Characterization of the effects of asparaginase from <i>escherichia coli</i> and a glutaminase‐free asparaginase from <i>vibrio succinogenes</i> on specific cell‐mediated cytotoxicity

Durden, Donald L.; Distasio, John A.

doi:10.1002/ijc.2910270110

Cited by 28 publications

(16 citation statements)

References 24 publications

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“…9,[41][42][43][44][45] A potential strength of glutaminase-deficient L-ASP variants is, therefore, the possibility of improved therapeutic index if the modified L-ASP remains active against the cancer cells. For example, glutaminasefree L-ASP variants such as Q59L might not induce pancreatitis.…”

Section: Discussionmentioning

confidence: 99%

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Chan

Lorenzi

Anishkin

et al. 2014

Blood

168

131

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• We used molecular dynamics, saturation mutagenesis, and enzymologic screening to develop a glutaminase-free mutant (Q59L) L-ASP.• We then used Q59L to show that glutaminase activity is not required for L-ASP activity against ASNS-negative cancer cells.L-Asparaginase (L-ASP) is a key component of therapy for acute lymphoblastic leukemia. Its mechanism of action, however, is still poorly understood, in part because of its dual asparaginase and glutaminase activities. Here, we show that L-ASP's glutaminase activity is not always required for the enzyme's anticancer effect. We first used molecular dynamics simulations of the clinically standard Escherichia coli L-ASP to predict what mutated forms could be engineered to retain activity against asparagine but not glutamine. Dynamic mapping of enzyme substrate contacts identified Q59 as a promising mutagenesis target for that purpose. Saturation mutagenesis followed by enzymatic screening identified Q59L as a variant that retains asparaginase activity but shows undetectable glutaminase activity. Unlike wild-type L-ASP, Q59L is inactive against cancer cells that express measurable asparagine synthetase (ASNS). Q59L is potently active, however, against ASNS-negative cells. Those observations indicate that the glutaminase activity of L-ASP is necessary for anticancer activity against ASNS-positive cell types but not ASNS-negative cell types.Because the clinical toxicity of L-ASP is thought to stem from its glutaminase activity, these findings suggest the hypothesis that glutaminase-negative variants of L-ASP would provide larger therapeutic indices than wild-type L-ASP for ASNS-negative cancers. (Blood. 2014;123(23):3596-3606) Introduction L-Asparaginase (L-ASP) is an enzyme drug used in combination with vincristine and a glucocorticoid (eg, dexamethasone) to treat acute lymphoblastic leukemia (ALL).1,2 We 3-6 and others 7 have reported a rationale for testing L-ASP against low-asparagine synthetase (ASNS) solid tumors as well. L-ASP's primary known enzymatic activity is deamidation of asparagine to aspartic acid and ammonia, but it also deamidates glutamine to glutamic acid and ammonia, although with lower affinity and lower maximal rate. L-ASP therapy is often limited by toxic side effects that are generally attributed to the glutaminase activity. 8,9 Those side effects often preclude completion of the full treatment regimen, resulting in poor outcome. 10 The question that arises, however, is whether the therapeutic index of L-ASP could be increased by decreasing its glutaminase activity 8,9 or whether that would also decrease the anticancer effect commensurately.One side of the debate hypothesizes that L-ASP's therapeutic index can be improved by increasing glutaminase activity. In support of that hypothesis, data collected over the last decade have suggested that glutaminase activity generally increases the efficacy of L-ASP and is sometimes required to achieve an anticancer effect. Those studies have reported asparaginase activity to be expendable. [11][12][13][...

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Section: Discussionmentioning

confidence: 99%

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

Chan

Lorenzi

Anishkin

et al. 2014

Blood

168

131

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“…Of note, the L-glutaminase activity of the clinically used L-asparaginases has been implicated in many of the side effects of this treatment, which include immunosuppression (2, 6, 7), hepatotoxicity (8), pancreatitis (9), and coagulation dysfunction (9, 10). Many of the side effects are believed to be due to the disruption of protein synthesis induced by the L-glutaminase activity of L-asparaginase drugs (2,7,8,11,12).To eliminate the toxic side effects related to Gln depletion, enzymes that have retained their L-asparaginase activity but have reduced L-glutaminase activity would be beneficial. To this goal, Derst et al (13) exploited crystal structures of the E. coli enzyme to design mutants with reduced L-glutaminase activity.…”

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confidence: 99%

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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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“…Although an absolute need of normal cells for L-asparagine could not be demonstrated so far, a wide spectrum of toxicity involving the liver, pancreas, brain, clotting mechanisms, and the immune system has been described after application of E. coli asparaginase [6-191. Several in-uiuo and in-vitro studies suggest that E. coli asparaginase, being able to deaminate not only L-asparagine but also L-glutamine, may be responsible for the drug-related toxicity and immunosuppression [6,7,16,20]. This is why we studied the influence of E. coli asparaginase [L-asparagine amidohydrolase (EC.3.5.1.1.)]…”

Section: Introductionmentioning

confidence: 99%

Asparaginase‐induced derangements of glutamine metabolism: the pathogenetic basis for some drug‐related side‐effects

Ollenschläger¹,

Roth²,

Linkesch³

et al. 1988

Eur J Clin Investigation

122

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Abstract. Several side-effects of asparaginase therapy have been said to be a consequence of the glutaminase activity of Escherichia coli asparaginase, especially the deleterious influence on the liver function. We report here the drug-induced impairments of asparagine and glutamine metabolism in correlation to concentrations changes of plasma proteins, synthesized in the liver, in patients with acute lymphatic leukaemia. One hour after asparaginase application, plasma glutamine decreased to 5% (0-39%: median, range) of the initial values, with a subsequent rise to concentrations slightly lower than those prior to therapy. During the 14 days of drug application the fasting plasma concentrations of glutamine fell to a median of 63% of the pre-therapeutic levels, indicating a depletion of the glutamine pools. Two days after the end of asparaginase application, in one patient the glutamine concentrations increased to the pre-therapeutic range. Plasma concentrations of fibrinogen and antithrombin I11 decreased to 46% and 56%, respectively, of the initial values, with a slight increase 2 days after the end of therapy. The changes of plasma protein concentrations followed the course of plasma glutamine and asparagine. From that we deduce that the hepatic synthesis of the plasma proteins might be influenced by asparagine and glutamine depletion as a consequence of the therapy with E. coli asparaginase.

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Characterization of the effects of asparaginase from escherichia coli and a glutaminase‐free asparaginase from vibrio succinogenes on specific cell‐mediated cytotoxicity

Cited by 28 publications

References 24 publications

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

The glutaminase activity of l-asparaginase is not required for anticancer activity against ASNS-negative cells

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

Asparaginase‐induced derangements of glutamine metabolism: the pathogenetic basis for some drug‐related side‐effects

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