Cysteine Protease Inhibitors Cure an Experimental <i>Trypanosoma cruzi</i> Infection

Engel, Juan C.; Doyle, Patricia S.; Hsieh, Ivy; McKerrow, James H.

doi:10.1084/jem.188.4.725

Cited by 372 publications

(369 citation statements)

References 30 publications

(41 reference statements)

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“…Enhanced NO generation resulted upon incubating cystatin with IFN-␥-activated mouse peritoneal macrophages in vitro, and the leishmanicidal activity acquired correlated with the induction of NO production. It may be mentioned that Engel et al (30) showed a parasiticidal effect of synthetic cysteine protease inhibitors on intracellular Trypanosoma cruzi by inactivation of cruzain, a major protease of the parasite. The reason cystatin may induce an increase in NO synthesis from activated macrophages remains unclear.…”

Section: Discussionmentioning

confidence: 99%

Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response

Das

Datta

Bandyopadhyay

et al. 2001

The Journal of Immunology

109

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The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4+ T cell differentiation. A 6-fold increase in NO production was observed with 0.5 μM chicken cystatin, a natural cysteine protease inhibitor, in IFN-γ-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-γ. In contrast to the case with promastigotes, cystatin and IFN-γ inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-γ or anti-TNF-α Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-γ treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4+ differentiation from Th2 to Th1, indicative of long-term resistance.

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Section: Discussionmentioning

confidence: 99%

Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response

Das

Datta

Bandyopadhyay

et al. 2001

The Journal of Immunology

109

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“…4,5,16,17 Vinyl sulfone inhibitor 38 was prepared via a Horner-Wadsworth-Emmons olefination (Scheme 3). Kinetic analysis of the vinyl sulfone inhibitor, surprisingly, indicated no time dependence and was consistent with competitive reversible inhibition (Figure 4a).…”

Section: Conversion Of Substrates Into Inhibitorsmentioning

confidence: 99%

Identification of a New Class of Nonpeptidic Inhibitors of Cruzain

et al. 2008

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Cruzain is the major cysteine protease of T. cruzi, which is the causative agent of Chagas' disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the Substrate Activity Screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored β-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl-and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group pK a , with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second order inactivation constant of 147,000 s −1 M −1 . This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas' disease.

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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%

“…[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. 27,28 In fact, to date, only irreversible inhibitors of cruzain have successfully cured parasitic infection, 23 implying that tight binding to the enzyme may be essential.…”

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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Cysteine Protease Inhibitors Cure an Experimental Trypanosoma cruzi Infection

Cited by 372 publications

References 30 publications

Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response

Successful Therapy of Lethal Murine Visceral Leishmaniasis with Cystatin Involves Up-Regulation of Nitric Oxide and a Favorable T Cell Response

Identification of a New Class of Nonpeptidic Inhibitors of Cruzain

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

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