In Silico and in Vitro Evaluation of Deamidation Effects on the Stability of the Fusion Toxin DAB389IL-2

Author(s): Nasrin Zarkar, Mohammad Ali Nasiri Khalili*, Fathollah Ahmadpour, Sirus Khodadadi, Mehdi Zeinoddini.

Journal Name: Current Proteomics

Volume 16 , Issue 4 , 2019

Submit Manuscript
Submit Proposal

Graphical Abstract:


Background: DAB389IL-2 (Denileukin diftitox) as an immunotoxin is a targeted pharmaceutical protein and is the first immunotoxin approved by FDA. It is used for the treatment of various kinds of cancer such as CTCL lymphoma, melanoma, and Leukemia but among all of these, treatment of CTCL has special importance. DAB389IL-2 consists of two distinct parts; the catalytic domain of Diphtheria Toxin (DT) that genetically fused to the whole IL-2. Deamidation is the most important reaction for chemical instability of proteins occurs during manufacture and storage. Deamidation of asparagine residues occurs at a higher rate than glutamine residues. The structure of proteins, temperature and pH are the most important factors that influence the rate of deamidation.

Methods: Since there is not any information about deamidation of DAB389IL-2, we studied in silico deamidation by Molecular Dynamic (MD) simulations using GROMACS software. The 3D model of fusion protein DAB389IL-2 was used as a template for deamidation. Then, the stability of deamidated and native form of the drug was calculated.

Results: The results of MD simulations were showed that the deamidated form of DAB389IL-2 is more unstable than the normal form. Also, deamidation was carried by incubating DAB389IL-2, 0.3 mg/ml in ammonium hydrogen carbonate for 24 h at 37o C in order to in vitro experiment.

Conclusion: The results of in vitro experiment were confirmed outcomes of in silico study. In silico and in vitro experiments were demonstrated that DAB389IL-2 is unstable in deamidated form.

Keywords: Deamidation, immunotoxin, protein stability, tumor, melanoma, lekemia.

Strebhardt, K.; Ullrich, A. Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat. Rev. Cancer, 2008, 8(6), 473.
Madhumathi, J.; Verma, R.S. Therapeutic targets and recent advances in protein immunotoxins. Curr. Opin. Microbiol., 2012, 15(3), 300-309.
Mindell, J.; Finkelstein, A.; Murphy, J. Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin. The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells. J. Boil. Chem., 1993, 268(16), 12077-12082.
Sutherland, J.A.; Ratnarathorn, M.; Howland, K.; Ciardelli, T.L.; Murphy, J.R. DAB389 interleukin-2 receptor binding domain mutations cytotoxic probes for studies of ligand-receptor interactions. J. Biol. Chem., 1996, 271(21), 12145-12149.
Potala, S.; Verma, R.S. Modified DT-IL2 fusion toxin targeting uniquely IL2Rα expressing leukemia cell lines-construction and characterization. J. Biotechnol., 2010, 148(2), 147-155.
Choe, S.; Bennett, M.J.; Fujii, G.; Curmi, P.M.; Kantardjieff, K.A.; Collier, R.J.; Eisenberg, D. The crystal structure of diphtheria toxin. Nature, 1992, 357(6375), 216.
Re, G.G.; Waters, C.; Poisson, L.; Willingham, M.C.; Sugamura, K.; Frankel, A.E. Interleukin 2 (IL-2) receptor expression and sensitivity to diphtheria fusion toxin DAB389IL-2 in cultured hematopoietic cells. Cancer Res., 1996, 56(11), 2590-2595.
Smith, K.A. The structure of IL2 bound to the three chains of the IL2 receptor and how signaling occurs. Med. Immunol., 2006, 5(1), 3.
Roderick, T.; Joren, C.; David, H.; Christene, A. Ontak-like human IL-2 fusion toxin. J. Immunol. Methods, 2017, 51-58.
Manning, M.C.; Chou, D.K.; Murphy, B.M.; Payne, R.W.; Katayama, D.S. Stability of protein pharmaceuticals: An update. Pharm. Res., 2010, 27(4), 544-575.
Gervais, D. Protein deamidation in biopharmaceutical manufacture: Understanding, control and impact. J. Chem. Technol. Biotechnol., 2016, 91(3), 569-575.
Malek-Sabet, N.; Masoumian, M.R.; Zeinali, M.; Khalilzadeh, R.; Mousaabadi, J.M. Production, purification, and chemical stability of recombinant human interferon-γ in low oxygen tension condition: A formulation approach. Prep. Biochem. Biotechnol., 2013, 43(6), 586-600.
Lewis, U.; Cheever, E.; Hopkins, W. Kinetic study of the deamidation of growth hormone and prolactin. Biochim. Biophys. Acta Protein Struct., 1970, 214(3), 498-508.
Fisher, B.V.; Porter, P.B. Stability of bovine insulin. J. Pharm. Pharmacol., 1981, 33(1), 203-206.
Minta, J.O.; Painter, R. Chemical and immunological characterization of the electrophoretic components of the Fc fragment of human immunoglobulin G. Immunochemistry, 1972, 9(8), 821-832.
Robinson, N.E.; Robinson, A.B. Prediction of protein deamidation rates from primary and three-dimensional structure. Proc. Natl. Acad. Sci. , 2001, 98(8), 4367-4372.
Robinson, N.E.; Robinson, A., Eds.; 1st, Molecular clocks: Deamidation of asparaginyl and glutaminyl residues in peptides and proteins; Althouse press: Cave Junction, OR, 2004, p. 419.
Pace, A.L.; Wong, R.L.; Zhang, Y.T.; Kao, Y-H.; Wang, Y.J. Asparagine deamidation dependence on buffer type, pH, and temperature. J. Pharm. Sci., 2013, 102(6), 1712-1723.
DeHart, M.P.; Anderson, B.D. The role of the cyclic imide in alternate degradation pathways for asparagine‐containing peptides and proteins. J. Pharm. Sci., 2007, 96(10), 2667-2685.
Moss, C.X.; Matthews, S.P.; Lamont, D.J.; Watts, C. Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules. J. Biol. Chem., 2005, 280(18), 18498-18503.
Karlsson, G.; Gellerfors, P.; Persson, A.; Norén, B.; Edlund, P.O.; Sandberg, C.; Birnbaum, S. Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography. J. Chromatogr. A, 1999, 855(1), 147-155.
Nilsson, M.R.; Driscoll, M.; Raleigh, D.P. Low levels of asparagine deamidation can have a dramatic effect on aggregation of amyloidogenic peptides: Implications for the study of amyloid formation. Protein Sci., 2002, 11(2), 342-349.
Chelius, D.; Rehder, D.S.; Bondarenko, P.V. Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies. Anal. Chem., 2005, 77(18), 6004-6011.
Linke, T.; Feng, J.; Yu, K.; Kim, H.J.; Wei, Z.; Wang, Y.; Wang, W.K.; Hunter, A.K. Process scale separation of an anti-CD22 immunotoxin charge variant. J. Chromatogr. A, 2012, 1260, 120-125.
Reubsaet, J.L.E.; Beijnen, J.H.; Bult, A.; van Maanen, R.J.; Marchal, J.D.; Underberg, W.J. Analytical techniques used to study the degradation of proteins and peptides: Chemical instability. J. Pharm. Biomed. Anal., 1998, 17(6), 955-978.
Takata, T.; Oxford, J.T.; Demeler, B.; Lampi, K.J. Deamidation destabilizes and triggers aggregation of a lens protein, βA3‐crystallin. Protein Sci., 2008, 17(9), 1565-1575.
Sasaoki, K.; Hiroshima, T.; Kusumoto, S.; Nishi, K. Oxidation of methionine residues of recombinant human interleukin 2 in aqueous solutions. Chem. Pharm. Bull., 1989, 37(8), 2160-2164.
Sasaoki, K.; Hiroshima, T.; Kusumoto, S.; Nishi, K. Deamidation at asparagine-88 in recombinant human interleukin 2. Chem. Pharm. Bull., 1992, 40(4), 976-980.
Talebi, S.; Saeedinia, A.; Zeinoddini, M.; Ahmadpour, F.; Sadeghizadeh, M. In Silico study of mutations on binding between interferon alpha 2b and IFNAR1 receptor. Curr. Proteomics, 2018, 15(1), 71-76.
Mandal, S.; Moudgil, M.N.; Mandal, S.K. Rational drug design. Eur. J. Pharmacol., 2009, 625(1-3), 90-100.
Durrant, J.D.; McCammon, J.A. Molecular dynamics simulations and drug discovery. BMC Boil., 2011, 9(1), 71.
Borhani, D.W.; Shaw, D.E. The future of molecular dynamics simulations in drug discovery. J. Comput. Aided Mol. Des., 2012, 26(1), 15-26.
Hao, G-F.; Yang, G-F.; Zhan, C-G. Structure-based methods for predicting target mutation-induced drug resistance and rational drug design to overcome the problem. Drug Discov. Today, 2012, 17(19-20), 1121-1126.
Yuan, P.M.; Talent, J.M.; Gracy, R.W. Molecular basis for the accumulation of acidic isozymes of triosephosphate isomerase on aging. Mech. Ageing Dev., 1981, 17(2), 151-162.
Geiger, T.; Clarke, S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. J. Biol. Chem., 1987, 262(2), 785-794.
Lewis, U.; Singh, R.; Bonewald, L.; Seavey, B. Altered proteolytic cleavage of human growth hormone as a result of deamidation. J. Biol. Chem., 1981, 256(22), 11645-11650.
Wilmarth, P.; Tanner, S.; Dasari, S.; Nagalla, S.; Riviere, M.; Bafna, V.; Pevzner, P.; David, L. Age-related changes in human crystallins determined from comparative analysis of post-translational modifications in young and aged lens: Does deamidation contribute to crystallin insolubility? J. Proteome Res., 2006, 5(10), 2554-2566.
Harrington, V.; McCall, S.; Huynh, S.; Srivastava, K.; Srivastava, O.P. Crystallins in water soluble-high molecular weight protein fractions and water insoluble protein fractions in aging and cataractous human lenses. Mol. Vis., 2004, 10(61), 476-489.
Takata, T.; Oxford, J.T.; Brandon, T.R.; Lampi, K.J. Deamidation alters the structure and decreases the stability of human lens βA3-crystallin. Biochemistry, 2007, 46(30), 8861-8871.
Flaugh, S.L.; Mills, I.A.; King, J. Glutamine deamidation destabilizes human γD-crystallin and lowers the kinetic barrier to unfolding. J. Biol. Chem., 2006, 281(41), 30782-30793.
Gupta, R.; Srivastava, K.; Srivastava, O. Truncation of motifs III and IV in human lens βA3-crystallin destabilizes the structure. Biochemistry, 2006, 45(33), 9964-9978.
Bhanuramanand, K.; Ahmad, S.; Rao, N. Engineering deamidation‐susceptible asparagines leads to improved stability to thermal cycling in a lipase. Protein Sci., 2014, 23(10), 1479-1490.
Yang, Y.; Zhao, J.; Geng, S.; Hou, C.; Li, X.; Lang, X.; Qiao, C.; Li, Y.; Feng, J.; Lv, M. Improving trastuzumab’s stability profile by removing the two degradation hotspots. J. Pharm. Sci., 2015, 104(6), 1960-1970.
Wakankar, A.A.; Borchardt, R.T. Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization. J. Pharm. Sci., 2006, 95(11), 2321-2336.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [307 - 313]
Pages: 7
DOI: 10.2174/1570164616666190131150033
Price: $58

Article Metrics

PDF: 19