Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

Abstract

Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design. (Blood. 2011; 117(5):1614-1621)

Mutations in Subunit Interface and B-cell Epitopes Improve Antileukemic Activities of Escherichia coli Asparaginase-II

“…In addition to Y176F, W66Y with both high glutaminase activity and higher cytotoxicity also appears to be an effective drug. Similar observations were reported by others for an EcA variant N24A mutation that showed stable glutaminase activity and also improved cell-killing properties (48).…”

In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow

“…As a result, the benefits compared with the use of native asparaginase are reduced. Several studies have shown that the structure of asparaginase permits the introduction of modifications without appreciable loss of enzymatic function (7,22,23). The data we present here may provide a rationale for the generation of novel modified versions of asparaginase.…”

Purification and Biochemical Characterization of Native and Pegylated Form of L-Asparaginase from <i>Aspergillus terreus</i> and Evaluation of Its Antiproliferative Activity

“…According to Panosyan et al [16] the effective deamination of glutamine by L-asparaginase appears to contribute to the decrease of asparagine depletion by depriving the asparagine synthetase of glutamine, the precursor asparagine biosynthesis. Bacterial L-asparaginase, used in therapeutic protocols, has low glutaminase activity but toxicity reactions are attributed to this activity [17,18]. Herein L-glutaminase activity was not detected in crude enzyme (concentrated or not) produced by A. terreus (PC-1.7.A) even after 60 min of the reaction.…”