Chagas'disease (American Trypanosomiasis) affects 16-18 million people in Latin America. It has a 25-30% death rate among infected people and causes between 45,000 and 50,000 deaths a year. The flagellate protozoan parasite Trypanosoma cruzi, the causative agent of Chagas'disease is transmitted to humans by bites of blood-feeding """"""""Assassin bugs"""""""". The most common agent is Triatoma infestans (vinchucas in Argentina, or barbeiros in Brazil). The enzyme trans-sialidase in T.cruzi catalyzes the transfer of sialic acids from host aloglycoconjugates to other parasite glycoconjugates and has a critical role in virulence of T.cruzi, the etiological agent of Chagas'disease. T.cruzi trans-sialidase (TcTS) is a validated target for inhibition with therapeutic possibilities for the cure for this lethal chronic disease. TcTS is also of interest due to its strong similarity in sequence and structure to a strict hydrolase, Trypanosoma rangeli sialidase (TrSA). An extensive comparison of the two will reveal structural requirements for sialyl-transferase activity and also be a guide for future efforts to transform sialidases into trans-sialidases that are of synthetic value. This theoretical study will focus on the reaction mechanisms for hydrolysis reactions of TcTS and TrSA and sialyl-transfer reaction of TcTS using hybrid (mixed quantum and classical, QM/MM) methods on entire enzyme structures. There is a methods development part of this proposal associated with two different QM/MM implementations, one native to the program Amber, and one that links Amber with the QM program Gaussian, via a program we designed name Pupil. Molecular dynamics (MD) simulations on various X-ray crystal structures of the enzymes which correspond to different points on the reaction paths will also be performed. Critical active site interactions and dynamical properties for sialyl-transfer reaction will be uncovered analyzing MD simulations of wild type and mutant enzymes and comparing the results. We will also perform extensive research into inhibitor design for TcTS, using a combination of docking, free energy calculations and mechanism-based leads. Public Health Relevance: A broader impact of the present research is the contribution to the understanding of the general principles of catalysis. From a synthetic point of view, trans-sialidases can be used to perform complex syntheses of O-linked and S-linked glycoconjugates of industrial and medicinal value. A deeper understanding of the mechanisms of the enzymes could be a guide for inhibitor design studies of the entire family.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI073674-02
Application #
7665461
Study Section
Macromolecular Structure and Function D Study Section (MSFD)
Program Officer
Rogers, Martin J
Project Start
2008-07-30
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
2
Fiscal Year
2009
Total Cost
$237,971
Indirect Cost
Name
University of Florida
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Bueren-Calabuig, Juan A; Pierdominici-Sottile, Gustavo; Roitberg, Adrian E (2014) Unraveling the differences of the hydrolytic activity of Trypanosoma cruzi trans-sialidase and Trypanosoma rangeli sialidase: a quantum mechanics-molecular mechanics modeling study. J Phys Chem B 118:5807-16
Miller 3rd, Bill R; Roitberg, Adrian E (2013) Design of e-pharmacophore models using compound fragments for the trans-sialidase of Trypanosoma cruzi: screening for novel inhibitor scaffolds. J Mol Graph Model 45:84-97
Miller 3rd, Bill R; Roitberg, Adrian E (2013) Trypanosoma cruzi trans-sialidase as a drug target against Chagas disease (American trypanosomiasis). Future Med Chem 5:1889-900
Di Russo, Natali V; Estrin, Dario A; Marti, Marcelo A et al. (2012) pH-Dependent conformational changes in proteins and their effect on experimental pK(a)s: the case of Nitrophorin 4. PLoS Comput Biol 8:e1002761
Sabri Dashti, Danial; Meng, Yilin; Roitberg, Adrian E (2012) pH-replica exchange molecular dynamics in proteins using a discrete protonation method. J Phys Chem B 116:8805-11
Pierdominici-Sottile, Gustavo; Horenstein, Nicole A; Roitberg, Adrian E (2011) Free energy study of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. From the Michaelis complex to the covalent intermediate. Biochemistry 50:10150-8
Meng, Yilin; Dashti, Danial Sabri; Roitberg, Adrian E (2011) Computing Alchemical Free Energy Differences with Hamiltonian Replica Exchange Molecular Dynamics (H-REMD) Simulations. J Chem Theory Comput 7:2721-2727
Pierdominici-Sottile, Gustavo; Roitberg, Adrian E (2011) Proton transfer facilitated by ligand binding. An energetic analysis of the catalytic mechanism of Trypanosoma cruzi trans-sialidase. Biochemistry 50:836-42
Meng, Yilin; Roitberg, Adrian E (2010) Constant pH replica exchange molecular dynamics in biomolecules using a discrete protonation model. J Chem Theory Comput 6:1401-1412
Swails, Jason M; Meng, Yilin; Walker, F Ann et al. (2009) pH-dependent mechanism of nitric oxide release in nitrophorins 2 and 4. J Phys Chem B 113:1192-201

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