Abstract
Arginine deiminase (ADI), a promising anticancer enzyme from Mycoplasma hominis, is currently in phase III of clinical trials for the treatment of arginine auxotrophic tumors. However, it has been associated with several drawbacks in terms of low stability at human physiological conditions, high immunogenicity, hypersensitivity and systemic toxicity. In our previous work, Pseudomonas furukawaii 24 was identified as a potent producer of ADI with optimum activity under physiological conditions. In the present study, phylogenetic analysis of microbial ADIs indicated P. furukawaii ADI (PfADI) to be closely related to experimentally characterized ADIs of Pseudomonas sp. with proven anticancer activity. Immunoinformatics analysis was performed indicating lower immunogenicity of PfADI than MhADI (M. hominis ADI) both in terms of number of linear and conformational B-cell epitopes and T-cell epitope density. Overall antigenicity and allergenicity of PfADI was also lower as compared to MhADI, suggesting the applicability of PfADI as an alternative anticancer biotherapeutic. Hence, in vitro experiments were performed in which the ADI coding arcA gene of P. furukawaii was cloned and expressed in E. coli BL21. Recombinant ADI of P. furukawaii was purified, characterized and its anticancer activity was assessed. The enzyme was stable at human physiological conditions (pH 7 and 37 °C) with Km of 1.90 mM. PfADI was found to effectively inhibit the HepG2 cells with an IC50 value of 0.1950 IU/ml. Therefore, the current in silico and in vitro studies establish PfADI as a potential anticancer drug candidate with improved efficacy and low immunogenicity.
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Acknowledgements
The authors are thankful to the Department of science and technology (DST), Government of India for financially supporting the study (SB/YS/LS-145/2014). The authors are also grateful to DST-FIST grant (1196 SR/FST/LS-I/2017/4) and Department of Biotechnology, Ministry of Science and Technology, BTISNET program grant [BT/BI/25/059/2012-BIF] for providing the infrastructure facility. RD expresses her gratitude to DST and MDU for providing the research fellowship. The authors sincerely thank and acknowledge Prof. Rakesh Bhatnagar for allowing us to carry out a part of this study in his laboratory at Jawaharlal Nehru University, New Delhi.
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This study was funded by the Department of Science and Technology (DST), Government of India (SB/YS/LS-145/2014).
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PG, RD,VG and AM conceived and planned the experiments. RD and VG performed the experiments. PG supervised the project and received the funding. RD wrote the manuscript. AM did the supervision of bioinformatics investigation. AK helped in bioinformatics studies. PG, AK, AM and VG did the reviewing and editing.
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Dhankhar, R., Kawatra, A., Gupta, V. et al. In silico and in vitro analysis of arginine deiminase from Pseudomonas furukawaii as a potential anticancer enzyme. 3 Biotech 12, 220 (2022). https://doi.org/10.1007/s13205-022-03292-2
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DOI: https://doi.org/10.1007/s13205-022-03292-2