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James H. Mckerrow - One of the best experts on this subject based on the ideXlab platform.
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identification of Cysteine Protease inhibitors as new drug leads against naegleria fowleri
Experimental Parasitology, 2018Co-Authors: Ingrid Zyserman, James H. Mckerrow, William R. Roush, Deboprosad Mondal, Francisco Sarabia, Anjan DebnathAbstract:Abstract Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3–7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that Cysteine Proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone Cysteine Protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against Cysteine Protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the Cysteine Protease activity of N. fowleri cell lysate with IC50 ranging between 3 nM and 6.6 μM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence of the activity of synthetic, small molecule Cysteine Protease inhibitors against N. fowleri.
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Synthesis and Evaluation of Oxyguanidine Analogues of the Cysteine Protease Inhibitor WRR-483 against Cruzain.
ACS Medicinal Chemistry Letters, 2015Co-Authors: Brian D. Jones, Anna Tochowicz, Yinyan Tang, Michael D. Cameron, Laura-isobel Mccall, Ken Hirata, Jair L. Siqueira-neto, Sharon L. Reed, James H. Mckerrow, William R. RoushAbstract:A series of oxyguanidine analogues of the Cysteine Protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major Cysteine Protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the Cysteine Protease, depending on the aryl moiety of the P1′ inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a Cysteine Protease by a vinyl sulfone inhibitor.
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Synthesis of macrocyclic trypanosomal Cysteine Protease inhibitors
Bioorganic & medicinal chemistry letters, 2008Co-Authors: Yen Ting Chen, James H. Mckerrow, Ricardo Lira, Elizabeth Hansell, William R. RoushAbstract:The importance of Cysteine Proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes Proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major Cysteine Protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related Cysteine Protease, rhodesain.
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Identification of the Major Cysteine Protease of Giardia and Its Role in Encystation
The Journal of biological chemistry, 2008Co-Authors: Kelly N. Dubois, James H. Mckerrow, Marla Abodeely, Judy A. Sakanari, Charles S. Craik, Malinda Lee, Mohammed SajidAbstract:Giardia lamblia is a protozoan parasite and the earliest branching clade of eukaryota. The Giardia life cycle alternates between an asexually replicating vegetative form and an infectious cyst form. Encystation and excystation are crucial processes for the survival and transmission of Giardia. Cysteine Proteases in Giardia have been implicated in proteolytic processing events that enable the continuance of the life cycle throughout encystation and excystation. Using quantitative real-time PCR, the expression of twenty-seven clan CA Cysteine Protease genes in the Giardia genome was measured during both vegetative growth and encystation. Giardia Cysteine Protease 2 was the most highly expressed Cysteine Protease during both life cycle stages measured, with a dramatic expression increase during encystation. The mRNA transcript for Giardia Cysteine Protease 2 was 7-fold up-regulated during encystation and was greater than 3-fold higher than any other Giardia Protease gene product. Recombinant Giardia Cysteine Protease 2 was expressed, purified, and biochemically characterized. The activity of the recombinant Cysteine Protease 2 protein was confirmed to be identical to the dominant Cysteine Protease activity found in G. lamblia lysates. Giardia Cysteine Protease 2 was co-localized with cyst wall protein in encystation-specific vesicles during encystation and processed cyst wall protein 2 to the size found in Giardia cyst walls. These data suggest that Giardia Cysteine Protease 2 is not only the major Cysteine endoProtease expressed in Giardia, but is also central to the encystation process.
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Cysteine Protease inhibitors as chemotherapy: Lessons from a parasite target
Proceedings of the National Academy of Sciences, 1999Co-Authors: Paul M. Selzer, Matthew Bogyo, Juan C. Engel, Judy A. Sakanari, Ivy Hsieh, Sabine Pingel, Bernhard Ugele, Victor J. Chan, David G. Russell, James H. MckerrowAbstract:Papain family Cysteine Proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of Cysteine Protease inhibitors in cell culture or in vivo. We now report that specific Cysteine Protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania Cysteine Protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-Protease antibodies with biotinylated surface proteins and accumulation of undigested debris and Protease in the flagellar pocket of treated parasites were consistent with a pathway of Protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.
Philip J. Rosenthal - One of the best experts on this subject based on the ideXlab platform.
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Falstatin, a Cysteine Protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion.
PLoS Pathogens, 2006Co-Authors: Kailash C. Pandey, Naresh Singh, Shirin Arastu-kapur, Matthew Bogyo, Philip J. RosenthalAbstract:Erythrocytic malaria parasites utilize Proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control Protease activity have not been established. We report here the identification of an endogenous Cysteine Protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi Cysteine Protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum Cysteine Proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived Cysteine Proteases, but it was a relatively weak inhibitor of the P. falciparum Cysteine Proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the Cysteine Protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite Cysteine Proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.
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Antimalarial Activities of Novel Synthetic Cysteine Protease Inhibitors
Antimicrobial Agents and Chemotherapy, 2003Co-Authors: Belinda J. Lee, Ajay Singh, Peggy Chiang, Scott J. Kemp, Erick A. Goldman, Michael I. Weinhouse, George P Vlasuk, Philip J. RosenthalAbstract:Among promising new targets for antimalarial chemotherapy are the Cysteine Protease hemoglobinases falcipain-2 and falcipain-3. We evaluated the activities of synthetic peptidyl aldehyde and α-ketoamide Cysteine Protease inhibitors against these Proteases, against cultured Plasmodium falciparum parasites, and in a murine malaria model. Optimized compounds inhibited falcipain-2 and falcipain-3, blocked hemoglobin hydrolysis, and prevented the development of P. falciparum at nanomolar concentrations. The compounds were equally active against multiple strains of P. falciparum with varied sensitivities to standard antimalarial agents. The peptidyl inhibitors were consistently less active against vinckepain-2, the putative falcipain-2 and falcipain-3 ortholog of the rodent malaria parasite Plasmodium vinckei. The lead compound morpholinocarbonyl-leucine-homophenylalanine aldehyde, which blocked P. falciparum development at low nanomolar concentrations, was tested in a murine P. vinckei model. When infused continuously at a rate of 30 mg/kg of body weight/day, the compound delayed the progression of malaria but did not eradicate infections. Our data demonstrate the potent antimalarial activities of novel Cysteine Protease inhibitors. Additionally, they highlight the importance of consideration of the specific enzyme targets of animal model parasites. In the case of falcipains, differences between P. falciparum and rodent parasites complicate the use of the rodent malaria model in the drug discovery process.
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Stage-specific antimalarial activity of Cysteine Protease inhibitors.
Biological Chemistry, 2002Co-Authors: Bhaskar R. Shenai, Andrey Semenov, Philip J. RosenthalAbstract:Cysteine Proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of Cysteine Proteases and sensitivity to Cysteine Protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine Protease activity was greatest in early trophozoites, while sensitivity to Cysteine Protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of Cysteine Protease inhibitors is due to a block in hemoglobin hydrolysis.
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Comparison of Efficacies of Cysteine Protease Inhibitors against Five Strains of Plasmodium falciparum
Antimicrobial Agents and Chemotherapy, 2001Co-Authors: Ajay Singh, Philip J. RosenthalAbstract:Falcipain-2, a Cysteine Protease and essential hemoglobinase of Plasmodium falciparum, is a potential antimalarial drug target. We compared the falcipain-2 sequences and sensitivities to Cysteine Protease inhibitors of five parasite strains that differ markedly in sensitivity to established antimalarial drugs. The sequence of falcipain-2 was highly conserved, and the sensitivities of all of the strains to falcipain-2 inhibitors were very similar. Thus, cross-resistance between Cysteine Protease inhibitors and other antimalarial agents is not expected in parasites that are now circulating and falcipain-2 remains a promising chemotherapeutic target.
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characterization of native and recombinant falcipain 2 a principal trophozoite Cysteine Protease and essential hemoglobinase of plasmodium falciparum
Journal of Biological Chemistry, 2000Co-Authors: Bhaskar R. Shenai, Ajay Singh, Puran Singh Sijwali, Philip J. RosenthalAbstract:Abstract Trophozoites of the malaria parasitePlasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine Protease inhibitors block hemoglobin degradation, indicating that a Cysteine Protease plays a key role in this process. A principal trophozoite Cysteine Protease was purified by affinity chromatography. Sequence analysis indicated that the Protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble Cysteine Protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the Protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this Protease is appropriate.
Jonathan A Ellman - One of the best experts on this subject based on the ideXlab platform.
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general solid phase method to prepare novel cyclic ketone inhibitors of the Cysteine Protease cruzain
Bioorganic & Medicinal Chemistry Letters, 2002Co-Authors: Lily Huang, Jonathan A EllmanAbstract:A series of constrained ketone-based inhibitors has been developed that show low nanomolar Ki values. These ketone inhibitors showed promising activity towards cruzain, the Cysteine Protease implicated in Chagas' disease. This series of constrained inhibitors, which can be accessed quickly and efficiently using a solid-phase combinatorial strategy, should be applicable to other members of the Cysteine Protease class.
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Parallel solution-phase synthesis of mechanism-based Cysteine Protease inhibitors.
Organic letters, 2001Co-Authors: Alice Lee, Jonathan A EllmanAbstract:A seven-step parallel solution-phase synthesis has been developed for access to ketone-containing mechanism-based Cysteine Protease inhibitors. The use of liquid−liquid extractions, volatile or solid-supported reagents, and resin-bound scavengers eliminates the need for intermediate column chromatographic purification during this synthesis sequence.
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General Solid-Phase Method for the Preparation of Mechanism-Based Cysteine Protease Inhibitors
Journal of the American Chemical Society, 1999Co-Authors: Alice Lee, Lily Jun Shen Huang, Jonathan A EllmanAbstract:The first general method has been developed for the expedient solid-phase synthesis of ketone-based Cysteine Protease inhibitors. The synthesis approach was designed to allow the introduction of diverse functionality at all variable sites about the ketone carbonyl using readily available precursors. The chloromethyl ketone scaffold 7 is attached to the solid support through the newly developed hydrazine linker 6. Successful nucleophilic displacement of the support-bound α-chloro hydrazones 8 with carboxylates, thiolates, and amines provides entry to the acyloxymethyl, mercaptomethyl, and amidomethyl ketone classes of Cysteine Protease inhibitors. Further transformations followed by cleavage from support provides the fully substituted ketone products in 40−100% overall yields after release from support. Significantly, racemization of the α-stereocenter does not occur during loading onto support, nucleophilic displacement, or cleavage from support.
William R. Roush - One of the best experts on this subject based on the ideXlab platform.
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identification of Cysteine Protease inhibitors as new drug leads against naegleria fowleri
Experimental Parasitology, 2018Co-Authors: Ingrid Zyserman, James H. Mckerrow, William R. Roush, Deboprosad Mondal, Francisco Sarabia, Anjan DebnathAbstract:Abstract Primary amebic meningoencephalitis (PAM) is a rapidly fatal infection caused by the free-living ameba Naegleria fowleri. PAM occurs principally in healthy children of less than 13 years old with a history of recent exposure to warm fresh water. While as yet not a reportable disease, the Centers for Disease Control and Prevention (CDC) documents a total of 143 cases in the United States. Only four patients have survived. Infection results from water containing N. fowleri entering the nose, followed by migration of the amebae to the brain. Within the brain, N. fowleri infection results in extensive necrosis, leading to death in 3–7 days. Mortality among patients with PAM is greater than 95%. The drugs of choice in treating PAM are the antifungal amphotericin B, and the antileishmanial, miltefosine. However neither drug is FDA-approved for this indication and the use of amphotericin B is associated with severe adverse effects. Moreover, very few patients treated with amphotericin B have survived PAM. Therefore, development of new, safe and effective drugs is a critical unmet need to avert future deaths of children. The molecular mechanisms underlying the pathogenesis of PAM are poorly understood but it is known that Cysteine Proteases of N. fowleri play a role in the progression of PAM. We therefore assessed the in vitro activity of the synthetic vinyl sulfone Cysteine Protease inhibitor, K11777, and 33 analogs with valine, phenylalanine or pyridylalanine at P2 position, against Cysteine Protease activity in the lysate of N. fowleri. Inhibitors with phenylalanine or pyridylalanine at P2 position were particularly effective in inhibiting the Cysteine Protease activity of N. fowleri cell lysate with IC50 ranging between 3 nM and 6.6 μM. Three of the 34 inhibitors also showed inhibitory activity against N. fowleri in a cell viability assay and were 1.6- to 2.5-fold more potent than the standard of care drug miltefosine. Our study provides the first evidence of the activity of synthetic, small molecule Cysteine Protease inhibitors against N. fowleri.
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Synthesis and Evaluation of Oxyguanidine Analogues of the Cysteine Protease Inhibitor WRR-483 against Cruzain.
ACS Medicinal Chemistry Letters, 2015Co-Authors: Brian D. Jones, Anna Tochowicz, Yinyan Tang, Michael D. Cameron, Laura-isobel Mccall, Ken Hirata, Jair L. Siqueira-neto, Sharon L. Reed, James H. Mckerrow, William R. RoushAbstract:A series of oxyguanidine analogues of the Cysteine Protease inhibitor WRR-483 were synthesized and evaluated against cruzain, the major Cysteine Protease of the protozoan parasite Trypanosoma cruzi. Kinetic analyses of these analogues indicated that they have comparable potency to previously prepared vinyl sulfone cruzain inhibitors. Co-crystal structures of the oxyguanidine analogues WRR-666 (4) and WRR-669 (7) bound to cruzain demonstrated different binding interactions with the Cysteine Protease, depending on the aryl moiety of the P1′ inhibitor subunit. Specifically, these data demonstrate that WRR-669 is bound noncovalently in the crystal structure. This represents a rare example of noncovalent inhibition of a Cysteine Protease by a vinyl sulfone inhibitor.
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Synthesis of macrocyclic trypanosomal Cysteine Protease inhibitors
Bioorganic & medicinal chemistry letters, 2008Co-Authors: Yen Ting Chen, James H. Mckerrow, Ricardo Lira, Elizabeth Hansell, William R. RoushAbstract:The importance of Cysteine Proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes Proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major Cysteine Protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related Cysteine Protease, rhodesain.
Matthew Bogyo - One of the best experts on this subject based on the ideXlab platform.
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Falstatin, a Cysteine Protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion.
PLoS Pathogens, 2006Co-Authors: Kailash C. Pandey, Naresh Singh, Shirin Arastu-kapur, Matthew Bogyo, Philip J. RosenthalAbstract:Erythrocytic malaria parasites utilize Proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control Protease activity have not been established. We report here the identification of an endogenous Cysteine Protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi Cysteine Protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum Cysteine Proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived Cysteine Proteases, but it was a relatively weak inhibitor of the P. falciparum Cysteine Proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the Cysteine Protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite Cysteine Proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.
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Activity-based probes that target diverse Cysteine Protease families
Nature Chemical Biology, 2005Co-Authors: Daisuke Kato, Kelly M Boatright, Alicia B Berger, Tamim Nazif, Galia Blum, Ciara Ryan, Kareem A H Chehade, Guy S Salvesen, Matthew BogyoAbstract:Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of Protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of Protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of Cysteine Protease families. These reagents selectively form covalent bonds with the active-site thiol of a Cysteine Protease, allowing direct biochemical profiling of Protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these Protease families.
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Cysteine Protease inhibitors as chemotherapy: Lessons from a parasite target
Proceedings of the National Academy of Sciences, 1999Co-Authors: Paul M. Selzer, Matthew Bogyo, Juan C. Engel, Judy A. Sakanari, Ivy Hsieh, Sabine Pingel, Bernhard Ugele, Victor J. Chan, David G. Russell, James H. MckerrowAbstract:Papain family Cysteine Proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of Cysteine Protease inhibitors in cell culture or in vivo. We now report that specific Cysteine Protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania Cysteine Protease activity was accompanied by defects in the parasite’s lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-Protease antibodies with biotinylated surface proteins and accumulation of undigested debris and Protease in the flagellar pocket of treated parasites were consistent with a pathway of Protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.
