{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,12]],"date-time":"2026-10-12T17:19:37Z","timestamp":1791825577451,"version":"4.3.4"},"reference-count":65,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2017,11,3]],"date-time":"2017-11-03T00:00:00Z","timestamp":1509667200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2017,11,3]],"date-time":"2017-11-03T00:00:00Z","timestamp":1509667200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p>L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the <jats:italic>Escherichia coli<\/jats:italic> (EcAII) and <jats:italic>Erwinia chrysanthemi<\/jats:italic> (ErAII) type II ASNases currently used in the clinics are characterized by high <jats:italic>in vivo<\/jats:italic> instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and <jats:italic>in vitro<\/jats:italic> antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations <jats:italic>in vivo<\/jats:italic> and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.<\/jats:p>","DOI":"10.1038\/s41598-017-15075-4","type":"journal-article","created":{"date-parts":[[2017,10,30]],"date-time":"2017-10-30T14:30:44Z","timestamp":1509373844000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":61,"title":["A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro"],"prefix":"10.1038","volume":"7","author":[{"given":"Maristella","family":"Maggi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven D.","family":"Mittelman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jean Hugues","family":"Parmentier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giorgio","family":"Colombo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3304-6104","authenticated-orcid":false,"given":"Massimiliano","family":"Meli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeannette Marie","family":"Whitmire","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"D. Scott","family":"Merrell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julian","family":"Whitelegge","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5790-1833","authenticated-orcid":false,"given":"Claudia","family":"Scotti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2017,11,3]]},"reference":[{"key":"15075_CR1","doi-asserted-by":"publisher","unstructured":"DeBoer, R. et al. Clinical impact of ABL1 kinase domain mutations and IKZF1 deletion in adults under age 60 with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL): molecular analysis of CALGB (Alliance) 10001 and 9665. Leuk Lymphoma 1\u20139, https:\/\/doi.org\/10.3109\/10428194.2016.1144881 (2016).","DOI":"10.3109\/10428194.2016.1144881"},{"key":"15075_CR2","first-page":"418","volume":"3","author":"D Covini","year":"2015","unstructured":"Covini, D. et al. Expanding Targets for a Metabolic Therapy of Cancer: L-Asparaginase. Topics in anti-cancer research \n                           3, 418\u2013445 (2015).","journal-title":"Topics in anti-cancer research"},{"key":"15075_CR3","doi-asserted-by":"publisher","first-page":"748","DOI":"10.3109\/10428194.2015.1101098","volume":"57","author":"N Hijiya","year":"2016","unstructured":"Hijiya, N. & van der Sluis, I. M. Asparaginase-associated toxicity in children with acute lymphoblastic leukemia. Leuk Lymphoma \n                           57, 748\u2013757, https:\/\/doi.org\/10.3109\/10428194.2015.1101098 (2016).","journal-title":"Leuk Lymphoma"},{"key":"15075_CR4","doi-asserted-by":"publisher","DOI":"10.1002\/pbc.25887","author":"M Tulstrup","year":"2015","unstructured":"Tulstrup, M. et al. Parents\u2019 and Adolescents\u2019 Preferences for Intensified or Reduced Treatment in Randomized Lymphoblastic Leukemia Trials. Pediatric blood \\& cancer. https:\/\/doi.org\/10.1002\/pbc.25887 (2015).","journal-title":"Pediatric blood \\& cancer"},{"key":"15075_CR5","doi-asserted-by":"publisher","first-page":"1080","DOI":"10.1172\/JCI106579","volume":"50","author":"RG Peterson","year":"1971","unstructured":"Peterson, R. G., Handschumacher, R. E. & Mitchell, M. S. Immunological responses to L-asparaginase. J Clin Invest \n                           50, 1080\u20131090, https:\/\/doi.org\/10.1172\/JCI106579 (1971).","journal-title":"J Clin Invest"},{"key":"15075_CR6","doi-asserted-by":"publisher","first-page":"1964","DOI":"10.1172\/JCI39590","volume":"119","author":"N Patel","year":"2009","unstructured":"Patel, N. et al. A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase. J Clin Invest \n                           119, 1964\u20131973, https:\/\/doi.org\/10.1172\/jci37977 (2009).","journal-title":"J Clin Invest"},{"key":"15075_CR7","doi-asserted-by":"publisher","first-page":"564","DOI":"10.1038\/sj.leu.2403629","volume":"19","author":"HM Robinson","year":"2005","unstructured":"Robinson, H. M. et al. Derivative chromosome 9 deletions are a significant feature of childhood Philadelphia chromosome positive acute lymphoblastic leukaemia. Leukemia \n                           19, 564\u2013571, https:\/\/doi.org\/10.1038\/sj.leu.2403629 (2005).","journal-title":"Leukemia"},{"key":"15075_CR8","doi-asserted-by":"publisher","first-page":"1383","DOI":"10.1182\/blood-2014-08-569228","volume":"125","author":"CJ Harrison","year":"2015","unstructured":"Harrison, C. J. Blood Spotlight on iAMP21 acute lymphoblastic leukemia (ALL), a high-risk pediatric disease. Blood \n                           125, 1383\u20131386, https:\/\/doi.org\/10.1182\/blood-2014-08-569228 (2015).","journal-title":"Blood"},{"key":"15075_CR9","doi-asserted-by":"publisher","first-page":"618","DOI":"10.1179\/1024533215Z.000000000402","volume":"20","author":"F Forghieri","year":"2015","unstructured":"Forghieri, F., Luppi, M. & Potenza, L. Philadelphia chromosome-positive Acute Lymphoblastic Leukemia. Hematology \n                           20, 618\u2013619, https:\/\/doi.org\/10.1179\/1024533215z.000000000402 (2015).","journal-title":"Hematology"},{"key":"15075_CR10","doi-asserted-by":"publisher","unstructured":"van der Meer, L. T. et al. In vivo imaging of antileukemic drug asparaginase reveals a rapid macrophage mediated clearance from the bone marrow. J Nucl Med, https:\/\/doi.org\/10.2967\/jnumed.116.177741 (2016).","DOI":"10.2967\/jnumed.116.177741"},{"key":"15075_CR11","doi-asserted-by":"publisher","first-page":"1219","DOI":"10.1016\/j.bbapap.2014.03.013","volume":"1844","author":"S Verma","year":"2014","unstructured":"Verma, S., Mehta, R. K., Maiti, P., R\u00f6hm, K. H. & Sonawane, A. Improvement of stability and enzymatic activity by site-directed mutagenesis of E. coli asparaginase II. Biochim Biophys Acta \n                           1844, 1219\u20131230, https:\/\/doi.org\/10.1016\/j.bbapap.2014.03.013 (2014).","journal-title":"Biochim Biophys Acta"},{"key":"15075_CR12","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1016\/j.enzmictec.2013.12.012","volume":"56","author":"J Vidya","year":"2014","unstructured":"Vidya, J., Ushasree, M. V. & Pandey, A. Effect of surface charge alteration on stability of L-asparaginase II from Escherichia sp. Enzyme Microb Technol \n                           56, 15\u201319, https:\/\/doi.org\/10.1016\/j.enzmictec.2013.12.012 (2014).","journal-title":"Enzyme Microb Technol"},{"key":"15075_CR13","doi-asserted-by":"publisher","first-page":"2867","DOI":"10.1016\/j.jmb.2015.06.017","volume":"427","author":"A Anishkin","year":"2015","unstructured":"Anishkin, A. et al. Catalytic Role of the Substrate Defines Specificity of Therapeutic l-Asparaginase. J Mol Biol \n                           427, 2867\u20132885, https:\/\/doi.org\/10.1016\/j.jmb.2015.06.017 (2015).","journal-title":"J Mol Biol"},{"key":"15075_CR14","doi-asserted-by":"publisher","first-page":"2009","DOI":"10.1110\/ps.9.10.2009","volume":"9","author":"C Derst","year":"2000","unstructured":"Derst, C., Henseling, J. & R\u00f6hm, K. H. Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein Sci \n                           9, 2009\u20132017, https:\/\/doi.org\/10.1110\/ps.9.10.2009 (2000).","journal-title":"Protein Sci"},{"key":"15075_CR15","doi-asserted-by":"publisher","first-page":"1614","DOI":"10.1182\/blood-2010-07-298422","volume":"117","author":"MN Offman","year":"2011","unstructured":"Offman, M. N. et al. Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity. Blood \n                           117, 1614\u20131621, https:\/\/doi.org\/10.1182\/blood-2010-07-298422 (2011).","journal-title":"Blood"},{"key":"15075_CR16","doi-asserted-by":"publisher","first-page":"1964","DOI":"10.1172\/JCI39590","volume":"119","author":"N Patel","year":"2009","unstructured":"Patel, N. et al. A dyad of lymphoblastic lysosomal cysteine proteases degrades the antileukemic drug L-asparaginase. J Clin Invest \n                           119, 1964\u20131973, https:\/\/doi.org\/10.1172\/JCI37977 (2009).","journal-title":"J Clin Invest"},{"key":"15075_CR17","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1016\/S0968-0004(01)02027-8","volume":"27","author":"I Schomburg","year":"2002","unstructured":"Schomburg, I. et al. BRENDA: a resource for enzyme data and metabolic information. 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Blood \n                           110, 4325 (2007).","journal-title":"Blood"},{"key":"15075_CR20","doi-asserted-by":"publisher","unstructured":"Cupp-Enyard, C. Sigma\u2019s Non-specific Protease Activity Assay - Casein as a Substrate. J Vis Exp, https:\/\/doi.org\/10.3791\/899 (2008).","DOI":"10.3791\/899"},{"key":"15075_CR21","doi-asserted-by":"crossref","unstructured":"Folin, O. & Ciocalcuteu, V. Tyrosine and tryptophan determinations in proteins J. Biol. Chem \n                           73 (1927).","DOI":"10.1016\/S0021-9258(18)84277-6"},{"key":"15075_CR22","doi-asserted-by":"publisher","first-page":"e1002433","DOI":"10.1371\/journal.pcbi.1002433","volume":"8","author":"G Morra","year":"2012","unstructured":"Morra, G., Potestio, R., Micheletti, C. & Colombo, G. Corresponding functional dynamics across the Hsp90 Chaperone family: insights from a multiscale analysis of MD simulations. 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Lancet Oncology \n                           16, 1580\u20131581, https:\/\/doi.org\/10.1016\/s1470-2045(15)00451-9 (2015).","journal-title":"Lancet Oncology"},{"key":"15075_CR27","first-page":"241","volume":"1","author":"VI Avramis","year":"2006","unstructured":"Avramis, V. I. & Tiwari, P. N. Asparaginase (native ASNase or pegylated ASNase) in the treatment of acute lymphoblastic leukemia. Int J Nanomedicine \n                           1, 241\u2013254 (2006).","journal-title":"Int J Nanomedicine"},{"key":"15075_CR28","doi-asserted-by":"publisher","first-page":"465","DOI":"10.1146\/annurev.genet.30.1.465","volume":"30","author":"S Gottesman","year":"1996","unstructured":"Gottesman, S. Proteases and their targets in Escherichia coli. Annu Rev Genet \n                           30, 465\u2013506, https:\/\/doi.org\/10.1146\/annurev.genet.30.1.465 (1996).","journal-title":"Annu Rev Genet"},{"key":"15075_CR29","doi-asserted-by":"crossref","unstructured":"Capasso, S., Mazzarella, L., Sorrentino, G., Balboni, G. & Kirby, A. J. Kinetics and mechanism of the cleavage of the peptide bond next to asparagine. Peptides \n                           17, 1075\u20131077, doi:0196-9781(96)00153-2 (1996).","DOI":"10.1016\/0196-9781(96)00153-2"},{"key":"15075_CR30","doi-asserted-by":"publisher","first-page":"880","DOI":"10.1016\/S1470-2045(09)70369-9","volume":"11","author":"T Szczepa\u0144ski","year":"2010","unstructured":"Szczepa\u0144ski, T., Harrison, C. J. & van Dongen, J. J. Genetic aberrations in paediatric acute leukaemias and implications for management of patients. 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A Phase 2 study of L-asparaginase encapsulated in erythrocytes in elderly patients with Philadelphia chromosome negative acute lymphoblastic leukemia: The GRASPALL\/GRAALL-SA2-2008 study. Am J Hematol \n                           90, 811\u2013818, https:\/\/doi.org\/10.1002\/ajh.24093 (2015).","journal-title":"Am J Hematol"},{"key":"15075_CR34","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1016\/S0378-5173(02)00046-7","volume":"237","author":"AL Soares","year":"2002","unstructured":"Soares, A. L., Guimar\u00e3es, G. M., Polakiewicz, B., de Moraes Pitombo, R. N. & Abrah\u00e3o-Neto, J. Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli L-asparaginase. Int J Pharm \n                           237, 163\u2013170 (2002).","journal-title":"Int J Pharm"},{"key":"15075_CR35","first-page":"942","volume":"31","author":"N Jaffe","year":"1971","unstructured":"Jaffe, N. et al. 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Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry \n                           6, 307\u2013316, https:\/\/doi.org\/10.1134\/s1990750812040026 (2012).","journal-title":"Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry"},{"key":"15075_CR38","doi-asserted-by":"publisher","first-page":"90","DOI":"10.5897\/BMBR11.027","volume":"7","author":"S Rakashanda","year":"2012","unstructured":"Rakashanda, S., Farukh, R., Shaista, R., Akbar, M. & Shajrul, A. Role of proteases in cancer: A review. Biotechnology and Molecular Biology Review \n                           7, 90\u2013101, https:\/\/doi.org\/10.5897\/bmbr11.027 (2012).","journal-title":"Biotechnology and Molecular Biology Review"},{"key":"15075_CR39","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1007\/BF00420905","volume":"96","author":"DA Kyriakidis","year":"1990","unstructured":"Kyriakidis, D. A., Tsirka, S. A., Tsavdaridis, I. K., Iliadis, S. N. & Kortsaris, A. H. 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