Functional and structural characterization and rational modification of ASNaseM: A new pharmaceutical for the acute lymphoblastic leukemia treatment?

Author(s)

Leonardo Schultz

• Document type: Doctoral Thesis
• Press: São Vicente. 2019-05-17.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências. São Vicente
• Defense date: 2019-04-10
• Advisor: Marcos Antônio de Oliveira

Abstract

Bacterial L-asparaginases (ASNases) are important biopharmaceuticals used in the treatment of acute lymphoid leukemia (ALL), since this tumor type is dependent on the availability of extracellular asparagine (Asn). Bacterial ASNases are able to efficiently hydrolyze Asn in aspartic acid (Asp) and ammonia (NH3), decreasing the availability of Asn to tumor cells and inducing apoptosis. Commercially, international pharmaceutical industries produce ASNases from Escherichia coli and Erwinia chrysanthemi, however none ASNase is produced by the Brazilian pharmaceutical companies. Additionally, the treatment with these enzymes can produce side effects, among them immunogenic ones, that are related to the high molecular weight of the enzyme (140kDa) and neurological, attributed to the glutaminase secondary activity (GLNase), being glutamine (Gln) the most abundant amino acid in the bloodstream. In this context, alternative sources of these enzymes as also the self-sufficiency of the production are important to mitigate side effects and avoid treatment failures due to international fluctuations in their manufacture. In this work, we performed the characterization of a yeast ASNase, called ASNaseM, which shares high homology (higher than 30% identity and 40% similarity) with the bacterial enzymes used in the treatment of ALL, and which has all the amino acids involved in the catalysis conserved, suggesting a potential alternative source for the treatment of ALL. Molecular exclusion chromatography experiments revealed an enzyme with retention time for a monomer (~ 45 kDa), and through circular dichroism spectroscopy, it was possible to observe that ASNaseM is heat-resistant, maintaining its secondary structure at temperatures higher than 40ºC, presenting Tm = 54.35 ± 0.37 °C, which may be the result of 4 disulfide bonds formed by 8 of 10 cysteines, as indicated by the free sulfhydryl group quantification assay using the DTNB reagent. The kinetic parameters were determined by spectrophotometric assay coupled to glutamate dehydrogenase, the enzyme presented allosteric behavior with Hill coefficient of 2.125 and S0.5 of ~ 0.35 mM and the catalytic efficiency was in the order of 101 M-1S-1 and activity specific of approximately 108 UI/mg. Additionally, a characteristic that is related to several side effects of the bacterial ASNases, observed in the commercial enzymes, the secondary GLNase activity was not detected to ASNaseM. Through cytotoxicity assays using ReH and Molt-4 (neoplastic cells) strains, it was possible to observe cytotoxic activity to neoplastic cells. Site-directed mutations were also performed to confer GLNase activity to ASNaseM based in known substitutions that confers or abolish this kind of activity to the bacterial enzymes. Our results revealed that the mutated enzymes showed no gain in GLNase activity, indicating that despite the similarities to the bacterial enzymes, gain of GLNase activity is a complex phenomenon and requires further investigation. In addition, we performed "in silico" analyzes of ASNase primary sequences, together with the evaluation of three-dimensional structures, to increase the knowledge of evolutionary aspects of these enzymes and propose a careful classification of ASNases. The results of the analysis reveal that yeast ASNase is evolutionarily distinct from other enzymes and also aided in the identification of ASNases that may possess important properties as alternative biopharmaceuticals having high ASNase activity or absence of GLNase activity. (AU)

• FAPESP's process: 14/22039-9 - Functional and structural characterization and rational modification of ASPaseM: a new pharmaceutical for the acute lymphoblastic leukemia treatment?
• Grantee: Leonardo Schultz da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)