Characterization of the Trypanosoma brucei Pteridine Reductase Active- Site using Computational Docking and Virtual Screening Techniques

Hina       Shamshad; Abdul       Hafiz; Ismail    I.    Althagafi; Maria       Saeed; Agha    Zeeshan    Mirza

Abstract

Background: Human African trypanosomiasis is a fatal disease prevalent in approximately 36 sub-Saharan countries. Emerging reports of drug resistance in Trypanosoma brucei are a serious cause of concern as only limited drugs are available for the treatment of the disease. Pteridine reductase is an enzyme of Trypanosoma brucei.

Methods: It plays a critical role in the pterin metabolic pathway that is absolutely essential for its survival in the human host. The success of finding a potent inhibitor in structure-based drug design lies within the ability of computational tools to efficiently and accurately dock a ligand into the binding cavity of the target protein. Here we report the computational characterization of Trypanosoma brucei pteridine reductase (Tb-PR) active-site using twenty-four high-resolution co-crystal structures with various drugs. Structurally, the Tb-PR active site can be grouped in two clusters; one with high Root Mean Square Deviation (RMSD) of atomic positions and another with low RMSD of atomic positions. These clusters provide fresh insight for rational drug design against Tb-PR. Henceforth, the effect of several factors on docking accuracy, including ligand and protein flexibility were analyzed using Fred.

Results: The online server was used to analyze the side chain flexibility and four proteins were selected on the basis of results. The proteins were subjected to small-scale virtual screening using 85 compounds, and statistics were calculated using Bedroc and roc curves. The enrichment factor was also calculated for the proteins and scoring functions. The best scoring function was used to understand the ligand protein interactions with top common compounds of four proteins. In addition, we made a 3D structural comparison between the active site of Tb-PR and Leishmania major pteridine reductase (Lm- PR). We described key structural differences between Tb-PR and Lm-PR that can be exploited for rational drug design against these two human parasites.

Conclusion: The results indicated that relying just on re-docking and cross-docking experiments for virtual screening of libraries isn’t enough and results might be misleading. Hence it has been suggested that small scale virtual screening should be performed prior to large scale screening.

Keywords: Tb-PTR1, re-docking, cross docking, virtual screening, african trypanosomiasis, Leishmania.

Graphical Abstract

[1] 
Ong, H.B.; Sienkiewicz, N.; Wyllie, S.; Fairlamb, A.H. Dissecting the metabolic roles of pteridine reductase 1 in Trypanosoma brucei and Leishmania major. J. Biol. Chem.,  2011, 286(12), 10429-10438.
[http://dx.doi.org/10.1074/jbc.M110.209593] [PMID:  21239486] 
[2] 
Setzer, W.N.; Ogungbe, I.V. In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plants. PLoS Negl. Trop. Dis.,  2012, 6(7),e1727.
[http://dx.doi.org/10.1371/journal.pntd.0001727] [PMID:  22848767] 
[3] 
Barrack, K.L.; Tulloch, L.B.; Burke, L.A.; Fyfe, P.K.; Hunter, W.N. Structure of recombinant Leishmania donovani pteridine reductase reveals a disordered active site. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.,  2011, 67(Pt 1), 33-37.
[http://dx.doi.org/10.1107/S174430911004724X] [PMID:  21206018] 
[4] 
Shanks, E.J.; Ong, H.B.; Robinson, D.A.; Thompson, S.; Sienkiewicz, N.; Fairlamb, A.H.; Frearson, J.A. Development and validation of a cytochrome c-coupled assay for pteridine reductase 1 and dihydrofolate reductase. Anal. Biochem.,  2010, 396(2), 194-203.
[http://dx.doi.org/10.1016/j.ab.2009.09.003] [PMID:  19748480] 
[5] 
Mpamhanga, C.P.; Spinks, D.; Tulloch, L.B.; Shanks, E.J.; Robinson, D.A.; Collie, I.T.; Fairlamb, A.H.; Wyatt, P.G.; Frearson, J.A.; Hunter, W.N.; Gilbert, I.H.; Brenk, R. One scaffold, three binding modes: novel and selective pteridine reductase 1 inhibitors derived from fragment hits discovered by virtual screening. J. Med. Chem.,  2009, 52(14), 4454-4465.
[http://dx.doi.org/10.1021/jm900414x] [PMID:  19527033] 
[6] 
Dawson, A.; Gibellini, F.; Sienkiewicz, N.; Tulloch, L.B.; Fyfe, P.K.; McLuskey, K.; Fairlamb, A.H.; Hunter, W.N. Structure and reactivity of Trypanosoma brucei pteridine reductase: inhibition by the archetypal antifolate methotrexate. Mol. Microbiol.,  2006, 61(6), 1457-1468.
[http://dx.doi.org/10.1111/j.1365-2958.2006.05332.x] [PMID:  16968221] 
[7] 
Spinks, D.; Ong, H.B.; Mpamhanga, C.P.; Shanks, E.J.; Robinson, D.A.; Collie, I.T.; Read, K.D.; Frearson, J.A.; Wyatt, P.G.; Brenk, R.; Fairlamb, A.H.; Gilbert, I.H. Design, synthesis and biological evaluation of novel inhibitors of Trypanosoma brucei pteridine reductase 1. ChemMedChem,  2011, 6(2), 302-308.
[http://dx.doi.org/10.1002/cmdc.201000450] [PMID:  21275054] 
[8] 
Schüttelkopf, A.W.; Hardy, L.W.; Beverley, S.M.; Hunter, W.N. Structures of Leishmania major pteridine reductase complexes reveal the active site features important for ligand binding and to guide inhibitor design. J. Mol. Biol.,  2005, 352(1), 105-116.
[http://dx.doi.org/10.1016/j.jmb.2005.06.076] [PMID:  16055151] 
[9] 
Cavazzuti, A.; Paglietti, G.; Hunter, W.N.; Gamarro, F.; Piras, S.; Loriga, M.; Allecca, S.; Corona, P.; McLuskey, K.; Tulloch, L.; Gibellini, F.; Ferrari, S.; Costi, M.P. Discovery of potent pteridine reductase inhibitors to guide antiparasite drug development. Proc. Natl. Acad. Sci. USA,  2008, 105(5), 1448-1453.
[http://dx.doi.org/10.1073/pnas.0704384105] [PMID:  18245389] 
[10] 
Gourley, D.G.; Schüttelkopf, A.W.; Leonard, G.A.; Luba, J.; Hardy, L.W.; Beverley, S.M.; Hunter, W.N. Pteridine reductase mechanism correlates pterin metabolism with drug resistance in trypanosomatid parasites. Nat. Struct. Biol.,  2001, 8(6), 521-525.
[http://dx.doi.org/10.1038/88584] [PMID:  11373620] 
[11] 
Tulloch, L.B.; Martini, V.P.; Iulek, J.; Huggan, J.K.; Lee, J.H.; Gibson, C.L.; Smith, T.K.; Suckling, C.J.; Hunter, W.N. Structure-based design of pteridine reductase inhibitors targeting African sleeping sickness and the leishmaniases. J. Med. Chem.,  2010, 53(1), 221-229.
[http://dx.doi.org/10.1021/jm901059x] [PMID:  19916554] 
[12] 
Dawson, A.; Tulloch, L.B.; Barrack, K.L.; Hunter, W.N. High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target. Acta Crystallogr. D Biol. Crystallogr.,  2010, 66(Pt 12), 1334-1340.
[http://dx.doi.org/10.1107/S0907444910040886] [PMID:  21123874] 
[13] 
Bkhaitan, M.M.; Mirza, A.Z.; Shamshad, H.; Ali, H.I. Identification of potent virtual leads and ADME prediction of isoxazolidine podophyllotoxin derivatives as topoisomerase II and tubulin inhibitors. J. Mol. Graph. Model.,  2017, 73, 74-93.
[http://dx.doi.org/10.1016/j.jmgm.2017.01.015] [PMID:  28242581] 
[14] 
RCSB PDB, University of New Jersey, Department of Chemistry and Chemical Biology 610 Taylor Road.  https://www.rcsb.org/
[15] 
McGann, M. Fred pose prediction and virtual screening accuracy. J. Chem. Inf. Model.,  2011, 51(3), 578-596.
[http://dx.doi.org/dx.doi:10.1021/ci100436p] 
[16] 
Zaheer-ul-Haq, Halim, S.A.; Uddin, R.; Madura, J.D. Benchmarking docking and scoring protocol for the identification of potential acetylcholinesterase inhibitors. J. Mol. Graph. Model.,  2010, 28(8), 870-882.
[http://dx.doi.org/10.1016/j.jmgm.2010.03.007] [PMID:  20447848] 
[17] 
Molecular graphics and analyses performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and 598 Current Computer-Aided Drug Design, 2020, Vol. 16, No. 5 Shamshad et al. Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. Chimera. 
[18] 
Sanschagrin, P.C.; Kuhn, L.A. Cluster analysis of consensus water sites in thrombin and trypsin shows conservation between serine proteases and contributions to ligand specificity. Protein Sci.,  1998, 7(10), 2054-2064.
[http://dx.doi.org/10.1002/pro.5560071002] [PMID:  9792092] 
[19] 
Azam, S.S.; Abbasi, S.W. Molecular docking studies for the identification of novel melatoninergic inhibitors for acetylserotonin-O-methyltransferase using different docking routines. Theor. Biol. Med. Model.,  2013, 10, 63.
[http://dx.doi.org/10.1186/1742-4682-10-63] [PMID:  24156411] 
[20] 
 Vida, OpenEye Scientific Software, OEDocking Santa Fe, New Mexico, 2010.http://www.eyesopen.com
[21] 
SYBYL Molecular Modeling Software version 6.9, 2003, Tripos Associates St. Louis, MO Sybyl 
[22] 
Paul, C.D. Hawkins, Anthony Nicholls, Conformer Generation with OMEGA: Learning from the Data Set and the Analysis of Failures. J. Chem. Inf. Model.,  2012, 52(11), 2919-2936.
[23] 
Molecular Operating Environment (MOE), 2013.08; Chemical Computing Group ULC; 1010 Sherbooke St. West, Suite #910, Montreal, QC: Canada, H3A 2R7, , 2018. 
[24] 
Babel, O. An open chemical toolbox, Noel M O’Boyle, Michael Banck, Craig A James, Chris Morley, Tim Vandermeersch and Geoffrey R Hutchison. J. Cheminform.,  20113, 33.
[25] 
Erickson, J.A.; Jalaie, M.; Robertson, D.H.; Lewis, R.A.; Vieth, M. Lessons in molecular recognition: the effects of ligand and protein flexibility on molecular docking accuracy. J. Med. Chem.,  2004, 47(1), 45-55.
[http://dx.doi.org/10.1021/jm030209y] [PMID:  14695819] 
[26] 
Gautier, R.; Camproux, A.C.; Tufféry, P. SCit: web tools for protein side chain conformation analysis. Nucleic Acids Res., 2004, 32(Web Server issue)W508-11, 
[http://dx.doi.org/10.1093/nar/gkh388] [PMID:  15215438] 
[27] 
Najmanovich, R.; Kuttner, J.; Sobolev, V.; Edelman, M. Side-chain flexibility in proteins upon ligand binding. Proteins,  2000, 39(3), 261-268.
[http://dx.doi.org/10.1002/(SICI)1097-0134(20000515)39:3<261:AID-PROT90>3.0.CO;2-4] [PMID:  10737948] 
[28] 
Truchon, J.F.; Bayly, C.I. Evaluating virtual screening methods: good and bad metrics for the “early recognition” problem. J. Chem. Inf. Model.,  2007, 47(2), 488-508.
[http://dx.doi.org/10.1021/ci600426e] [PMID:  17288412] 
[29] 
Kitchen, D.B.; Decornez, H.; Furr, J.R.; Bajorath, J. Docking and scoring in virtual screening for drug discovery: methods and applications. Nat. Rev. Drug Discov.,  2004, 3(11), 935-949.
[http://dx.doi.org/10.1038/nrd1549] [PMID:  15520816] 
[30] 
Warren, G.L.; Andrews, C.W.; Capelli, A.M.; Clarke, B.; LaLonde, J.; Lambert, M.H.; Lindvall, M.; Nevins, N.; Semus, S.F.; Senger, S.; Tedesco, G.; Wall, I.D.; Woolven, J.M.; Peishoff, C.E.; Head, M.S. A critical assessment of docking programs and scoring functions. J. Med. Chem.,  2006, 49(20), 5912-5931.
[http://dx.doi.org/10.1021/jm050362n] [PMID:  17004707] 
[31] 
Borsari, C.; Luciani, R.; Pozzi, C.; Poehner, I.; Henrich, S.; Trande, M.; Cordeiro-da-Silva, A.; Santarem, N.; Baptista, C.; Tait, A.; Di Pisa, F.; Dello Iacono, L.; Landi, G.; Gul, S.; Wolf, M.; Kuzikov, M.; Ellinger, B.; Reinshagen, J.; Witt, G.; Gribbon, P.; Kohler, M.; Keminer, O.; Behrens, B.; Costantino, L.; Tejera Nevado, P.; Bifeld, E.; Eick, J.; Clos, J.; Torrado, J.; Jiménez-Antón, M.D.; Corral, M.J.; Alunda, J.M.; Pellati, F.; Wade, R.C.; Ferrari, S.; Mangani, S.; Costi, M.P. Profiling of Flavonol Derivatives for the Development of Antitrypanosomatidic Drugs. J. Med. Chem.,  2016, 59(16), 7598-7616.
[http://dx.doi.org/10.1021/acs.jmedchem.6b00698] [PMID:  27411733] 
[32] 
Setzer, W.N.; Ogungbe, I.V. In-silico investigation of antitrypanosomal phytochemicals from Nigerian medicinal plants. PLoS Negl. Trop. Dis.,  2012, 6(7),e1727.
[http://dx.doi.org/10.1371/journal.pntd.0001727] [PMID:  22848767] 
[33] 
 Flavonoid Standard Reference Material. https://www.extrasynthese.com/flavonoid.html?p=2

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