Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-Asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi)

• Grant number: 16/25896-5
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: April 01, 2017
• End date: January 19, 2022
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Gisele Monteiro
• Grantee: Iris Munhoz Costa
• Host Institution: Faculdade de Ciências Farmacêuticas (FCF). Universidade de São Paulo (USP). São Paulo , 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): 19/09953-7 - Molecular biology-leading edge technologies for the characterization of resistance to asparaginase, BE.EP.DR

Abstract

Acute lymphoblastic leukemia (ALL) is characterized by the uncontrolled production of lymphoid blasts and by blocking the production of normal hematopoiesis, and neoplasia is more frequent in children and adolescents. Treatment of the disease is done with L-asparaginase (L-ASNase), an enzyme obtained from the bacteria Escherichia coli and Dickeya chrysanthemi (formerly known as Erwinia chrysanthemi). L-ASNase hydrolyzes L-asparagine (Asn) in aspartic acid (Asp) and ammonia and prevents tumor cells from obtaining Asn from the bloodstream for protein synthesis and proliferation, leading to cell death by apoptosis. Since the 1970s the enzyme has been the main agent for the remission of ALL. However, both formulations are associated with a high rate of adverse effects, mainly drug resistance caused by the production of anti-asparaginase antibodies and marked hypersensitivity, which compromise the efficacy of the treatment. In addition, patients suffer from various periods of drug shortage on the world market; Here in Brazil the lack of commercialization occurs since 2013. This way it is necessary to search for new alternatives for the treatment of ALL. The development of mutant isoforms from commercially available bacterial enzymes may contribute to the reduction of adverse effects and be an alternative for national marketing. Our research group using the L-ASNase from D. chrysanthemi has created a library of mutants from random mutagenesis. From this library, ten mutants were selected that presented in preliminary tests an improvement in the specific activity of up to 285% in relation and wild enzyme. This project aims to characterize the ten isoforms of D. chrysanthemi and to select those that present the best biochemical characteristics to evaluate the cytotoxic potential.

Scientific publications

(7)

(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)

EFFER, BRIAN; LIMA, GUILHERME MEIRA; CABARCA, SINDY; PESSOA, ADALBERTO; FARIAS, JORGE G.; MONTEIRO, GISELE. L-Asparaginase from E. chrysanthemi expressed in glycoswitch: effect of His-Tag fusion on the extracellular expression. PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY, v. 49, n. 7, APR 2019. (17/20384-9, 16/25896-5, 15/07749-2, 13/08617-7, 16/15787-4)

EFFER, BRIAN; KLEINGESINDS, EDUARDO KREBS; LIMA, GUILHERME MEIRA; COSTA, IRIS MUNHOZ; SANCHEZ-MOGUEL, IGNACIO; PESSOA, ADALBERTO; SANTIAGO, VERONICA FEIJOLI; PALMISANO, GIUSEPPE; FARIAS, JORGE G.; MONTEIRO, GISELE. Glycosylation of Erwinase results in active protein less recognized by antibodies. Biochemical Engineering Journal, v. 163, p. 11-pg., 2020-11-15. (13/08617-7, 15/07749-2, 17/20384-9, 18/15104-0, 16/15787-4, 16/25896-5)

LIMA, GUILHERME M.; MENEZES, MILENE C.; COSTA, IRIS M.; SERRANO, SOLANGE M. T.; MONTEIRO, GISELE. Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, JUN 2021. (13/08617-7, 18/15041-8, 16/25896-5, 13/07467-1, 18/15104-0)

RODRIGUES, MARIANE A. D.; PIMENTA, MARCELA V.; COSTA, IRIS M.; ZENATTI, PRISCILA P.; MIGITA, NATACHA A.; YUNES, JOSE A.; RANGEL-YAGUI, CARLOTA O.; DE SA, MATHEUS M.; PESSOA, ADALBERTO; COSTA-SILVA, TALES A.; et al. Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase. Biochemical Pharmacology, v. 182, DEC 2020. (14/06863-3, 18/18257-1, 13/08617-7, 16/25896-5, 15/07749-2, 18/15104-0, 13/08139-8, 18/15549-1)

LIMA, GUILHERME M.; MENEZES, MILENE C.; COSTA, IRIS M.; SERRANO, SOLANGE M. T.; MONTEIRO, GISELE. Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 96, n. 9, p. 2659-2666, SEP 2021. (18/15041-8, 13/08617-7, 13/07467-1, 18/15104-0, 16/25896-5)

COSTA, IRIS MUNHOZ; MOURA, DEBORA CUSTODIO; LIMA, GUILHERME MEIRA; PESSOA, ADALBERTO; DOS SANTOS, CAMILA ORESCO; DE OLIVEIRA, MARCOS A.; MONTEIRO, GISELE. Engineered asparaginase from Erwinia chrysanthemi enhances asparagine hydrolase activity and diminishes enzyme immunoreactivity - a new promise to treat acute lymphoblastic leukemia. JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, v. 97, n. 1, OCT 2021. (16/25896-5, 15/07749-2, 18/15104-0, 13/08617-7)

COSTA-SILVA, T. A.; COSTA, I. M.; BIASOTO, H. P.; LIMA, G. M.; SILVA, C.; PESSOA, A.; MONTEIRO, G.. Critical overview of the main features and techniques used for the evaluation of the clinical applicability of L-asparaginase as a biopharmaceutical to treat blood cancer. BLOOD REVIEWS, v. 43, p. 17-pg., 2020-09-01. (18/15041-8, 13/08617-7, 16/25896-5, 15/07749-2)

Academic Publications

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

COSTA, Iris Munhoz. Biochemical characterization and cytotoxic evaluation of mutant isoforms of L-asparaginase II from Dickeya chrysanthemi (Erwinia chrysanthemi). 2022. Doctoral Thesis - Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) São Paulo.