Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi

Harth, Günter; Andrews, Norma W.; Mills, Alea A.; Engel, Juan C.; Smith, Robert E.; McKerrow, James H.

doi:10.1016/0166-6851(93)90086-d

Cited by 158 publications

(127 citation statements)

References 18 publications

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“…The process, therefore, requires fast and extensive protein degradation and recycling of building blocks for the synthesis of new macromolecules. Initial studies suggested that cruzipain, the most abundant lysosomal/reservosomal cysteine peptidase of T. cruzi belonging to the CA clan (21) of proteases, is critical for the differentiation as the cell-permeable cruzipain inhibitors effectively inhibited the process (19,51), whereas overexpression of this enzyme is associated with enhanced metacyclogenesis (52). Our results clearly demonstrate that autophagy is also involved in cell remodeling most probably by delivering large parts of the cytoplasm and organelles to lysosomes/reservosomes.…”

Section: Discussionmentioning

confidence: 51%

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

Álvarez

Kosec

Sant’Anna

et al. 2008

Journal of Biological Chemistry

138

173

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Autophagy is the major mechanism used by eukaryotic cells to degrade and recycle proteins and organelles. Bioinformatics analysis of the genome of the protozoan parasite Trypanosoma cruzi revealed the presence of all components of the Atg8 conjugation system, whereas Atg12, Atg5, and Atg10 as the major components of the Atg12 pathway could not be identified. The two TcATG4 (autophagin) homologs present in the genome were found to correctly process the two ATG8 homologs after the conserved Gly residue. Functional studies revealed that both ATG4 homologues but only one T. cruzi ATG8 homolog (TcATG8.1) complemented yeast deletion strains. During starvation of the parasite, TcAtg8.1, but not TcAtg8.2, was found by immunofluorescence to be located in autophagosome-like vesicles. This confirms its function as an Atg8/LC3 homolog and its potential to be used as an autophagosomal marker. Most importantly, autophagy is involved in differentiation between developmental stages of T. cruzi, a process that is essential for parasite maintenance and survival. These findings suggest that the autophagy pathway could represent a target for a novel chemotherapeutic strategy against Chagas disease.

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Section: Discussionmentioning

confidence: 51%

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

Álvarez

Kosec

Sant’Anna

et al. 2008

Journal of Biological Chemistry

138

173

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“…[11][12][13][14] Addition of a cruzain inhibitor to cultures of mammalian cells exposed to trypomastigotes or to mammalian cells already infected with T. cruzi amastigotes blocks replication and differentiation of the parasite, thus interrupting the parasite life cycle. [15][16][17][18][19][20][21] Several groups have demonstrated that irreversible inhibition of cruzain by small molecules eradicates infection of the parasite in cell culture and animal models. 17,[22][23][24][25] Irreversible inhibitors that contain an electrophilic functional group, such as vinyl sulfones, fluoro methyl ketones, aziridines or nitriles, covalently bind to cruzain via nucleophilic attack of the active site cysteine, 26 showing good inhibition activity.…”

Section: Introductionmentioning

confidence: 99%

“…Inhibition of cruzain activity with fluoromethyl ketone-based inhibitors seems to be correlated with the loss of feasibility of parasites in both ex-vivo tissue culture and in vivo mouse models. 19,32,33 Information derived from the study of the molecular mechanism of hydrolysis catalyzed by cysteine proteases could be used as starting point to explore the inhibition mechanism at atomistic level. Early studies revealed a participation of the residues Cys25 and His159 (cruzain numbering) from the active site of theses proteases.…”

Section: Introductionmentioning

confidence: 99%

First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones

2015

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Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics−molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen−carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.

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“…Knowledge of the Cz crystal structure has promoted the design of a variety of cysteine peptidase inhibitors including peptidyl and non-peptidyl inhibitors. Among peptidyl inhibitors, the irreversible inhibitor, N-acylhydrazone has been studied (Ifa, 2000;dos Santos Filho, 2009), as well as the halomethyl ketone based (Ashall, 1990;Harth, 1993), diazomethane ketones (Shaw, 1994;Lalmanach, 1996), vinyl sulfones (Roush, 2001;Engel, 1998;Barr, 2005;Jacobsen, 2000), and reversible inhibitors such as oxadiazoles (Ferreira,2009), and aryl ureas (Du, 2000). In the non-peptidyl group thiosemicarbazones (Du, 2002), triazole, triazine nitriles (Brak, 2010;Mott, 2010) and non-peptidic vinylsulfones ) can be found some of which are presented in Table 4.…”

Section: Peptidasesmentioning

confidence: 99%

Novel Therapeutic Strategies for Chagas` Disease

Vermelho¹,

Fraga²,

Carvalho³

et al. 2011

Drug Development - A Case Study Based Insight Into Modern Strategies

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Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of Trypanosoma cruzi

Cited by 158 publications

References 18 publications

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

Autophagy Is Involved in Nutritional Stress Response and Differentiation in Trypanosoma cruzi

First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones

Novel Therapeutic Strategies for Chagas` Disease

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