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

• Grant number: 14/22039-9
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Effective date (Start): March 01, 2015
• Effective date (End): April 30, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Marcos Antonio de Oliveira
• Grantee: Leonardo Schultz da Silva
• Host 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)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DE MOURA, WERNER ALFINITO FEIO; SCHULTZ, LEONARDO; BREYER, CARLOS ALEXANDRE; DE OLIVEIRA, ANA LAURA PIRES; TAIRUM, CARLOS ABRUNHOSA; FERNANDES, GABRIELLA COSTA; TOYAMA, MARCOS HIKARI; PESSOA-JR, ADALBERTO; MONTEIRO, GISELE; DE OLIVEIRA, MARCOS ANTONIO. Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains. Biotechnology Letters, JUL 2020. (13/08617-7, 17/19942-7, 17/25272-4, 19/04054-4, 14/22039-9, 16/19245-1, 17/20291-0, 11/13500-6)

DA SILVA, LEONARDO SCHULTZ; DOONAN, LIAM B.; PESSOA, JR., ADALBERTO; DE OLIVEIRA, MARCOS ANTONIO; LONG, PAUL F.. Structural and functional diversity of asparaginases: Overview and recommendations for a revised nomenclature. Biotechnology and Applied Biochemistry, MAR 2021. (18/04685-1, 13/08617-7, 14/22039-9, 17/19942-7)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

SILVA, Leonardo Schultz da. Functional and structural characterization and rational modification of ASNaseM: A new pharmaceutical for the acute lymphoblastic leukemia treatment?. 2019. Doctoral Thesis - Universidade Estadual Paulista (Unesp). Instituto de Biociências. São Vicente São Vicente.