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. 2013 Dec;57(12):6063-73.
doi: 10.1128/AAC.00734-13. Epub 2013 Sep 23.

A Cysteine Protease Inhibitor Rescues Mice From a Lethal Cryptosporidium Parvum Infection

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Free PMC article

A Cysteine Protease Inhibitor Rescues Mice From a Lethal Cryptosporidium Parvum Infection

Momar Ndao et al. Antimicrob Agents Chemother. .
Free PMC article

Abstract

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, can stunt infant growth and can be lethal in immunocompromised individuals. The most widely used drugs for treating cryptosporidiosis are nitazoxanide and paromomycin, although both exhibit limited efficacy. To investigate an alternative approach to therapy, we demonstrate that the clan CA cysteine protease inhibitor N-methyl piperazine-Phe-homoPhe-vinylsulfone phenyl (K11777) inhibits C. parvum growth in mammalian cell lines in a concentration-dependent manner. Further, using the C57BL/6 gamma interferon receptor knockout (IFN-γR-KO) mouse model, which is highly susceptible to C. parvum, oral or intraperitoneal treatment with K11777 for 10 days rescued mice from otherwise lethal infections. Histologic examination of untreated mice showed intestinal inflammation, villous blunting, and abundant intracellular parasite stages. In contrast, K11777-treated mice (210 mg/kg of body weight/day) showed only minimal inflammation and no epithelial changes. Three putative protease targets (termed cryptopains 1 to 3, or CpaCATL-1, -2, and -3) were identified in the C. parvum genome, but only two are transcribed in infected mammals. A homology model predicted that K11777 would bind to cryptopain 1. Recombinant enzymatically active cryptopain 1 was successfully targeted by K11777 in a competition assay with a labeled active-site-directed probe. K11777 exhibited no toxicity in vitro and in vivo, and surviving animals remained free of parasites 3 weeks after treatment. The discovery that a cysteine protease inhibitor provides potent anticryptosporidial activity in an animal model of infection encourages the investigation and development of this biocide class as a new, and urgently needed, chemotherapy for cryptosporidiosis.

Figures

Fig 1
Fig 1
K11777 eliminates oocyst shedding from C. parvum-infected mice. Animals (8 mice/group) were infected with 1,500 sporulated oocysts and 4 days later were treated with K11777 BID for 10 days p.o. (A) or i.p. (B). The mice were euthanized on day 35 after infection. (A) P.o. treatment at 35 mg/kg (●), 70 mg/kg (■), and 105 mg/kg (▲) and BID treatment with paromomycin p.o. at 125 mg/kg (◆). Infected controls were mock treated (+) and treated with 0.5% DMSO (asterisks). (B) I.p. treatment at 35 mg/kg (▼), 52.5 mg/kg (◆), and 70 mg/kg (○). Infected controls were mock treated (+) and treated with 0.5% DMSO (×). The results are expressed as the mean number of oocysts shed per group ± standard deviations (SD); n = 3 independent experiments. The number of oocysts was determined by flow cytometry.
Fig 2
Fig 2
K11777 rescues mice from otherwise lethal infections by C. parvum. Animals (8 mice/group) were infected with 1,500 sporulated oocysts and 4 days later were treated with K11777 BID for 10 days p.o. (A) or i.p. (B). The mice were euthanized on day 35 after infection. (A) P.o. treatment at 35 mg/kg (●), 70 mg/kg (■), and 105 mg/kg (▲) and BID treatment with paromomycin p.o. at 125 mg/kg (◆). Infected controls were mock treated (+) and treated with 0.5% DMSO (asterisks). Uninfected controls were mock treated (□) and treated with 0.5% DMSO p.o. (▽). (B) I.p. treatment at 35 mg/kg (▼), 52.5 mg/kg (◆), and 70 mg/kg (○). Infected controls were mock treated (+) and treated with 0.5% DMSO i.p. (×). Uninfected controls were mock treated (□) and treated with 0.5% DMSO (▽).
Fig 3
Fig 3
K11777 treatment resolves intestinal pathology due to C. parvum infection. (A and B) Small intestines from C. parvum-infected mice either untreated (A) or treated p.o. with 105 mg/kg K11777 BID as described for Fig. 1 and 2 (B). (C) Small intestine from uninfected mice that were treated p.o. with 105 mg/kg K11777 BID. (D) Small intestine from C. parvum-infected mice that were treated p.o. with 125 mg/kg paromomycin BID. The boxed areas are shown enlarged below (A to C) and to the right (D). Note the marked acute inflammation and abundance of C. parvum (yellow arrows) in panel A. Small intestines were harvested at 14 days (A) and 35 days (B, C, and D) after infection. Transverse sections of the small intestine were stained with hematoxylin and eosin. Magnification, ×400. Scale bars = 24 μm.
Fig 4
Fig 4
Cryptopains 1 and 2, but not 3, are transcribed in C. parvum. RNA preparations were DNase treated and reverse transcribed, and the cDNA was subjected to PCR using specific cryptopain primers. Arrows indicate cryptopain.
Fig 5
Fig 5
K11777 reacts with the active site of cryptopain 1. Lane 1, recombinant cryptopain 1, expressed in P. pastoris, labeled with the active-site-directed probe 125I-DCG-04 (43), and then subjected to SDS-PAGE and autoradiography; lane 2, same as lane 1 except that recombinant cryptopain was preincubated with 5 μM K11777, which subsequently prevented the reaction of the enzyme with the affinity probe.
Fig 6
Fig 6
(A) Redocking of K11777 to cruzain crystal structure (PDB ID 2OZ2). (B) Docking of K11777 to crypto1M model. The docking results are shown as gray carbon, and crystal structure pose is shown as orange carbon. Nitrogen, oxygen, hydrogen, and sulfur atoms are shown in blue, red, white, and yellow, and binding-pocket residues are shown in a surface representation with the same color scheme.
Fig 7
Fig 7
MSA of family C01 peptidases from C. parvum showing active-site residue regions. All five C01 C. parvum sequences, as well as PDB IDs for cruzain (2OZ2) and human cathepsin L (1FH0), were used as input into PROMALS3D (see Materials and Methods), which incorporates predicted secondary structure and available 3D structure in calculating the MSA. The active-site residues (Q, C, H, and N, in blue boxes) are strictly conserved, but the S2 pocket Glu in cryptopain 1 is in a variable region and appears to be conserved only in cruzain (pink box). Cathepsin L-like sequences (cryptopains) are cgd6 (cryptopain 1), cgd3 (cryptopain 2), and cgd7 (cryptopain 3); cathepsin C-like sequences are cgd2 and cgd4. Higher conservation is shown by higher numbers above the alignments (Conservation). Strictly conserved amino acids are shown as boldface uppercase letters (Consensus_aa). “Consensus_ss” indicates where conserved secondary structure is predicted: e, beta strand (blue letters); h, alpha helix (red letters).

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