Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity.
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.
Biomolecular Modelling Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London, UK.
Marc N
MN
Offman
Marcin
M
Krol
Naina
N
Patel
Shekhar
S
Krishnan
JiZhong
J
Liu
Vaskar
V
Saha
Paul A
PA
Bates
D001215:Asparaginase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D049109:Cell Proliferation; D003198:Computer Simulation; D005972:Glutaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D001215:Asparaginase; D005972:Glutaminase