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Paul S. Gaynon - One of the best experts on this subject based on the ideXlab platform.
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Asparaginase antibody and Asparaginase activity in children with higher risk acute lymphoblastic leukemia children s cancer group study ccg 1961
Journal of Pediatric Hematology Oncology, 2004Co-Authors: Eduard H Panosyan, Harland N. Sather, Lawrence J. Ettinger, Janet Franklin, Paul S. Gaynon, Nita L Seibel, Sagrario Martinaragon, Ioannis A Avramis, James B Nachman, Peter G SteinherzAbstract:Abstract:We investigated the anti-Asparaginase antibody (Ab) and Asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL). Patients received nine doses of native Escherichia coli Asparaginase during induction. Half of rapid early r
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A randomized comparison of native Escherichia coli Asparaginase and polyethylene glycol conjugated Asparaginase for treatment of children with newly diagnosed standard-risk acute lymphoblastic leukemia: a children's Cancer Group Study
Blood, 2002Co-Authors: Vassilios I. Avramis, Bruce Bostrom, Susan Sencer, Antonia P. Periclou, Harland N. Sather, Lewis J. Cohen, Alice G. Ettinger, Lawrence J. Ettinger, Janet Franklin, Paul S. GaynonAbstract:For this study, 118 children with standard-risk acute lymphoblastic leukemia (ALL) were given randomized assignments to receive native or pegylated Escherichia coli Asparaginase as part of induction and 2 delayed intensification phases. Patients treated with pegaspargase had more rapid clearance of lymphoblasts from day 7 and day 14 bone marrow aspirates and more prolonged Asparaginase activity than those treated with native Asparaginase. In the first delayed intensification phase, 26% of native Asparaginase patients had high-titer antibodies, whereas 2% of pegaspargase patients had those levels. High-titer antibodies were associated with low Asparaginase activity in the native arm, but not in the pegaspargase arm. Adverse events, infections, and hospitalization were similar between arms. Event-free survival at 3 years was 82%. A population pharmacodynamic model using the nonlinear mixed effects model (NONMEM) program was developed that closely fit the measured enzyme activity and asparagine concentrations. Half-lives of Asparaginase were 5.5 days and 26 hours for pegaspargase and native Asparaginase, respectively. There was correlation between Asparaginase enzymatic activity and depletion of asparagine or glutamine in serum. In cerebrospinal fluid asparagine, depletion was similar with both enzyme preparations. Intensive pegaspargase for newly diagnosed ALL should be tested further in a larger population.
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acute lymphoblastic leukaemia a guide to Asparaginase and pegaspargase therapy
BioDrugs, 1997Co-Authors: Lawrence J. Ettinger, Vassilios I. Avramis, Alice G. Ettinger, Paul S. GaynonAbstract:The cure rate for children with acute lymphoblastic leukaemia (ALL) has increased to approximately 70%, in part related to the use of the protein synthesis inhibitor drug Asparaginase in multiagent chemotherapy regimens. Its lack of haematological toxicity allows its incorporation into phases of therapy in which myelosuppression would be expected either from the disease itself (induction therapy) or secondary to other chemotherapeutic agents (consolidation, intensification or reinduction phases of therapy). Its antileukaemic effect is related to the degree and duration of asparagine depletion. The 2 native forms of L-Asparaginase are derived from Escherichia coli and Ewinia chrysantherni. The half-lives (t½) of these forms are approximately 1.2 and 0.6 days, respectively. In order to increase the biological t½, pegaspargase was synthesised by the covalent attachment of monomethoxypolyethylene glycol (PEG) to native E. coli L-Asparaginase: it has a t½, of approximately 5.7 days. The duration of asparagine depletion, the substrate amino acid of the drug, is directly related to Asparaginase t½. Asparaginase is associated with several unique toxicities, including hyperglycaemia, hypolipoproteinaemia, hypoalbuminaemia, coagulation factor deficiencies, hepatotoxicity and pancreatitis. Since Asparaginase is a protein, it may induce hypersensitivity reactions. The incidence of these reactions increases with use. In addition, silent hypersensitivity, i.e. the development of IgG antibodies without clinical reactions, results in a decreased t½ of Asparaginase, shortened duration of asparagine depletion, and probably decreased efficacy. The use of pegaspargase allows continued treatment with Asparaginase in patients with clinical hypersensitivity reactions. In addition, its use in patients with silent hypersensitivity may maintain the efficacy of Asparaginase. So far, the optimal use of the 3 forms of Asparaginase has not been determined in children with ALL, partly due to the lack of appropriate pharmacokinetic monitoring methods. As the technology has become available, it has been demonstrated that there is little rationale for the dosage and administration schedules presently in use. Studies are required to determine appropriate dosages and administration methods (intravenous or intramuscular) and schedules for each form of Asparaginase, based upon pharmacokinetic parameters. The incidence and time to onset of hypersensitivity (clinical or silent) reactions and the appropriate means of continuing Asparaginase therapy with therapeutic effect needs to be evaluated. Pharmacokinetic studies are now available as a research tool. These will allow further investigation to determine if failure to maintain asparagine depletion is a remediable cause of treatment failure.
Kjeld Schmiegelow - One of the best experts on this subject based on the ideXlab platform.
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peg Asparaginase allergy in children with acute lymphoblastic leukemia in the nopho all2008 protocol
Pediatric Blood & Cancer, 2015Co-Authors: Louise Tram Henriksen, Kjeld Schmiegelow, Goda Vaitkeviciene, Jonas Abrahamsson, Arja Harilasaari, Ellen Ruud, Kaie Pruunsild, Olafur G Jonsson, Mats HeymanAbstract:Background L-Asparaginase is an effective drug in the treatment of childhood acute lymphoblastic leukemia (ALL). The use of L-Asparaginase may be limited by serious adverse events of which allergy is the most frequent. The objective of this study was to describe the clinical aspects of PEG-Asparaginase allergy in children treated according to the Nordic Society of Paediatric Haematology and Oncology (NOPHO) ALL2008 protocol. Procedure Children (1–17 years) enrolled in the NOPHO ALL2008 protocol between July 2008 and August 2011, who developed PEG-Asparaginase allergy were identified through the NOPHO ALL2008 toxicity registry. In the NOPHO ALL2008 protocol, patients are randomized to 8 or 15 doses of intramuscular PEG-Asparaginase (Oncaspar®) 1,000 IU/m2/dose administered at 2 or 6 weeks intervals during a total period of 30 weeks. (Clinical trials.gov no: NCT00819351). Results Of 615 evaluable patients, 79 patients developed clinical PEG-Asparaginase allergy (cumulative risk; 13.2%) and discontinued PEG-Asparaginase therapy for that reason. PEG-Asparaginase allergy occurred after a median of two doses (75% range 2–4, max 14). In 58% of PEG-Asparaginase hypersensitive patients, the clinical allergic reactions appeared within 2 hr after PEG-Asparaginase administration and ranged from mild symptoms to systemic anaphylaxis. Nine patients experienced an anaphylactic reaction within 1 hr and 50 min from Asparaginase administration; none were fatal. Four of 68 patients (6%) who subsequently received Erwinase therapy also reacted allergic to Erwinase. Conclusion Clinical allergy to PEG-Asparaginase occurred early in treatment, was in general moderate in severity, and mostly developed within 2 hr after PEG-Asparaginase administration. The risk of subsequent Erwinase allergic reactions was low. Pediatr Blood Cancer 2015;62:427–433. © 2014 Wiley Periodicals, Inc.
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Asparaginase associated pancreatitis in children with acute lymphoblastic leukaemia in the nopho all2008 protocol
British Journal of Haematology, 2014Co-Authors: Raheel Altaf Raja, Kjeld Schmiegelow, Birgitte Klug Albertsen, Kaie Prunsild, Bernward Zeller, Goda Vaitkeviciene, Jonas Abrahamsson, Mats Heyman, Mervi TaskinenAbstract:SummaryL-Asparaginase is an important drug in the treatment of childhood acutelymphoblastic leukaemia (ALL). Treatment is associated with several toxici-ties, including acute pancreatitis. Clinical course, presentation, re-exposureto L-asparginase after pancreatitis and risk of recurrent pancreatitis withinan Asparaginase-intensive protocol has been poorly reported. Children (1–17 years) on the ongoing Nordic Society of Paediatric Haematology andOncology (NOPHO) ALL2008 protocol with Asparaginase-associated pan-creatitis (AAP) diagnosed between 2008 and 2012 were identified throughthe online NOPHO ALL toxicity registry. NOPHO ALL2008 includes eightor 15 doses of intramuscular pegylated L-asparginase (PEG-Asparaginase)1000 iu/m 2 /dose at 2–6 weeks intervals, with a total of 30 weeks of expo-sure to PEG-Asparaginase (clinicaltrials.gov no: NCT00819351). Of 786children, 45 were diagnosed with AAP with a cumulative risk of AAP of5 9%. AAP occurred after a median of five doses (range 1–13), and 11 d(median) from the latest administration of PEG-Asparaginase. Thirteenpatients developed pseudocysts (30%) and 11 patients developed necrosis(25%). One patient died from pancreatitis. Twelve AAP patients werere-exposed to L-asparginase, two of whom developed mild AAP once more,after four and six doses respectively. In conclusion, re-exposure to PEG-Asparaginase in ALL patients with mild AAP seems safe.Keywords: L-Asparaginase, leukaemia, pancreatitis, toxicity, risk factors.
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Asparaginase associated pancreatitis in children
British Journal of Haematology, 2012Co-Authors: Raheel Altaf Raja, Kjeld Schmiegelow, Thomas FrandsenAbstract:l-Asparaginase has been an element in the treatment for acute lymphoblastic leukaemia (ALL) and non-Hodgkin lymphoma since the late 1960s and remains an essential component of their combination chemotherapy. Among the major toxicities associated with l-Asparaginase therapy are pancreatitis, allergic reactions, thrombotic events, hepatotoxicity and hyperlipidaemia. Acute pancreatitis is one of the most common reasons for stopping treatment with l-Asparaginase. Short-term complications of Asparaginase-associated pancreatitis include development of pseudocysts and pancreatic necrosis. Long-term complications include chronic pancreatitis and diabetes. The pathophysiology of Asparaginase-associated pancreatitis remains to be uncovered. Individual clinical and genetic risk factors have been identified, but they are only weak predictors of pancreatitis. This review explores the definition, possible risk factors, treatment and complications of Asparaginase-associated pancreatitis.
Barbara L Asselin - One of the best experts on this subject based on the ideXlab platform.
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intravenous pegylated Asparaginase versus intramuscular native escherichia coli l Asparaginase in newly diagnosed childhood acute lymphoblastic leukaemia dfci 05 001 a randomised open label phase 3 trial
Lancet Oncology, 2015Co-Authors: Lynda M Vrooman, Jeffrey G Supko, Andrew E Place, Kristen E Stevenson, Marian H Harris, Sarah K Hunt, Jane E Obrien, Barbara L AsselinAbstract:Summary Background l-Asparaginase is a universal component of treatment for childhood acute lymphoblastic leukaemia, and is usually administered intramuscularly. Pegylated Escherichia coli Asparaginase (PEG-Asparaginase) has a longer half-life and is potentially less immunogenic than the native Escherichia coli ( E coli ) preparation, and can be more feasibly administered intravenously. The aim of the Dana-Farber Cancer Institute Acute Lymphoblastic Leukaemia Consortium Protocol 05-001 (DFCI 05-001) was to compare the relative toxicity and efficacy of intravenous PEG-Asparaginase and intramuscular native E coli l-Asparaginase in children with newly diagnosed acute lymphoblastic leukaemia. Methods DFCI 05-001 enrolled patients aged 1–18 years with newly diagnosed acute lymphoblastic leukaemia from 11 consortium sites in the USA and Canada. Patients were assigned to an initial risk group on the basis of their baseline characteristics and then underwent 32 days of induction therapy. Those who achieved complete remission after induction therapy were assigned to a final risk group and were eligible to participate in a randomised comparison of intravenous PEG-Asparaginase (15 doses of 2500 IU/m 2 every 2 weeks) or intramuscular native E coli l-Asparaginase (30 doses of 25 000 IU/m 2 weekly), beginning at week 7 after study entry. Randomisation (1:1) was unmasked, and was done by a statistician-generated allocation sequence using a permuted blocks algorithm (block size of 4), stratified by final risk group. The primary endpoint of the randomised comparison was the overall frequency of Asparaginase-related toxicities (defined as allergy, pancreatitis, and thrombotic or bleeding complications). Predefined secondary endpoints were disease-free survival, serum Asparaginase activity, and quality of life during therapy as assessed by PedsQL surveys. All analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00400946. Findings Between April 22, 2005, and Feb 12, 2010, 551 eligible patients were enrolled. 526 patients achieved complete remission after induction, of whom 463 were randomly assigned to receive intramuscular native E coli l-Asparaginase (n=231) or intravenous PEG-Asparaginase (n=232). The two treatment groups did not differ significantly in the overall frequency of Asparaginase-related toxicities (65 [28%] of 232 patients in the intravenous PEG-Asparaginase group vs 59 [26%] of 231 patients in the intramuscular native E coli l-Asparaginase group, p=0·60), or in the individual frequency of allergy (p=0·36), pancreatitis (p=0·55), or thrombotic or bleeding complications (p=0·26). Median follow-up was 6·0 years (IQR 5·0–7·1). 5-year disease-free survival was 90% (95% CI 86–94) for patients assigned to intravenous PEG-Asparaginase and 89% (85–93) for those assigned to intramuscular native E coli l-Asparaginase (p=0·58). The median nadir serum Asparaginase activity was significantly higher in patients who received intravenous PEG-Asparaginase than in those who received intramuscular native E coli l-Asparaginase. Significantly more anxiety was reported by both patients and parent-proxy in the intramuscular native E coli l-Asparaginase group than in the intravenous PEG-Asparaginase group. Scores for other domains were similar between the groups. The most common grade 3 or worse adverse events were bacterial or fungal infections (47 [20%] of 232 in the intravenous PEG-Asparaginase group vs 51 [22%] of 231 patients in the intramuscular E coli l-Asparaginase group) and Asparaginase-related allergic reactions (14 [6%] vs 6 [3%]). Interpretation Intravenous PEG-Asparaginase was not more toxic than, was similarly efficacious to, and was associated with decreased anxiety compared with intramuscular native E coli l-Asparaginase, supporting its use as the front-line Asparaginase preparation in children with newly diagnosed acute lymphoblastic leukaemia. Funding National Cancer Institute and Enzon Pharmaceuticals.
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Asparaginase pharmacokinetics and implications of therapeutic drug monitoring
Leukemia & Lymphoma, 2015Co-Authors: Barbara L Asselin, Carmelo RizzariAbstract:Asparaginase is widely used in chemotherapeutic regimens for the treatment of acute lymphoblastic leukemia (ALL) and has led to a substantial improvement in cure rates, especially in children. Optimal therapeutic effects depend on a complete and sustained depletion of serum asparagine. However, pronounced interpatient variability, differences in pharmacokinetic properties between Asparaginases and the formation of Asparaginase antibodies make it difficult to predict the degree of asparagine depletion that will result from a given dose of Asparaginase. The pharmacological principles underlying Asparaginase therapy in the treatment of ALL are summarized in this article. A better understanding of the many factors that influence Asparaginase activity and subsequent asparagine depletion may allow physicians to tailor treatment to the individual, maximizing therapeutic effect and minimizing treatment-related toxicity. Therapeutic drug monitoring provides a means of assessing a patient's current depletion status and can be used to better evaluate the potential benefit of treatment adjustments.
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administration of erwinia Asparaginase erwinase following allergy to peg Asparaginase in children and young adults with acute lymphoblastic leukemia treated on aall07p2 achieves therapeutic nadir serum Asparaginase activity a report from the children
Blood, 2010Co-Authors: Wanda L Salzer, Jeffrey G Supko, Barbara L Asselin, Meenakshi Devidas, Elizabeth A Raetz, Paul V. Plourde, Nicole Kaiser, Naomi J Winick, Gregory H Reaman, William L CarrollAbstract:Abstract 2134 Introduction: L-Asparaginase is a vital component of multi-agent chemotherapy for children and young adults with acute lymphoblastic leukemia (ALL). In the United States, there are 2 Asparaginase preparations approved by the Food and Drug Administration, native E. coli (Elspar®) and PEG-Asparaginase (Oncaspar®). PEG-Asparaginase is the most commonly utilized Asparaginase product due to its longer half-life and decreased immunogenicity. However, the incidence of clinical allergy to PEG-Asparaginase approaches 20%, with repeated administration. Due to cross reactivity with native E. coli Asparaginase, there is no FDA-approved preparation available for patients who develop clinical allergy to PEG-Asparaginase. A third preparation, Erwinia Asparaginase (Erwinase®), derived from Erwinia chrysanthemi, is not commercially available in the United States. The optimal dosing of Erwinase® necessary to obtain nadir Asparaginase activity > 0.1 IU/mL similar to that obtained after conventional dosing of PEG-Asparaginase is unknown. Patients and Methods: We hypothesized that substitution of Erwinase® 25,000 IU/m2 × 6 doses intramuscularly (IM) on a Monday/Wednesday/Friday schedule in children and young adults with ALL would provide a 48 hour nadir serum Asparaginase activity ≥ 0.1 IU/mL, and effectively deplete plasma asparagine, a surrogate marker of Asparaginase activity. Eligible patients on COG study AALL07P2 were >1 to Results: A total of 55 eligible/evaluable patients were enrolled from February 2008 to April 2010. Blood samples were obtained at scheduled time points during Erwinase® therapy and assayed for serum Asparaginase activity and asparagine concentration in plasma. Nadir serum Asparaginase activity ≥ 0.1 IU/mL was achieved in 49/53 patients (92.5%) at 48 hours after dosing and in 46/52 patients (88.5%) at 72 hours after dosing. Plasma asparagine was significantly depleted ( Conclusion: Erwinase® as administered using the AALL07P2 regimen was well tolerated and achieved nadir serum Asparaginase activity at both 48 and 72 hours after dosing that was similar to that achieved with PEG-Asparaginase. We conclude that following allergy to PEG-Asparaginase, Erwinase® 25,000 IU/m2 × 6 doses IM on a Monday/Wednesday/Friday schedule can be substituted for a single dose of PEG-Asparaginase. Disclosures: Supko: EUSA Pharma: Research Funding. Plourde: EUSA Pharma: Employment. Winick: EUSA Pharma: EUSA Advisory Board.
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intravenous peg Asparaginase during remission induction in children and adolescents with newly diagnosed acute lymphoblastic leukemia
Blood, 2010Co-Authors: Lewis B. Silverman, Lynda M Vrooman, Jeffrey G Supko, Donna Neuberg, Barbara L Asselin, Uma H Athale, Luis A Clavell, Kristen E Stevenson, Christina Woodward, Peter D ColeAbstract:Over the past several decades, L-Asparaginase, an important component of therapy for acute lymphoblastic leukemia (ALL), has typically been administered intramuscularly rather than intravenously in North America because of concerns regarding anaphylaxis. We evaluated the feasibility of giving polyethylene glycosylated (PEG)–Asparaginase, the polyethylene glycol conjugate of Escherichia coli L-Asparaginase, by intravenous infusion in children with ALL. Between 2005 and 2007, 197 patients (age, 1-17 years) were enrolled on Dana-Farber Cancer Institute ALL Consortium Protocol 05-01 and received a single dose of intravenous PEG-Asparaginase (2500 IU/m2) over 1 hour during remission induction. Serum Asparaginase activity more than 0.1 IU/mL was detected in 95%, 88%, and 7% of patients at 11, 18, and 25 days after dosing, respectively. Toxicities included allergy (1.5%), venous thrombosis (2%), and pancreatitis (4.6%). We conclude that intravenous administration of PEG-Asparaginase is tolerable in children with ALL, and potentially therapeutic enzyme activity is maintained for at least 2 weeks after a single dose in most patients. This trial was registered at www.clinicaltrials.gov as #NCT00400946.
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erwinia Asparaginase after allergy to e coli Asparaginase in children with acute lymphoblastic leukemia
Pediatric Blood & Cancer, 2010Co-Authors: Lynda M Vrooman, Jeffrey G Supko, Donna Neuberg, Barbara L Asselin, Uma H Athale, Luis A Clavell, Kara M Kelly, Caroline Laverdiere, Bruno MichonAbstract:Background Escherichia coli Asparaginase is an important component of treatment for childhood acute lymphoblastic leukemia (ALL); however, hypersensitivity develops in up to 30% of patients. We assessed the nadir enzyme activity and tolerability of Erwinia Asparaginase, an alternative preparation, in E. coli Asparaginase-allergic patients. Patients and Methods Between 2000 and 2002, 215 children with newly diagnosed ALL were enrolled on Dana-Farber Cancer Institute ALL Consortium Protocol 00-01 and were to receive 30 weekly doses of intramuscular E. coli Asparaginase. If E. coli Asparaginase allergy developed, patients were switched to twice-weekly intramuscular Erwinia Asparaginase (25,000 IU/m2). Nadir serum Asparaginase activity (NSAA) was measured every 3 weeks. Results Forty-two patients (20%) developed E. coli Asparaginase allergy and switched to Erwinia. Of 38 patients with evaluable samples, 34 (89%) Erwinia-treated patients had at least one therapeutic NSAA (≥0.1 IU/ml). The median NSAA was 0.247 IU/ml 3 days and 0.077 IU/ml 4 days after an Erwinia dose. Associated toxicities included allergy in 14 (33%) and pancreatitis in 3 patients (7%). At a median follow-up of 5.4 years, event-free survival (±standard error) of the 42 patients who switched to Erwinia was 86 ± 5% compared with 81 ± 3% for the 170 patients without E. coli Asparaginase allergy (P = 0.55). Conclusions Twice-weekly Erwinia Asparaginase was well tolerated and achieved a therapeutically effective NSAA in most E. coli Asparaginase-allergic patients. Development of E. coli allergy and subsequent treatment with twice-weekly Erwinia did not adversely impact event-free survival. Erwinia Asparaginase should be considered for E. coli Asparaginase-allergic patients. Pediatr Blood Cancer 2010;54:199–205. © 2009 Wiley-Liss, Inc.
Naeem Rashid - One of the best experts on this subject based on the ideXlab platform.
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heterologous gene expression and characterization of tk2246 a highly active and thermostable plant type l Asparaginase from thermococcus kodakarensis
International Journal of Biological Macromolecules, 2020Co-Authors: Shahid Mahmood Chohan, Muhammad Sajed, Sabeel Un Naeem, Naeem RashidAbstract:Abstract The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l -Asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l -Asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 μmol min−1 mg−1 towards l -asparagine. The enzyme exhibited a 10% activity towards d -asparagine as compared to 100% against l -asparagine. No detectable activity was observed towards l - or d -glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 μmol min−1 mg−1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol−1. To the best of our knowledge, this is the first characterization of a plant-type l -Asparaginase from class Thermococci of phylum Euryarchaeota.
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heterologous gene expression and characterization of tk2246 a highly active and thermostable plant type l Asparaginase from thermococcus kodakarensis
International Journal of Biological Macromolecules, 2020Co-Authors: Shahid Mahmood Chohan, Muhammad Sajed, Sabeel Un Naeem, Naeem RashidAbstract:Abstract The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l -Asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l -Asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 μmol min−1 mg−1 towards l -asparagine. The enzyme exhibited a 10% activity towards d -asparagine as compared to 100% against l -asparagine. No detectable activity was observed towards l - or d -glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 μmol min−1 mg−1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol−1. To the best of our knowledge, this is the first characterization of a plant-type l -Asparaginase from class Thermococci of phylum Euryarchaeota.
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structure and function of the thermostable l Asparaginase from thermococcus kodakarensis
Acta Crystallographica Section D-biological Crystallography, 2017Co-Authors: Alun R. Coker, S P Wood, Shahid Mahmood Chohan, Naeem Rashid, J B Cooper, M. AkhtarAbstract:l-Asparaginases catalyse the hydrolysis of asparagine to aspartic acid and ammonia. In addition, l-Asparaginase is involved in the biosynthesis of amino acids such as lysine, methionine and threonine. These enzymes have been used as chemotherapeutic agents for the treatment of acute lymphoblastic leukaemia and other haematopoietic malignancies since the tumour cells cannot synthesize sufficient l-asparagine and are thus killed by deprivation of this amino acid. l-Asparaginases are also used in the food industry and have potential in the development of biosensors, for example for asparagine levels in leukaemia. The thermostable type I l-Asparaginase from Thermococcus kodakarensis (TkA) is composed of 328 amino acids and forms homodimers in solution, with the highest catalytic activity being observed at pH 9.5 and 85°C. It has a Km value of 5.5 mM for l-asparagine, with no glutaminase activity being observed. The crystal structure of TkA has been determined at 2.18 A resolution, confirming the presence of two α/β domains connected by a short linker region. The N-terminal domain contains a highly flexible β-hairpin which adopts `open' and `closed' conformations in different subunits of the solved TkA structure. In previously solved l-Asparaginase structures this β-hairpin was only visible when in the `closed' conformation, whilst it is characterized with good electron density in all of the subunits of the TkA structure. A phosphate anion resides at the active site, which is formed by residues from both of the neighbouring monomers in the dimer. The high thermostability of TkA is attributed to the high arginine and salt-bridge content when compared with related mesophilic enzymes.
Hien Anh Nguyen - One of the best experts on this subject based on the ideXlab platform.
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a novel l Asparaginase with low l glutaminase coactivity is highly efficacious against both t and b cell acute lymphoblastic leukemias in vivo
Cancer Research, 2018Co-Authors: Hien Anh Nguyen, Amanda M. Schalk, Li Liu, Jenny Zhang, Aleksandar Antanasijevic, Michael Caffrey, Damiano Rondelli, Dolores Mahmud, Maarten C Bosland, Andre KajdacsyballaAbstract:Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-Asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-Asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-Asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-Asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-Asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.
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the differential ability of asparagine and glutamine in promoting the closed active enzyme conformation rationalizes the wolinella succinogenes l Asparaginase substrate specificity
Scientific Reports, 2017Co-Authors: Hien Anh Nguyen, Donald L Durden, Arnon LavieAbstract:Many side effects of current FDA-approved L-Asparaginases have been related to their secondary L-glutaminase activity. The Wolinella succinogenes L-Asparaginase (WoA) has been reported to be L-glutaminase free, suggesting it would have fewer side effects. Unexpectedly, the WoA variant with a proline at position 121 (WoA-P121) was found to have L-glutaminase activity in contrast to Uniprot entry P50286 (WoA-S121) that has a serine residue at this position. Towards understanding how this residue impacts the L-glutaminase property, kinetic analysis was coupled with crystal structure determination of these WoA variants. WoA-S121 was confirmed to have much lower L-glutaminase activity than WoA-P121, yet both showed comparable L-Asparaginase activity. Structures of the WoA variants in complex with L-aspartic acid versus L-glutamic acid provide insights into their differential substrate selectivity. Structural analysis suggests a mechanism by which residue 121 impacts the conformation of the conserved tyrosine 27, a component of the catalytically-important flexible N-terminal loop. Surprisingly, we could fully model this loop in either its open or closed conformations, revealing the roles of specific residues of an evolutionary conserved motif among this L-Asparaginase family. Together, this work showcases critical residues that influence the ability of the flexible N-terminal loop for adopting its active conformation, thereby effecting substrate specificity.
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Discovery of human-like L-Asparaginases with potential clinical use by directed evolution
Nature Publishing Group, 2017Co-Authors: Coraline Rigouin, Hien Anh Nguyen, Amanda M. Schalk, Arnon LavieAbstract:Abstract L-Asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-Asparaginases is micromolar KM for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-Asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-Asparaginases have millimolar KM for asparagine. We recently identified the low KM guinea pig L-Asparaginase (gpASNase1). Because gpASNase1 and human L-Asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low KM chimeras we developed a suitable bacterial selection system (E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low KM clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63N-hC and 65N-hC, share respectively 85.7% and 87.1% identity with the hASNase1 but have a KM similar to gpASNase1. These clones possess 100–140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-Asparaginases maintain their in vitro ALL killing potential
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design and characterization of erwinia chrysanthemi l Asparaginase variants with diminished l glutaminase activity
Journal of Biological Chemistry, 2016Co-Authors: Hien Anh Nguyen, Arnon LavieAbstract:Current FDA-approved l-Asparaginases also possess significant l-glutaminase activity, which correlates with many of the toxic side effects of these drugs. Therefore, l-Asparaginases with reduced l-glutaminase activity are predicted to be safer. We exploited our recently described structures of the Erwinia chrysanthemi l-Asparaginase (ErA) to inform the design of mutants with diminished ability to hydrolyze l-glutamine. Structural analysis of these variants provides insight into the molecular basis for the increased l-asparagine specificity. A primary role is attributed to the E63Q mutation that acts to hinder the correct positioning of l-glutamine but not l-asparagine. The substitution of Ser-254 with either an asparagine or a glutamine increases the l-asparagine specificity but only when combined with the E63Q mutation. The A31I mutation reduces the substrate Km value; this is a key property to allow the required therapeutic l-asparagine depletion. Significantly, an ultra-low l-glutaminase ErA variant maintained its cell killing ability. By diminishing the l-glutaminase activity of these highly active l-Asparaginases, our engineered ErA variants hold promise as l-Asparaginases with fewer side effects.
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structural insight into substrate selectivity of erwinia chrysanthemi l Asparaginase
Biochemistry, 2016Co-Authors: Hien Anh Nguyen, Arnon LavieAbstract:l-Asparaginases of bacterial origin are a mainstay of acute lymphoblastic leukemia treatment. The mechanism of action of these enzyme drugs is associated with their capacity to deplete the amino acid l-asparagine from the blood. However, clinical use of bacterial l-Asparaginases is complicated by their dual l-Asparaginase and l-glutaminase activities. The latter, even though representing only ∼10% of the overall activity, is partially responsible for the observed toxic side effects. Hence, l-Asparaginases devoid of l-glutaminase activity hold potential as safer drugs. Understanding the key determinants of l-Asparaginase substrate specificity is a prerequisite step toward the development of enzyme variants with reduced toxicity. Here we present crystal structures of the Erwinia chrysanthemi l-Asparaginase in complex with l-aspartic acid and with l-glutamic acid. These structures reveal two enzyme conformations—open and closed—corresponding to the inactive and active states, respectively. The binding of lig...
