Luca Salvati

Inhibition of Cysteine Protease Activity by NO-donors

Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

Cysteine protease as a target for nitric oxide in parasitic organisms

Nitric oxide (NO) is an important cytotoxic and cytostatic mediator for several parasites, including intracellular (e.g. Trypanosoma, Leishmania, Plasmodium, Toxoplasma) and extracellular (e.g. Entamoeba) protozoa and the helminth Schistosoma 1xNitric oxide and parasitic disease. Clark, I.A. and Rockett, K.A. Adv. Parasitol. 1996; 37: 1–56Crossref | PubMedSee all References1. Increasing evidence suggests that parasitic cysteine proteases could represent NO targets, providing molecular bases for the parasiticidal effect of NO (2xNitric oxide inhibits falcipain, the Plasmodium falciparum trophozoite cysteine protease. Venturini, G. et al. Biochem. Biophys. Res. Commun. 2000; 267: 190–193Crossref | PubMed | Scopus (34)See all References, 3xNitric oxide inhibits cruzipain, the major papain-like cysteine protease from Trypanosoma cruzi. Venturini, G. et al. Biochem. Biophys. Res. Commun. 2000; 270: 437–441Crossref | PubMed | Scopus (47)See all References, 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References, 5xNO donors inhibit Leishmania infantum cysteine proteinase activity. Salvati, L. et al. Biochim. Biophys. Acta. 2001; 1545: 357–366Crossref | PubMed | Scopus (49)See all References). NO-donors (e.g. S-nitroso-acetyl-penicillamine, SNAP) inhibit the catalytic activity of cruzipain, falcipain and Leishmania infantum cysteine protease in vitro. L. infantum cysteine protease is inhibited following incubation of promastigotes with SNAP, which leads to parasite killing. Predictably, NO-deprived NO-donors (e.g. N-acetyl-penicillamine) affect neither the enzyme action nor the parasite viability. Moreover, reducing agents (e.g. dithiothreitol) prevent inhibition of parasite cysteine protease and restore enzyme activity 5xNO donors inhibit Leishmania infantum cysteine proteinase activity. Salvati, L. et al. Biochim. Biophys. Acta. 2001; 1545: 357–366Crossref | PubMed | Scopus (49)See all References5.The prevalence and higher reactivity of thiols over other nucleophiles account for the propensity of S-nitrosylation and mixed disulfide bridge formation. The NO-mediated chemical modification(s) of the Cys catalytic residue in cysteine proteases is assisted by neighboring basic and acid amino-acid residues (e.g. the invariant His residue forming the Cys–His catalytic dyad). In particular, S-nitrosylation of the Cys25 catalytic residue of cruzipain is facilitated by His159, which stabilizes the reactive deprotonated form of the Cys25 S atom 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4 (Fig. 1Fig. 1).Fig. 1The three-dimensional structure of cruzipain. The cysteine-25 catalytic residue, undergoing the nitric-oxide-mediated S-nitrosylation, in addition to the histidine-159 residue, facilitating the Cys25 chemical modification, are shown in ball-and-stick.View Large Image | Download PowerPoint SlideThe NO-mediated chemical modification(s) of cysteine proteases depends on the chemical properties of the NO-donor, in addition to the presence of NO scavengers (e.g. albumin and hemoglobin), reducing agents (e.g. reduced glutathione, GSH), oxidants (e.g. oxygen and related reactive species) and metals. Hemoglobin could also act as a NO carrier because NO release is apparently facilitated by oxygen release in the venous capillaries 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4. Plasmodium falciparum in blood cultures with low-oxygen tensions similar to those found in the venous capillaries is extremely susceptible to NO (Ref. 6xSensitivity of malaria parasites to nitric oxide at low oxygen tensions. Taylor-Robinson, A.W. and Looker, M. Lancet. 1998; 351: 1630Abstract | Full Text | Full Text PDF | PubMed | Scopus (31)See all ReferencesRef. 6). Flavo-hemoglobin, truncated-hemoglobin and myoglobin could detoxify NO pseudo-enzymatically. The NO-mediated chemical modification(s) of endogenous reducing agents could be induced by exogenous and endogenous NO(-donors). At the physiological GSH concentration, S-nitroso-glutathione (GSNO) formation represents the significant metabolic fate of N2O3. Most of N2O3, obtained from the reaction between O2 with NO is consumed by GSH-nitrosylation. Then, GSNO oxidizes protein thiol(s) by NO-transfer reaction. Alternatively, a direct nucleophilic attack of protein thiol(s) on GSNO, which does not require nitrosothiol cleavage, has been suggested as a potential mechanism for GSNO-mediated protein thiolation. However, reducing agents might revert in part the NO-induced oxidation of Cys residues 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References4. Interestingly, trypanosomes containing low levels of endogenous reducing agents (e.g. trypanothione and GSH) display an increased sensitivity to oxidative stress 7xTrypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Krieger, S. et al. Mol. Microbiol. 2000; 35: 542–552Crossref | PubMed | Scopus (227)See all References7, indicating that GSH is essential for protection against NO cytotoxicity in macrophages and parasites (e.g. Leishmania) 8xGlutathione protects macrophages and Leishmania major against nitric oxide-mediated cytotoxicity. Romao, P.R. et al. Parasitology. 1999; 118: 559–566Crossref | PubMed | Scopus (26)See all References8.Although NO-mediated chemical modification(s) of cysteine proteases accounts for the loss of enzyme activity and the parasiticidal effect of NO, the binding of NO to other parasite molecular targets should be taken into account. In particular, ribonucleotide reductase inhibition has been suggested to explain the cytostatic effect of NO on Trypanosoma brucei gambiense and Trypanosoma brucei brucei. Moreover, the NO-mediated chemical modification(s) of host cysteine-containing proteins could also influence parasite survival 1xNitric oxide and parasitic disease. Clark, I.A. and Rockett, K.A. Adv. Parasitol. 1996; 37: 1–56Crossref | PubMedSee all References, 4xInhibition of cysteine protease activity by NO-donors. Ascenzi, P. et al. Curr. Protein Peptide Sci. 2001; 2: 137–153Crossref | PubMed | Scopus (51)See all References.Considering that cysteine proteases appear as promising targets for anti-parasite chemotherapy 9xDevelopment of cysteine protease inhibitors as chemotherapy for parasitic diseases: insights on safety, target validation, and mechanism of action. McKerrow, J.H. Int. J. Parasitol. 1999; 29: 833–837Crossref | PubMed | Scopus (115)See all References9, NO-releasing drugs could have an enhancing role in the therapeutic treatment of parasitic diseases 10xA topical nitric oxide-generating therapy for cutaneous leishmaniasis. Davidson, R.N. et al. Trans. Roy. Soc. Trop. Med. Hyg. 2000; 94: 319–322PubMedSee all References10.

Inhibition of Azotobacter vinelandii rhodanese by NO-donors.

Nitric oxide (NO) is a versatile regulatory molecule that affects enzymatic activity through chemical modification of reactive amino acid residues (e.g., Cys and Tyr) and by binding to metal centers. In the present study, the inhibitory effect of the NO-donors S-nitroso-glutathione (GSNO), (+/-)E-4-ethyl-2-[E-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), and S-nitroso-N-acetyl-penicillamine (SNAP) on the catalytic activity of Azotobacter vinelandii rhodanese (RhdA) has been investigated. GSNO, NOR-3, and SNAP inhibit RhdA sulfurtransferase activity in a concentration- and time-dependent fashion. The absorption spectrum of the NOR-3-treated RhdA displays a maximum at 335 nm, indicating NO-mediated S-nitrosylation. RhdA inhibition by NO-donors correlates with S-nitrosothiol formation. The reducing agent dithiothreitol prevents RhdA inhibition by NO-donors, fully restores the catalytic activity, and reverts the NOR-3-induced RhdA absorption spectrum to that of the active enzyme. These results indicate that RhdA inhibition occurs via NO-mediated S-nitrosylation of the unique Cys230 catalytic residue.

Does inhibition of Trypanosoma cruzi key enzymes affect parasite life cycle and geographic distribution

The hemoflagellate protozoan parasite Trypanosoma cruzi is the causative agent of the Chagas disease, a progressive fatal cardiomyopathy that afflicts more than 20 million people in Central and South America. The T. cruzi life cycle is affected by changes in temperature and pH, the parasite replication being facilitated in mammalian hosts rather than in insect vectors. Here, we postulate that the modulation of key enzymes by pH‐ and temperature‐dependent substrate inhibition may affect the T. cruzi life cycle and limit the geographic range covered by the parasite.

Inhibition of Saccharomyces cerevisiae phosphomannose isomerase by the NO-donor S-nitroso-acetyl-penicillamine.

Phosphomannose isomerase (PMI; EC. 5.3.1.8) is an essential metalloenzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. The Cysl50 residue (according to Candida albicans PMI numbering) is conserved in the active centre of mammalian and yeast PMI, but not in bacterial species where it is replaced by Asn. Here, the dose- and time-dependent inhibitory effect of the NO-donor S-nitroso-acetyl-penicillamine on the Saccharomyces cerevisiae PMI catalytic activity is reported. The analysis of the X-ray crystal structure of C. albicans PMI and of the molecular model of S. cerevisiae PMI provides a rationale for the low reactivity of Cysl50 towards alkylating and nitrosylating agents.