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Design of e-pharmacophore models using compound fragments for the trans-sialidase of Trypanosoma cruzi: screening for novel inhibitor scaffolds.

ABSTRACT: Chagas' is a fatal disease that affects millions of people worldwide. The lack of safe and effective treatments for Chagas' highlights the need for the discovery of new drugs to fight the disease. Trypanosoma cruzi, the parasitic cause of Chagas' disease, synthesizes a trans-sialidase (TcTS) enzyme responsible for the transfer of sialic acids from the host cell surface to glycoconjugates on the parasitic cell surface. TcTS has no human analogs and is vital to the life cycle of T. cruzi, making TcTS an important enzyme for drug design against Chagas' disease. We use fragment docking to generate various e-pharmacophore hypotheses depicting protein residues important for ligand binding. Virtual screening of the ZINC Clean Leads database with more than 4 million compounds using the e-pharmacophore models found 82 potential inhibitors of TcTS. Molecular dynamics and free energy of binding calculations were used to rank the compounds based on their affinity for TcTS. Two compounds-ZINC13359679 and ZINC02576132-were found to be the most promising lead candidates for TcTS inhibition, and their binding modes are analyzed in detail.

SUBMITTER: Miller BR

PROVIDER: S-EPMC3824395 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Design of e-pharmacophore models using compound fragments for the trans-sialidase of Trypanosoma cruzi: screening for novel inhibitor scaffolds.

Miller Bill R BR Roitberg Adrian E AE

Journal of molecular graphics & modelling 20130816

Chagas' is a fatal disease that affects millions of people worldwide. The lack of safe and effective treatments for Chagas' highlights the need for the discovery of new drugs to fight the disease. Trypanosoma cruzi, the parasitic cause of Chagas' disease, synthesizes a trans-sialidase (TcTS) enzyme responsible for the transfer of sialic acids from the host cell surface to glycoconjugates on the parasitic cell surface. TcTS has no human analogs and is vital to the life cycle of T. cruzi, making T ...[more]

PMID: 24012872

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Project description:Trans-sialidase (TS), a virulence factor from Trypanosoma cruzi, is an enzyme playing key roles in the biology of this protozoan parasite. Absent from the mammalian host, it constitutes a potential target for the development of novel chemotherapeutic drugs, an urgent need to combat Chagas' disease. TS is involved in host cell invasion and parasite survival in the bloodstream. However, TS is also actively shed by the parasite to the bloodstream, inducing systemic effects readily detected during the acute phase of the disease, in particular, hematological alterations and triggering of immune cells apoptosis, until specific neutralizing antibodies are elicited. These antibodies constitute the only known submicromolar inhibitor of TS's catalytic activity. We now report the identification and detailed characterization of a neutralizing mouse monoclonal antibody (mAb 13G9), recognizing T. cruzi TS with high specificity and subnanomolar affinity. This mAb displays undetectable association with the T. cruzi superfamily of TS-like proteins or yet with the TS-related enzymes from Trypanosoma brucei or Trypanosoma rangeli. In immunofluorescence assays, mAb 13G9 labeled 100% of the parasites from the infective trypomastigote stage. This mAb also reduces parasite invasion of cultured cells and strongly inhibits parasite surface sialylation. The crystal structure of the mAb 13G9 antigen-binding fragment in complex with the globular region of T. cruzi TS was determined, revealing detailed molecular insights of the inhibition mechanism. Not occluding the enzyme's catalytic site, the antibody performs a subtle action by inhibiting the movement of an assisting tyrosine (Y???), whose mobility is known to play a key role in the trans-glycosidase mechanism. As an example of enzymatic inhibition involving non-catalytic residues that occupy sites distal from the substrate-binding pocket, this first near atomic characterization of a high affinity inhibitory molecule for TS provides a rational framework for novel strategies in the design of chemotherapeutic compounds.

Project description:Oral transmission of T. cruzi is probably the most frequent mechanism among animals in the wild. In this context, there is a high prevalence of human infections in regions where triatomine infection with T. cruzi is low. This led to the hypothesis that the consumption of raw or undercooked meat from animals infected with T. cruzi could be responsible for the transmission of the infection. Therefore, the general objective of this study was to demonstrate the role of meat consumption from infected animals in the oral transmission of T. cruzi infection. Groups of five female mice Balb/c were fed with muscles obtained from mice in the acute phase of infection by the clone H510 C8C3hvir of T. cruzi, and the infection of the fed mice was monitored by a parasitemia curve. Similarly, we assessed the infective capacity of T. cruzi trypomastigotes and amastigotes by infecting groups of five mice Balb/c females, which were infected orally using a nasogastric probe, and the infection was monitored by parasitemia curve. Finally, different trypomastigote and amastigote inoculums were used to determine their infective capacity. Adhesion assays of T. cruzi proteins to AGS stomach cells were performed and the adhered proteins were detected by western blotting using monoclonal or polyclonal antibodies. In all cases, 60–100% of animals were fed meat from mice infected in the acute phase or infected by means of a nasogastric probe with trypomastigotes or amastigotes. These animals developed high parasitemia, and 80% died around day 40 post-infection. The adhesion tests showed that cruzipain is a molecule of trypomastigotes and amastigotes that binds to AGS cells. LC-MS/MS also confirmed that transialidase may be involved in TCT attachment of TCT or invasion of stomach cells. It was concluded that the consumption of meat from infected animals in the acute phase of T. cruzi infection allows transmission of the infection. Similarly, trypomastigotes and amastigotes were able to infect mice when administered orally, while cruzipain was a relevant molecule in this infective process.

Dataset Information

Design of e-pharmacophore models using compound fragments for the trans-sialidase of Trypanosoma cruzi: screening for novel inhibitor scaffolds.

Publications

Design of e-pharmacophore models using compound fragments for the trans-sialidase of Trypanosoma cruzi: screening for novel inhibitor scaffolds.

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