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

Grant number: 14/22039-9
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): March 01, 2015
Effective date (End): April 30, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Marcos Antonio de Oliveira
Grantee:Leonardo Schultz da Silva
Home Institution: Instituto de Biociências (IB-CLP). Universidade Estadual Paulista (UNESP). Campus Experimental do Litoral Paulista. São Vicente , SP, Brazil
Associated research grant:13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical, AP.TEM
Associated scholarship(s):18/04685-1 - Biological diversity of ASNAses: evaluation of enzyme evolution and determination of the crystallographic structure of the ASNaseM, BE.EP.DD

Abstract

L-asparaginases (L-ASPases) are important bacterial biopharmaceuticals used to treat Acute Lymphoblastic Leukemia (ALL), since this tumor type is dependent of asparagine availability of extracellular asparagine (Asn). Bacterial L-ASPases are able to hydrolyse efficiently Asn in Aspartic acid (Asp) and ammonia (NH3), thus reducing the Asn availability for tumor cells and inducing apoptosis. Despite appearing to be an alternative, human isoforms could not be used in the ALL treatment since the Km lies in the millimolar range, whereas bacterial enzymes presents a Km ~ 7.5 × 10-6 M. International pharmaceutical industries commercializes L-ASPases of Eschericchia coli and Erwinia chrysantemi, but the biopharmaceutical is not produced by pharmaceutical Brazilian industries. In this context, alternative sources of this enzyme and self-sufficiency in its production are important to avoid treatment failures due to fluctuations in international manufacturing. In this work, we aim to characterize a new L-ASPase, called ASPaseM that shares high homology (> 30% identity and 40% similarity) with the bacterial enzymes used in the ALL treatment and has all the amino acids involved in catalysis preserved, suggesting a potential alternative source for the treatment of ALL. Among our objectives relies in the structural characterization of ASPaseM by X-ray crystallography, as also, the enzyme engineering in order to improve its physicochemical and pharmacological characteristics. Initially, we will standardize methods for expression and purification and functional assays to determine enzymatic activity. Additionally to x ray crystallography, we also apply another methodologies to structural evaluation, as spectroscopic methods involving Circular Dichroism (CD), Dynamic Light Scattering (DLS), X-ray scattering at low angle (SAXS) and Size Exclusion Chromatography (SEC). Additionally, we will perform approaches to the rational protein engineering based on the enzyme structure by site directed mutagenesis in order to produce a L-ASPase with better characteristics suited for therapies with high asparaginase catalytic activity and low glutaminase activity. Engineering protocols will also apply to modify protein packing and folding or deletion of proteolytic sites. Throughout the project will be carried out the ASPaseM crystallization (and mutants of interest) and as also the co-crystallization with ligands (Asp, Glu or succninato) aiming at the determination of crystallographic structures of ASPaseM or mutants, with or without ligands in the active site. Additionally, the cytotoxicity of the ASPaseM and mutants enzyme will evaluated in SHR monocytic cells from acute lymphoblastic leukemia (ATCCCRL-8286). The current project is already initialized and preliminary data are presented at the end of the research proposal. (AU)

Articles published in Agência FAPESP about the scholarship:
Protein isolated from baker's yeast shows potential against leukemia cells