Characterization and protein engineering of L- asparaginase 1 from Saccharomyces cerevisiae to evaluate its use as biopharmaceutical Prof. Dr. Gisele Monteiro Department of Biochemical and Pharmaceutical Technology Faculty 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: ©2014 by American Society of Hematology High ASNS Low ASNS ASNS-negative

L-ASNase IIEscherichia coliErwinia chrysanthemi KMKM 15  M58  M k cat 2,7x10 3 s -1 23,8x10 3 s -1 k cat /K M 5,1x10 5 M -1 s -1 4,1x10 5 M -1 s -1 Specific activity200U/mg120U/mg Dose in the treatment6.000UI/m UI/m 2 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

K 0,5 mM V max μmol/min K cat s -1 K cat / S 0,5 M/s nHnH ScASNase ± ± 0, ± x ± 0.3 No glutaminase activity detected

Amino acids – catalytic site Mutant Specific Activity (U/mg) Residual Activity % K215A0,0240,012 T141A0,0430,022 T64A0,10,051 Y78A0,1720,088

Asp1-WT Y78A T141A K215A T64A Circular Dichroisms Prof. Dr. Marcos Oliveira - Unesp Results

Cytotoxicity assay – MOLT-4 leukemia cells ScASNaseIKidrolase - 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 activity Antitumor activity

Thanks to authors Financial support. Grants 2013/ and 2013/