In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation

Seyed Mehdi Sajjadi, Hamzeh Rahimi, Saeed Mohammadi, Mohammad Faranoush, Hasan Mirzahoseini, Gholamreza Toogeh

Abstract


Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results.


Keywords


In silico; Hirudin variant 3; PEGylation

Full Text:

PDF

References


Lazar JB, Winant RC, Johnson PH. Hirudin: amino-terminal residues play a major role in the interaction with thrombin. J Biol Chem. 1991;266(2):685-688.

Markwardt F. Hirudin: the promising antithrombotic. Cardiovasc Drug Rev. 1992;10(2):211-232.

Liu CC, Schultz PG. Recombinant expression of selectively sulfated proteins in Escherichia coli. Nat Biotechnol. 2006;24(11):1436-1440.

Greinacher A, Lubenow N. Recombinant hirudinin clinical practice focus on lepirudin. Circulation. 2001;103(10):1479-1484.

Hou B, LiS, Li X, Xiu Z. Design; preparation and in vitro bioactivity of mono-PEGylated recombinant hirudin. Chin J Chem Eng. 2007;15(6):775-780.

Warkentin TE. Bivalent direct thrombin inhibitors: hirudin and bivalirudin. Best Pract Res ClinHaematol. 2004;17(1):105-125.

Wang X, Hu J, Pan D, Teng H, Xiu Z. PEGylation kinetics of recombinant hirudin and its application for the production of PEGylated HV2 species. BiochemEng J. 2014;85:38-48.

Avgerinos GC, Turner BG, Gorelick KJ, Papendieck A, Weydemann U, Gellissen G. Production and clinical development of a Hansenulapolymorpha-derived PEGylated hirudin. Semin Thromb Hemost. 2001;27(4):357-372.

Pasut G, Veronese FM. PEGylation for improving the effectiveness of therapeutic biomolecules. Drugs Today (Barc). 2009;45(9):687-695.

Fee CJ, Van Alstine JM. PEG-proteins: reaction engineering and separation issues. Chem Eng Sci. 2006;61(3):924-939.

Colonna C, Conti B, Perugini P, Pavanetto F, Modena T, Dorati R, et al. Site-directed PEGylation as successful approach to improve the enzyme replacement in the case of prolidase. Int J Pharm. 2008;358(1-2):230-237.

Gaberc-Porekar V, Zore I, Podobnik B, Menart V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr Opin Drug Discov Devel. 2008;11(2):242-250.

Roberts MJ, Bentley MD, Harris JM. Chemistry for peptide and protein PEGylation. Adv Drug Deliv Rev. 2002;54(4):459-476.

Cohan RA, Madadkar-sobhani A, Khanahmad H, Roohvand F, Aghasadeghi MR, Hedayati MH, et al. Design, modeling, expression and chemoselective PEGylation of a new nanosize cysteine analog of erythropoietin. Int J Nanomedicine. 2011;6:1217-1227.

Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi M, Valentini M, et al. Site‐directed enzymatic PEGylation of the human granulocyte colony‐stimulating factor. FEBS J. 2009;276(22):6741-6750.

Jadhav AN, Dash RC, Hirwani RR. Relative stability of thrombin-hirudin complex is illustrated using molecular dynamics. J Comput Meth Mol Des. 2014;4(4):54-62.

Šali A, Blundell TL. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993;234(3):779-815.

Söding J, Biegert A, Lupas AN. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res.2005;33(Web Server issue):W244-248.

Eramian D, Shen My, Devos D, Melo F, Sali A, Marti‐Renom MA. A composite score for predicting errors in protein structure models. Protein Sci. 2006;15(7):1653-1666.

Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Cryst. 1993;26(2):283-291.

Eisenberg D, Lüthy R, Bowie JU. VERIFY3D: assessment of protein models with three-dimensional profiles. Methods Enzymol. 1997;277:396-404.

Comeau SR, Gatchell DW, Vajda S, Camacho CJ. ClusPro: a fully automated algorithm for protein-protein docking. Nucleic Acids Res. 2004;32(Web Server issue):W96-99.

Comeau SR, Gatchell DW, Vajda S, Camacho CJ. ClusPro: an automated docking and discrimination method for the prediction of protein complexes. Bioinformatics. 2004;20(1):45-50.

Laskowski RA, Swindells MB. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J ChemInf Model. 2011;51(10):2778-2786.

DeLano W. The PyMOL molecular graphics system. 2002, DeLano Scientific LLC. San Carlos,CA,USA. http://pymol.sourceforge.net.

UniProt Consortium. The Universal Protein resource (UniProt). Nucleic Acids Res. 2007;35(Database issue):D193-197.

Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ. Jalview Version 2--a multiple sequence alignment editor and analysis workbench. Bioinformatics. 2009;25(9):1189-1191.

Cole C, Barber JD, Barton GJ. The Jpred 3 secondary structure prediction server. Nucleic Acids Res. 2008;36(Web Server issue):W197-201.

Cheng J, Saigo H, Baldi P. Large-Scale Prediction of disulphide bridges using kernel methods, two-dimensional recursive neural networks, and weighted graph matching. Proteins. 2006;62(3):617-629.

Fariselli P, Riccobelli P, Casadio R. Role of evolutionary information in predicting the disulfide-bonding state of cysteine in proteins. Proteins. 1999;36(3):340-346.

Vullo A, Frasconi P. Disulfide connectivity prediction using recursive neural networks and evolutionary information. Bioinformatics. 2004;20(5): 653-659.

Shi W, Punta M, Bohon J, Sauder JM, D'Mello R, Sullivan M, et al. Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy. Genome Res. 2011;21(6):898-907.

Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi M, Valentini M, et al. Site-directed enzymatic PEGylation of the human granulocyte colony-stimulating factor. FEBS J. 2009;276(22):6741–6750.

Golshani M, Rafati S, Jahanian-Najafabadi A, Nejati-Moheimani M, Siadat SD, Shahcheraghi F, et al. In silico design, cloning and high level expression of L7/L12-TOmp31 fusion protein of Brucella antigens.Res Pharm Sci. 2015;10(5):436–445.

Muhammad SA, Ali A, Ismail T, Zafar R, Ilyas U, Ahmad J. In silico study ofanti-carcinogenic lysyloxidase-like2 inhibitors. Comput Biol Chem. 2014;(51):71–82.

Fontana A, Spolaore B, Mero A, Veronese FM. Site-specific modification and PEGylation of pharmaceutical proteins mediated by transglutaminase. Adv Drug Deliv Rev. 2008;60(1):13-28.

Cong Y, Pawlisz E, Bryant P, Balan S, Laurine E, Tommasi R, et al. Site-specific PEGylation at histidine tags. Bioconjug Chem. 2012;23(2):248-263.

Shaunak S, Godwin A, Choi JW, Balan S, Pedone E, Vijayarangam D, et al. Site-specific PEGylation of native disulfide bonds in therapeutic proteins. Nat Chem Biol. 2006;2(6):312-313.

Rosendahl MS, Doherty DH, Smith DJ, Carlson SJ, Chlipala EA, Cox GN. A long-acting; highly potent interferon α-2 conjugate created using site-specific PEGylation. Bioconjug Chem. 2005;16(1):200-207.

Doherty DH, Rosendahl MS, Smith DJ, Hughes JM, Chlipala EA, Cox GN. Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor. Bioconjug Chem. 2005;16(5):1291-1298.

Alibeik S, Zhu S, Brash JL. Surface modification with PEG and hirudin for protein resistance and thrombin neutralization in blood contact. Colloids Surf B Biointerfaces. 2010;81(2):389-396.

Li X, Xiu Z, Zhao J, Li S, Li X, Su Z. An integrated process of PEGylation and separation of hirudin on an anion exchange column. J Biotechnol. 2008;136:S501.

Wang XD, Teng H, Hu JJ, Xiu ZL. PEGylation of recombinant hirudin in mixed aqueous-organic solutions. Procbiochem. 2015;50(3):367–377.

Qin H, Xiu Z, Zhang D, BaoY, LiX, Han G. PEGylation of hirudin and analysis of its antithrombin activity in vitro. Chin J Chem Eng. 2007;15(4):586-590.

Otto A, Seckler R. Characterization, stability and refolding of recombinant hirudin. Eur J Biochem. 1991;202(1):67-73.


Refbacks

  • There are currently no refbacks.


Creative Commons Attribution-NonCommercial 3.0

This work is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.