinc ions which are 3.3 A apart. This distance is bridged by two shared zinc ligands, an aspartic acid residue and a hydroxyl ion. Conclusions: We find that the CPG 2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica , where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.
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a new crystal form of Carboxypeptidase G2 from pseudomonas sp strain rs 16 which is more amenable to structure determination
Acta Crystallographica Section D-biological Crystallography, 1996Co-Authors: A D Tucker, R G Melton, S Rowsell, Richard A PauptitAbstract:Carboxypeptidase G2 is a zinc-dependent exopeptidase which has applications in cancer therapy. Crystallization of Carboxypeptidase G2, first achieved more than a decade ago, yields large crystals; however, problems with non-isomorphism between native crystals as well as failure to obtain any useful heavy-atom derivatives have precluded structure solution. A modification of the crystallization protocol leading to a promising new crystal form which diffracts beyond 3.0 A resolution on a rotating-anode source is now reported. These crystals are readily indexed on an apparent C-centred orthorhombic lattice with a = 81.35, b = 230.9 and c = 105.5 A, but the correct crystal system is monoclinic. The crystals have space group P21, with a = 81.35, b = 105.5, c = 122.4 A and β = 109.3°. There are two possible non-equivalent monoclinic indexings with these lattice constants. A partial native data set collected at the SRS, Daresbury, indicates that 1.9 A diffraction is attainable. Structure determination using MIR methods is in progress.
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Anti-tumour effects of an antibody-Carboxypeptidase G2 conjugate in combination with phenol mustard prodrugs
British Journal of Cancer, 1995Co-Authors: D. C. Blakey, C J Springer, S. East, P. J. Burke, S K Sharma, Dh Davies, Ri Dowell, Ab Mauger, R G MeltonAbstract:ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4-[N,N-bis(2-chloroethyl)amino]-phenoxycarbonyl-L- glutamic acid (PGP) and (S)-2-[N-[4-[N,N-bis(2-chloroethyl)amino]- phenoxycarbonyl]amino]-4-(5-tetrazoyl)butyric acid (PTP), which are cleaved by the bacterial enzyme CPG2 to release the 4-[N,N-bis(2-chloroethyl)amino] phenol drug. In vitro, both prodrugs are approximately 100- to 200-fold less potent than the parent drug (1 h IC50 = 1.4 microM) in LoVo colorectal tumour cells. These prodrugs have been evaluated for utility in ADEPT when used in combination with a conjugate of CPG2 and the F(ab')2 fragment of the anti-CEA monoclonal antibody, A5B7. The conjugate was shown to localise specifically to established LoVo tumour xenografts growing in nude mice and optimal tumour-normal tissue ratios were achieved after 72 h. Administration of either prodrug, at doses which cause 6-8% body weight loss, 72 h after administration of the A5B7-CPG2 conjugate to the LoVo tumour-bearing mice resulted in tumour regressions and growth delays of 14-28 days. The PTP prodrug in combination with a high dose of conjugate (10 mg kg-1) gave the best anti-tumour activity despite being a 10-fold worse substrate for CPG2 than PGP. Prodrug alone, active drug alone or prodrug in combination with a non-specific conjugate had minimal anti-tumour activity in this tumour model.
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Galactosylated antibodies and antibody-enzyme conjugates in antibody-directed enzyme prodrug therapy
Cancer, 1994Co-Authors: Surinder K. Sharma, Joan A. Boden, G T Rogers, R G Melton, C J Springer, Kenneth D. Bagshawe, Philip J. Burke, R. F. SherwoodAbstract:Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two- and three-phase system in which an antibody to a tumor-associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic-antigen antibody fragment A5B7-F(ab') 2 conjugated to a bacterial enzyme, Carboxypeptidase G2 (CPG2), in LS174T xenografted mice
Roger F. Sherwood - One of the best experts on this subject based on the ideXlab platform.
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282 – Glutamate Carboxypeptidase
Handbook of Proteolytic Enzymes, 2020Co-Authors: Roger F. SherwoodAbstract:Publisher Summary This chapter describes the activity, specificity and structural chemistry of glutamate Carboxypeptidase. Glutamate Carboxypeptidases are characterized as Zn2+ requiring exopeptidases with specificity for glutamate and this specificity is not absolute as Carboxypeptidase G was reported as exhibiting activity against pteroyl compounds with C-terminal aspartic acid. Physicochemical properties and substrate affinities for folic acid, and a variety of analogs are compared for the G1 and G2 enzymes. Carboxypeptidase G2 is a dimeric protein of 83,600 kDa containing two atoms of zinc per subunit. There are no disulfide bonds and the complete nucleotide and amino acid sequences were resolved following cloning of the gene into Escherichia coli. In both native and recombinant form the enzyme is located in the periplasmic space, targeted by a 22 amino acid signal peptide. Carboxypeptidase G2 has been crystallized and the crystal structure determined at 2.5 A resolutions. Each subunit of the molecular dimer consists of a larger catalytic domain containing two zinc ions at the active site and a separate smaller domain that forms the dimer interface.
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Covalent linkage of Carboxypeptidase G2 to soluble dextrans—II: in vivo distribution and fate of conjugates
Biochemical Pharmacology, 2002Co-Authors: Roger G Melton, C N Wiblin, R L Foster, A Baskerville, Roger F. SherwoodAbstract:Abstract The in vivo fate of the therapeutic enzyme, Carboxypeptidase G2 (CPG2) in native form and covalently-linked to soluble dextrans was studied in the mouse using radiolabelled compounds. Clearance, from the blood, of all compounds tested was found to be as intact, active material, whilst excreted radiolabel was associated in all cases with low molecular weight substances. The clearance and excretion rates of native CPG2 were found to balance, but this was not so for dextran-CPG2 conjugate or CNBr-activated dextran. Tissue distribution studies demonstrated that there was little or no tissue uptake of native CPG2, whereas dextran-CPG2 conjugate, and CNBr-activated dextran were retained in the liver. Within the liver, the CPG2 component of dextran-CPG2 conjugate was degraded more rapidly than the dextran moiety. Blockade of reticulo-endothelial system (RES) led to increased half-lives of dextran CPG2 conjugate and CNBr-activated dextran, demonstrating the involvement of the RES in the clearance of these compounds. Impairment of RES activity did not affect the clearance rate of native CPG2. These results are discussed in relation to the potential use of dextran-CPG2 conjugates in cancer chemotherapy.
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In vitro and in vivo characterisation of a recombinant Carboxypeptidase G2::anti-CEA scFv fusion protein.
Immunotechnology, 1996Co-Authors: N. Paul Michael, Roger G Melton, William Nicholas, R.barbara Pedley, Richard H.j. Begent, Joan A. Boden, Kerry A. Chester, Roger F. Sherwood, L Robson, Nigel P MintonAbstract:Abstract Background: There is considerable interest in the specific targeting of therapeutic agents to cancer cells. Of particular promise is a technique known as Antibody-Directed Enzyme Prodrug Therapy (ADEPT). In this approach an enzyme is targeted to the tumour by its conjugation to a tumour specific-antibody tumour. After allowing sufficient time for the conjugate to localise at the tumour and clear from the circulatory system, a relatively non-toxic prodrug is administered. This prodrug is converted to a highly cytotoxic drug by the action of the targeted enzyme localised at the tumour site. Objectives: To construct gene fusions between the pseudomonad Carboxypeptidase G2 (CPG2) gene and DNA encoding MFE-23 (an anti-carcinoembryonic antigen (CEA) single-chain Fv (scFv) molecule), derived from a phage display library. To overexpress the resultant gene fusions in Escherichia coli, and assess the in vitro and in vivo properties of the purified fusion proteins. Study design: To introduce unique cloning restriction sites into the 5′-end of the CPG2 gene by site-directed mutagenesis to facilitate fusion to the 3′-end of the gene encoding MFE-23 (constructs with or without a flexible (Gly4Ser)3 linker-encoding sequence were designed). To overexpress the resultant gene fusions under transcriptional control of the lac promoter and to direct the fusion proteins produced to the periplasmic space of E. coli through translational coupling to the pelB signal peptide. Results: Biologically active recombinant CPG2::MFE-23 scFv fusion proteins were produced in E. coli and shown to possess enzyme and anti-EA activity. Affinity chromatography followed by size exclusion gel filtration yielded approximately 0.7 – 1.4 mg/l from shake flask culture. The fusion protein in which the enzyme and antibody moieties were joined by a linker peptide was shown to be effectively localised in nude mice bearing human colon tumour xenografts, giving favourable tumour to blood ratios. Conclusion: MFE-23 scFv serves as an ideal candidate for the antibody arm of a bacterially expressed fusion protein with CPG2. The biological properties of this recombinant protein suggest that it may be employed for tumour specific prodrug activation. However, further assessment of its stability and pharmokinetics is required if genetic fusion is to be considered as an alternative to chemical conjugation.
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optimisation of small scale coupling of a5b7 monoclonal antibody to Carboxypeptidase G2
Journal of Immunological Methods, 1993Co-Authors: R G Melton, G T Rogers, J M B Boyle, Kenneth D. Bagshawe, Philip J. Burke, Roger F. SherwoodAbstract:Abstract Conjugates of F(ab′) 2 fragment of the monoclonal antibody A5B7 coupled to Carboxypeptidase G 2 (CPG 2 ) have been produced using the heterobifunctional reagents 2-mercapto-[ S -acetyl]acetic acid, N -hydroxysuccinimide ester (SATA) and m -maleimidobenzoyl- N -hydroxysuccinimide ester (SMPB). The effect of various levels of modifying reagent on enzyme activity and antigen binding activity were determined, and it was shown that whilst CPG 2 is relatively sensitive to modification, insertion of three maleimide groups per CPG 2 resulted in the loss of 30% of enzyme activity; 5B7 F(ab′) 2 was insensitive to modification, little or no activity being lost. The coupling efficiency of the reation was shown to be fairly constant over a wide range of substitution levels. There was thus no advantage to be gained in using high substitution levels, which may result in loss of enzyme activity. The formation of undesired high molecular weight aggregates could be controlled by adjustment of the protein concentration during the final coupling step.
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crystallization and preliminary crystallographic analysis of Carboxypeptidase G2 from pseudomonas sp strain rs 16
Journal of Molecular Biology, 1991Co-Authors: Lesley F Lloyd, Charles A Collyer, Roger F. SherwoodAbstract:Abstract Carboxypeptidase G2 a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and l -glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A , b = 82 A , c = 116 A and β = 118 ° . The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3·0 A and are suitable for X-ray structure analysis.
Frank Friedlos - One of the best experts on this subject based on the ideXlab platform.
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a novel technique to monitor Carboxypeptidase G2 expression in suicide gene therapy using 19f magnetic resonance spectroscopy
NMR in Biomedicine, 2009Co-Authors: Laura Mancini, Caroline J. Springer, Frank Friedlos, Lawrence Davies, Martin O Leach, Geoffrey S Payne, Maria Falckminiotis, Andrzej S K DzikjuraszAbstract:Development and evaluation of new anticancer drugs are expedited when minimally invasive biomarkers of pharmacokinetic and pharmacodynamic behaviour are available. Gene-directed enzyme prodrug therapy (GDEPT) is a suicide gene therapy in which the anticancer drug is activated in the tumor by an exogenous enzyme previously targeted by a vector carrying the gene. GDEPT has been evaluated in various clinical trials using several enzyme/prodrug combinations. The key processes to be monitored in GDEPT are gene delivery and expression, as well as prodrug delivery and activation. {4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoyl}-L-glutamic acid, a prodrug for the GDEPT enzyme Carboxypeptidase-G2 (CPG2; K-m = 1.71 mu M; k(cat) = 732 s(-1)), was measured with F-19 magnetic resonance spectroscopy (MRS). The 1 ppm chemical shift separation found between the signals of prodrug and activated drug (4-[bis(2-chloroethyl)amino]-3,5-difluorobenzoic acid) is sufficient for the detection of prodrug activation in vivo. However, these compounds hydrolyze rapidly, and protein binding broadens the MR signals. A new CPG2 substrate was designed with hydroxyethyl instead of chloroethyl groups (K-m = 3.5 mu M, k(cat) = 747 s(-1)). This substrate is nontoxic and stable in solution, has a narrow MRS resonance in the presence of bovine and foetal bovine albumin, and exhibits a 1.1 ppm change in chemical shift upon cleavage by CPG2. In cells transfected to express CPG2 in the cytoplasm (MDA MB 361 breast carcinoma cells and WiDr colon cancer cells), well-resolved F-19 MRS signals were observed from clinically relevant concentrations of the new substrate and its nontoxic product. The MRS conversion half-life (470 min) agreed with that measured by HPLC (500 min). This substrate is, therefore, suitable for evaluating gene delivery and expression prior to administration of the therapeutic agent. Copyright (C) 2009 John Wiley & Sons, Ltd.
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attenuated salmonella targets prodrug activating enzyme Carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy
Clinical Cancer Research, 2008Co-Authors: Frank Friedlos, Lawrence Davies, Richard Marais, Jan Martin, Panos Lehouritis, Douglas Hedley, Lesley Ogilvie, David Bermudes, Ivan King, Caroline J. SpringerAbstract:PURPOSE: We engineered the oncolytic Salmonella typhimurium-derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme Carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. EXPERIMENTAL DESIGN: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity. We assessed prodrug activation by high-performance liquid chromatography. Target human tumor cell and bacterial vector cell cytotoxicity was measured by flow cytometry and colony-forming assays. Therapy was shown in two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. RESULTS: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 x 10(7)/g to 2 x 10(8)/g at 40 days postinoculation. The concentration of CPG2 in these tumors increases steadily to therapeutic levels of 1 to 6 units/g. The bacteria alone reduce the growth of the tumors. Subsequent administration of prodrugs further reduces significantly the growth of the xenografts. CONCLUSIONS: The bacteria multiply within tumors, resulting in a selective expression of CPG2. The CPG2-expressing bacteria alone reduce the growth of tumors. However, in the presence of prodrugs activated by CPG2, this oncolytic effect is greatly increased. We conclude that bacterial oncolytic therapy, combined with CPG2-mediated prodrug activation, has great potential in the treatment of a range of cancers.
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suicide gene therapy of human colon carcinoma xenografts using an armed oncolytic adenovirus expressing Carboxypeptidase G2
Cancer Research, 2007Co-Authors: Silke Schepelmann, Janet L Martin, Frank Friedlos, Richard Marais, Douglas Hedley, Lesley Ogilvie, Ian Scanlon, Ping Chen, Caroline J. SpringerAbstract:We have designed a targeted systemic suicide gene therapy that combines the advantages of tumor-selective gene expression, using the human telomerase promoter (hTERT), with the beneficial effects of an oncolytic adenovirus to deliver the gene for the prodrug-activating enzyme Carboxypeptidase G2 (CPG2) to tumors. Following delivery of the vector (AdV.hTERT-CPG2) and expression of CPG2 in cancer cells, the prodrug ZD2767P was administered for conversion by CPG2 to a cytotoxic drug. This system is sometimes termed gene-directed enzyme prodrug therapy (GDEPT). Here, we have shown that it is applicable to 10 human colorectal carcinoma cell lines with a direct correlation between viral toxicity and CPG2 production. SW620 xenografts were selected for analysis and were significantly reduced or eradicated after a single administration of AdV.hTERT-CPG2 followed by a prodrug course. The oncolytic effect of adenovirus alone did not result in DNA cross-links or apoptosis, whereas DNA cross-links and apoptosis occurred following prodrug administration, showing the combined beneficial effects of the GDEPT system. The apoptotic regions extended beyond the areas of CPG2 expression in the tumors, indicative of significant bystander effects in vivo . Higher concentrations of vector particles and CPG2 were found in the AdV.hTERT-CPG2 plus prodrug–treated tumors compared with the virus alone, showing an unexpected beneficial and cooperative effect between the vector and GDEPT. This is the first time that a tumor-selective GDEPT vector has been shown to be effective in colorectal carcinoma and that apoptosis and significant bystander effects have been identified as the mechanisms of cytotoxicity within the tumor. [Cancer Res 2007;67(10):4949–55]
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novel fluorinated prodrugs for activation by Carboxypeptidase G2 showing good in vivo antitumor activity in gene directed enzyme prodrug therapy
Journal of Medicinal Chemistry, 2005Co-Authors: Lawrence Davies, Frank Friedlos, Jan Martin, Douglas Hedley, Lesley Ogilvie, Ian Scanlon, Caroline J. SpringerAbstract:Sixteen novel polyfluorinated benzoic acid mustards have been synthesized for use in gene-directed enzyme prodrug therapy (GDEPT). Eight of these were benzoic acid l-glutamate mustards for evaluation as prodrugs and the other eight were the active drugs formed by the action of the bacterial enzyme Carboxypeptidase G2 (CPG2). All of the di- and trifluorinated prodrugs were efficiently cleaved by the enzyme. In contrast, the tetrafluorinated prodrugs were found to be competitive inhibitors of CPG2, the first such inhibitors to have been described. The di- and trifluorinated prodrugs were differentially cytotoxic to human breast carcinoma cells (MDA MB 361) expressing CPG2, compared to control cells that did not express the enzyme. The difluorinated prodrug {4-[bis(2-bromoethyl)amino]-3,5-difluorobenzoyl}-l-glutamic acid and its iodoethylamino analogue were effective substrates for the enzyme and showed excellent therapeutic activity in CPG2-expressing MDA MB 361 xenografts, either curing or greatly inhibiti...
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systemic gene directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with Carboxypeptidase G2
Cancer Research, 2005Co-Authors: Silke Schepelmann, Janet L Martin, Frank Friedlos, Richard Marais, Douglas Hedley, Lesley Ogilvie, Ian Scanlon, Paul L Hallenbeck, Lynda K Hawkins, Caroline J. SpringerAbstract:Hepatocellular carcinoma is the fifth most common cancer worldwide, and there is no effective therapy for unresectable disease. We have developed a targeted systemic therapy for hepatocellular carcinoma. The gene for a foreign enzyme is selectively expressed in the tumor cells and a nontoxic prodrug is then given, which is activated to a potent cytotoxic drug by the tumor-localized enzyme. This approach is termed gene-directed enzyme prodrug therapy (GDEPT). Adenoviruses have been used to target cancer cells, have an intrinsic tropism for liver, and are efficient gene vectors. Oncolytic adenoviruses produce clinical benefits, particularly in combination with conventional anticancer agents and are well tolerated. We rationalized that such adenoviruses, if their expression were restricted to telomerase-positive cancer cells, would make excellent gene vectors for GDEPT therapy of hepatocellular carcinoma. Here we use an oncolytic adenovirus to deliver the prodrug-activating enzyme Carboxypeptidase G2 (CPG2) to tumors in a single systemic administration. The adenovirus replicated and produced high levels of CPG2 in two different hepatocellular carcinoma xenografts (Hep3B and HepG2) but not other tissues. GDEPT enhanced the adenovirus-alone therapy to elicit tumor regressions in the hepatocellular carcinoma models. This is the first time that CPG2 has been targeted and expressed intracellularly to effect significant therapy, showing that the combined approach holds enormous potential as a tumor-selective therapy for the systemic treatment of hepatocellular carcinoma.
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Ultrasound enhances ZD2767P‒Carboxypeptidase G2 against chemoresistant ovarian cancer cells by altering the intracellular pharmacokinetics of ZD2767D.
Molecular Pharmaceutics, 2020Co-Authors: Xiaocui Zhong, Ying Zhang, Xinya Li, Guanhua Qian, Tinghe YuAbstract:Prodrug‒Carboxypeptidase G2 (e.g., ZD2767P+CPG2) can realize a targeted treatment where the specific advantage is a lack of CPG2 analogs in humans, but it is limited by low efficacy. Here ultrasoun...
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ultrasound enhances zd2767p Carboxypeptidase G2 against chemoresistant ovarian cancer cells by altering the intracellular pharmacokinetics of zd2767d
Molecular Pharmaceutics, 2020Co-Authors: Xiaocui Zhong, Ying Zhang, Xinya Li, Guanhua Qian, Tinghe YuAbstract:Prodrug‒Carboxypeptidase G2 (e.g., ZD2767P+CPG2) can realize a targeted treatment where the specific advantage is a lack of CPG2 analogs in humans, but it is limited by low efficacy. Here ultrasoun...
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biphasically modulating the activity of Carboxypeptidase G2 with ultrasound
Cellular Physiology and Biochemistry, 2017Co-Authors: Ying Zhang, Xueling Zheng, Tinghe YuAbstract:BACKGROUND/AIMS: Carboxypeptidase G2 (CPG2) has been used for cancer prodrug therapy to realize the targeted release of active drugs, but there yet lacks a means to modulate the CPG2 activity. Here ultrasound was used to modulate the CPG2 activity. METHODS: The activity of insonated CPG2 was determined, and then underlying biochemical (i.e., monomer, dimer and conformation) and ultrasonic (i.e., heat and cavitation) mechanisms were explored. RESULTS: Ultrasound (1.0 MHz) increased or decreased the enzymatic activity; the activity decreased as zero- or first-order kinetics, depending on the intensity. L1 (10 W/cm2 for 200 s) improved the activity via increasing the specific activity. L2 or L3 (20 W/cm2 for 1200 or 3000 s) decreased the activity via disassembling the dimer, degrading the monomer, inducing glycosylation, transforming conformation and decreasing the specific activity. An increase or a slight decrease of activity attributable to 10 W/cm2 was reversible, but the activity decrease due to 20 W/cm2 was irreversible. The enzymatic modulation was realized via cavitation. CONCLUSION: Ultrasound can biphasically modulate the CPG2 activity, and can be employed in the CPG2-prodrug therapy to adjust the release and moles of active drugs.
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nanosecond electric pulses deprive zinc ions of Carboxypeptidase G2
Bioelectrochemistry, 2015Co-Authors: Tinghe Yu, Xiao FuAbstract:Abstract Nanosecond electric pulses (nsEP, 10 kV/cm with a pulse duration of 8, 16 or 24 ns) inhibited the activity of Carboxypeptidase G2 (CPG2), a zinc-dependent homodimer; the relative activity was 120 s. No alterations were detected in electrophoresis, chromatography, mass spectroscopy and circular dichroism, thus demonstrating intactness of the apoenzyme. Inductively coupled plasma-mass spectrometry indicated that zinc levels were 3.30 μg/mg protein in control CPG2, and decreased to 0.40, 0.12 or 0.38 μg/mg protein after 240 s of 8-, 16- or 24-ns pulses, respectively. In CPG2 exposed to 240 s of 8-, 16- and 24-ns pulses, the reloading of zinc with redialysis recovered the activity to 94.7 ± 3.4%, 84.0 ± 5.2% and 81.7 ± 7.0%, respectively (p = 0.0853, 0.0741, 0.0668). These data demonstrated that nsEP inhibited CPG2 via removal of zinc, and that nsEP can be used to modulate CPG2.