l-Asparaginase as Potent Anti-leukemic Agent and Its Significance of Having Reduced Glutaminase Side Activity for Better treatment of Acute Lymphoblastic Leukaemia

Ramya, L. N.; Doble, Mukesh; Rekha, V. P. B.; Pulicherla, K. K.

doi:10.1007/s12010-012-9755-z

Cited by 62 publications

(36 citation statements)

References 137 publications

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“…Compelling evidence (2,8,12,20,21) suggests that the thromboembolic coagulopathy (10,(22)(23)(24)(25), pancreatitis (26,27), and liver dysfunction (8,28) are direct results of the L-glutaminase side activity present in the Food and Drug Administration-approved bacterial L-asparaginases and not from the cancer-killing L-asparaginase activity. Therefore, an enzyme with high L-asparaginase activity (and here a low Asn K m value is critical) and low L-glutaminase activity may be advantageous clinically.…”

Section: Discussionmentioning

confidence: 99%

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Nguyen

Lavie

2016

Journal of Biological Chemistry

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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 K m 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.L-Asparaginases are amidohydrolases that catalyze the hydrolysis of the amino acid L-asparagine (Asn) 2 to L-aspartate (Asp) and ammonia. Currently, the Food and Drug Administration has approved the L-asparaginase from Escherichia coli (EcA) and Erwinia chrysanthemi (ErA) for treatment of select blood cancers. These enzyme drugs function by depleting Asn from the blood, thereby affecting certain blood cancers that are dependent on exogenous Asn. Sensitivity to L-asparaginase has been attributed to a reduced ability of those cancers to synthesize this amino acid de novo (1). Because the concentration of Asn in human blood is ϳ50 M (2), for an L-asparaginase to be clinically useful, its Asn K m value must be in the low micromolar range. This property is present in EcA (K m ϭ 15 M) and ErAPatients treated with L-asparaginase often confront two types of adverse effects. The first is due to an immune response against the bacterial enzymes. To decrease the immunogenicity, the current standard of care in the United States employs a PEGylated version of EcA (3, 4). If immunogenicity still arises, ErA is used as second line treatment (5).The second source of adverse effects of L-asparaginase treatment relates to the enzyme's ability to hydrolyze the amino acid L-glutamine (Glu). That is, in addition to catalyzing the hydrolysis of Asn (L-asparaginase activity), both EcA and ErA also catalyze the hydrolysis of Gln to L-glutamate (Glu) and ammonia (L-glutaminase activity). L-Glutamine is the most abundant amino acid in the blood with a concentration range of 400 -650 M (2). The L-glutaminase activity of these bacterial L-asparaginases is significant, ϳ2% of the EcA activity and as high as 10% for ErA (3). Of note, the L-glutaminase activity of the clinically used L-asparaginases has been implicated in many of the side effe...

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Section: Discussionmentioning

confidence: 99%

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Nguyen

Lavie

2016

Journal of Biological Chemistry

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“…However, the L-asparaginases from these two bacterial species have side effects, which are attributed in part to the presence of L-glutaminase activity or allergic responses to L-asparaginases from these sources (6,11). Thus, L-asparaginases with low L-glutaminase activity or from different sources are of interest (7,35). RmAsnase, the fungal L-asparaginase from R. miehei, showed antiproliferative activity against cells from several human leukemia cell lines and displayed very low L-glutaminase activity.…”

Section: Discussionmentioning

confidence: 99%

“…RmAsnase, the fungal L-asparaginase from R. miehei, showed antiproliferative activity against cells from several human leukemia cell lines and displayed very low L-glutaminase activity. Thus, RmAsnase may also be useful as an alternative to bacterial L-asparaginases in leukemia treatment (2,6,11,32,35).…”

Section: Discussionmentioning

confidence: 99%

Biochemical Characterization of a Novel l -Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

Huang

Liu

Sun

et al. 2014

Appl Environ Microbiol

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A novel fungal gene encoding the Rhizomucor miehei L-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported L-asparaginases. The purified L-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this L-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

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“…In the present study, the purified recombinant ansA3 exhibited very negligible glutaminase activity of 0.6 % of that of L-asparaginase which is quite lower than reported in earlier studies. L-Asparaginase having reduced glutaminase activity is better for ALL treatment [29].…”

Section: Substrate Specificitymentioning

confidence: 99%

Characterization of a Recombinant Glutaminase-Free l-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

Sudhir

Dave

Prajapati

et al. 2014

Appl Biochem Biotechnol

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L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

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l-Asparaginase as Potent Anti-leukemic Agent and Its Significance of Having Reduced Glutaminase Side Activity for Better treatment of Acute Lymphoblastic Leukaemia

Cited by 62 publications

References 137 publications

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Design and Characterization of Erwinia Chrysanthemi l-Asparaginase Variants with Diminished l-Glutaminase Activity

Biochemical Characterization of a Novel l -Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

Characterization of a Recombinant Glutaminase-Free l-Asparaginase (ansA3) Enzyme with High Catalytic Activity from Bacillus licheniformis

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