Characterization and protein engineering of L-asparaginase 1 from Saccharomyces cerevisiae to
evaluate its use as biopharmaceutical
Prof. Dr. Gisele MonteiroDepartment of Biochemical and Pharmaceutical
TechnologyFaculty of Pharmaceutical Sciences
University of São Paulo - USP
Leukemia
• Blood cancer• Prevalence in childhood – 80% cases• Treatment – chemotherapy and L-
asparaginase
Complete remission and cure
Discovery of L-asparaginase (L-ASNase)
Kidd 1953
Catalytic action
Antitumor activity
Adapted from Wai Kin Chan et al. Blood 2014;123:3596-3606
©2014 by American Society of Hematology
High ASNS
Low ASNS
ASNS-negative
L-ASNase II Escherichia coli Erwinia chrysanthemi
KM 15M 58M
kcat 2,7x103 s-1 23,8x103 s-1
kcat/KM 5,1x105 M-1 s-1 4,1x105M-1 s-1
Specific activity 200U/mg 120U/mg
Dose in the treatment 6.000UI/m2 25.000UI/m2
Source of L-ASNase
Problems• Allergic reactions
• Hyperammonemia
• Immune reactions– Antibodies and
proteases• Silence inactivation
Objective
• Test the potencial of ScASNaseI as biopharmaceutical to treat Acute
lymphoblastic leukemia - ALL
Results
K0,5
mM
Vmax
μmol/min
Kcat
s-1
Kcat/ S0,5
M/s
nH
ScASNase1 0.075 ± 0.027 0.0083 ± 0,0002290 35 ± 0.9 4.65x105 2.2 ± 0.3
No glutaminase activity detected
Amino acids – catalytic site
Mutant Specific Activity (U/mg)
Residual Activity %
K215A 0,024 0,012T141A 0,043 0,022T64A 0,1 0,051Y78A 0,172 0,088
Asp1-WT
Y78A
T141A
K215A
T64A
• Circular Dichroisms
Prof. Dr. Marcos Oliveira - Unesp
Results
Cytotoxicity assay – MOLT-4 leukemia cells
ScASNaseI Kidrolase - EcASNaseII
Concluding Remarks
• ScASNase has potential to be a new biopharmaceutical to treat ALL
• High specific activity• No glutaminase activity• Eukaryotic protein• Allosteric enzyme• High substrate affinity• Antitumor activityAntitumor activity
Thanks to authors
Financial support. Grants 2013/08617-7 and 2013/16685-2
