1. 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

2. Leukemia Blood cancer Prevalence in childhood – 80% cases Treatment – chemotherapy and L- asparaginase Complete remission and cure

3. Discovery of L-asparaginase (L-ASNase) Kidd 1953

4. Catalytic action

5. 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

6. 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 2 25.000UI/m 2 Source of L-ASNase

7. Problems Allergic reactions Hyperammonemia Immune reactions – Antibodies and proteases Silence inactivation

8. Objective Test the potencial of ScASNaseI as biopharmaceutical to treat Acute lymphoblastic leukemia - ALL

9. Results

11. K 0,5 mM V max μmol/min K cat s -1 K cat / S 0,5 M/s nHnH ScASNase10.075 ± 0.0270.0083 ± 0,000229035 ± 0.94.65x10 5 2.2 ± 0.3 No glutaminase activity detected

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

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

14. Cytotoxicity assay – MOLT-4 leukemia cells ScASNaseIKidrolase - EcASNaseII

15. 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

16. Thanks to authors Financial support. Grants 2013/08617-7 and 2013/16685-2