L-asparaginases (EC 3.5.1.1) are a family of enzymes that catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. These proteins with different biochemical, physicochemical and pharmacological properties are found in many organisms, including bacteria, fungi, algae, plants and mammals. To date, asparaginases from E. coli and Dickeya dadantii (formerly known as Erwinia chrysanthemi) are widely used in hematology for the treatment of lymphoblastic leukemias. However, their medical use is limited by side effects associated with the ability of these enzymes to hydrolyze L-glutamine, as well as the development of immune reactions. To solve these issues, gene-editing methods to introduce amino-acid substitutions of the enzyme are implemented. In this review, we focused on molecular analysis of the mechanism of enzyme action and to optimize the antitumor activity.

E.coli type II L-asparaginase is widely used for treatment of acute lymphoblastic leukemia. However, serious side effects such as allergic or hypersensitivity reactions are common for L-asparaginase treatment. Methods for minimizing immune response on L-asparaginase treatment in human include bioengeneering of less immunogenic version of the enzyme or utilizing the homologous enzymes of different origin. To rationalize these approaches we compared immunogenicity of L-asparaginases from five bacterial organisms and performed sequence-structure analysis of the presumable epitope regions. IgG and IgM immune response in C57B16 mice after immunization with Wollinella succinogenes type II (WsA), Yersinia pseudotuberculosis type II (YpA), Erwinia carotovora type II (EwA), and Rhodospirillum rubrum type I (RrA) and Escherichia coli type II (EcA) L-asparaginases was evaluated using standard ELISA method. The comparative bioinformatics analysis of structure and sequence of the bacterial L-asparaginases presumable epitope regions was performed. We showed different immunogenic properties of five studied L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin as a second-line treatment. Studied L-asparaginases might be placed in the following order based on the immunogenicity level: YpA > RrA, WsA ≥ EwA > EcA. Most significant cross-immunogenicity was shown between EcA and YpA. We propose that a long N-terminus of YpA enzyme enriched with charged aminoacids and tryptophan could be a reason of higher immunogenicity of YpA in comparison with other considered enzymes. Although the recognized structural and sequence differences in putative epitope regions among five considered L-asparaginases does not fully explain experimental observation of the immunogenicity of the enzymes, the performed analysis set the foundation for further research in this direction. The performed studies showed different immunogenic properties of L-asparaginases and confirmed the possibility of replacement of EcA with L-asparaginase from different origin. The preferable enzymes for the second line treatment are WsA, RrA, or EwA.

L-Lysine α-oxidase (LO) from a novel Trichoderma strain: Trichoderma cf. aureoviride Rifai shows favorable biochemical and kinetic properties (K_m for L-lysine of 17.9 µmol/l, optimum pH 8.0, high stability) and significant antiproliferative activity both in vitro and in vivo. The molecular weight of LO was determined to be 115–116 kDa; the active dimer consists of two identical 57–58 kDa subunits. LO shows considerable cytotoxicity against the following tumor cell lines: K562, LS174T, HT29, SCOV3, PC3, and MCF7, with the inhibition concentration (IC₅₀) ranging from 3.0×10^–6 to 7.8×10^–2 U/ml (3.2×10^–8 to 8.2×10^–4 mg/ml). Two human colon cancer xenografts HCT116 and LS174T and breast adenocarcinoma T47D implanted subcutaneously into Balb/c nude mice showed high sensitivity to LO with a T/C of 12, 37, and 36%, respectively (P<0.05). The antitumor efficacy of LO was observed in the absence of pronounced morbidity or toxicity in vivo. Taken together, these data suggest that LO may be considered as an effective anticancer agent for the treatment of solid tumors in vivo. This study presents promising data on the possible application of LO in clinical oncology for patients with colorectal cancer.