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Antimicrobial Agents and Chemotherapy, August 2006, p. 2886-2888, Vol. 50, No. 8
0066-4804/06/$08.00+0     doi:10.1128/AAC.01060-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cysteine Proteinase Inhibitors in Murine Cysticercosis

Salman Baig,1,3 Raymond T. Damian,1 Jorge Morales-Montor,1,2 Rupal Thazhath,1 Amr Ghaleb,1 Meredith Welch,1 Jamil Talhouk,4 and A. Clinton White Jr.3*

Department of Cellular Biology, University of Georgia, Biological Sciences Building, Athens, Georgia 30603,1 Departamento de Inmunología, Instituto de Investigaciones Biomédicas, UNAM AP 70228, Mexico City DF 04510, México,2 Infectious Diseases Section, Department of Medicine, Baylor College of Medicine, 1 Baylor Plaza, 535EA, Houston, Texas 77030,3 Enzyme Systems Products, Livermore, California4

Received 15 August 2005/ Returned for modification 2 September 2005/ Accepted 8 December 2005


   ABSTRACT
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We explored the prophylactic efficacies of two novel protease inhibitors in murine cysticercosis. Our results demonstrated a 95% and 80% reduction in parasite burden for mice injected with Z-LLL-FMK and Z-LLY-FMK, respectively. Further studies are merited on the role of cysteine proteinase inhibitors in treatment of cysticercosis.


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Taenia solium causes neurocysticercosis, a common neurologic infection in the Third World. Although good porcine husbandry and meat inspection have interrupted transmission of T. solium in developed countries, these approaches have been problematic in developing countries (2). Current treatments for neurocysticercosis employ compounds developed for other indications (8, 9), and these treatments remain suboptimal (4). For example, in a recent trial demonstrating the efficacy of albendazole, only 56% of those treated with albendazole were free of seizures and only 38% had radiologic resolution at 6 months of follow-up (5).

Protease inhibitors are key components of human immunodeficiency virus therapy (11). Protease inhibitors have also demonstrated activity in a variety of parasite models. For example, fluoromethylketone (FMK)-derivatized dipeptides have been shown to cure murine malaria (7), vinylsulfone (VS)-derivatized dipeptides work in animal models of leishmaniasis and Chagas' disease (3), and cysteine protease inhibitors are reported to arrest or cure murine schistosomiasis (8). The current study investigated two specifically designed protease inhibitors that were selected for activity against a prominent T. solium cysteine protease (1, 10).

In vivo studies. Protease inhibitors were selected based on their activity against a prominent cyst wall cysteine protease (1, 10). BALB/c mice were divided into the following groups (five mice per group): solvent plus buffer (placebo), untreated, and treated. The treatments included the following compounds: carboxybenzoxy (Z)-LLL-FMK, Z-LLY-FMK, Z-LLL-VS, Z-LF-VS, and Z-FA-FMK. Each mouse in a treated group or placebo group was injected intraperitoneally daily (2.2 µg of inhibitor in 150 µl of dimethyl sulfoxide or solvent control) beginning 2 days prior to infection and continuing for 30 days postchallenge. Each animal was challenged with 10 Taenia crassiceps cysts (in 200 µl phosphate-buffered saline [PBS]). After completing the treatment, the mice were left untreated for an additional 4.5 months to allow the parasites to multiply. Subsequently, the peritoneal cavity was washed and the numbers of cysts were determined. The results were confirmed in a second experiment, with the parasite burden counted at the end of the treatment period. Statistical analysis of variance was performed using Prism 2.01 software (GraphPad Software Inc.), comparing the group means using the Tukey test for the sum of residual and two-factor interactions and an analysis of variance.

Electron microscopy. Cysticerci were removed from the peritoneal cavity, transferred to 3% glutaraldehyde in 0.1 M cacodylate buffer, and fixed for 2 h. After washing, the cysticerci were dehydrated with ethanol (graded concentrations of 30 to 100%), washed in 100% ethanol, and dried in a critical point drier (Samdri) using ethanol as the intermediate fluid and CO2 as the transitional fluid. The dried cysts were then coated with approximately 300-Å gold in a sputter coater (SPI) for 2 min at 2 x 108 kPa/15 mA and observed using a scanning electron microscope (JEOL JSM 5800).

Spleen lymphocyte proliferation and trypan blue exclusion. Splenocytes were suspended in RPMI 1640 containing 10% fetal bovine serum, adjusted to 106 cells/ml, and plated in 96-well culture plates (Costar, California). Then, the cells were stimulated with concanavalin A (10 µg/ml) plus Z-LLY-FMK, Z-LLL-FMK, or both inhibitors (0, 1, 5, 10, 20, and 50 µg/ml) and incubated (54 h, 5% CO2, 37°C). Cells were pulsed with [3H]thymidine (1 µCi/well) and harvested 18 h later. Cell viability was determined by staining with trypan blue (10% [vol/vol]).

In vivo study results. Z-FA-FMK, Z-LF-VS, and Z-LLL-VS demonstrated no or only modest antiparasitic activity. By contrast, the inhibitors most potent in vitro, Z-LLL-FMK and Z-LLY-FMK, greatly reduced parasite numbers (Fig. 1) (Z-LLL-FMK, 95%, and Z-LLY-FMK, 80%; P < 0.05). No obvious toxicity was noted with the FMK inhibitors. However, the Z-LLL-VS group was marked by swollen necks and ruffled coats.


Figure 1
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  		FIG. 1. Prevention of murine cysticercosis with cysteine proteinase inhibitors. BALB/c mice (five mice per group) were injected intraperitoneally daily beginning 2 days prior to infection with inhibitors (1.48 x 10–2 µg/µl dissolved in 150 µl of dimethyl sulfoxide), solute control, or no injection. Subsequently, all groups were challenged intraperitoneally with 10 T. crassiceps cysts in 200 µl of 0.15 M PBS. Mice continued to receive inhibitor daily for 4 weeks. Mice were kept for an additional 4.5 months. Cysts in all groups were removed by washing with sterile 0.15 M PBS, pH 7.2, and the number of cysts was counted. Percent protection was calculated by comparing the numbers of cysts in treated controls with numbers of cysts in untreated groups. Means of protection are shown, along with standard deviations, and were determined to be statistically significant (P < 0.05) by Tukey test and analysis of variance.

 
Scanning electron microscopy results. Scanning electron microscopy examination of cysticerci from mice not treated with the inhibitors revealed healthy organisms, with a healthy-appearing surface tegument and microtriches (Fig. 2A). The cysts recovered from mice treated with Z-LLY-FMK were covered by an intense but focal inflammatory reaction, which was located where the cyst wall was degenerating (Fig. 2B). Microtriches were either sloughed or significantly shortened (Fig. 2B). We identified fibroblasts and collagen, neutrophils, and macrophages on the cyst surfaces from treated mice. Breaks in the integrity of the cyst surface were also noted for the cysts recovered from treated mice (Fig. 2B). Higher magnification revealed tegumental erosion, particularly adjacent to inflammatory cells.


Figure 2
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  		FIG. 2. Scanning electron microscopy (SEM) of the surface of Taenia crassiceps cysts removed from BALB/c mice treated with either solvent control (A) or with Z-LLY-FMK (B). Microtriches (M) in the cysts from untreated mice were found to be longer than those from treated mice. SEM results of cysts from treated mice revealed fibroblasts, holes in the tegument, and sloughed microtriches. Magnification, x11,000.

 
In vitro toxicity studies. Preliminary toxicity studies showed that Z-LLL-FMK and Z-LLY-FMK were not toxic to normal BALB/c mouse lymphocytes in vitro by trypan blue exclusion (viability of 85% to 92% with Z-LLL-FMK and Z-LLY-FMK). The inhibitors did not affect the responsiveness to concanavalin A, even at doses three times those needed to control infection in mice (data not shown).

Discussion. We tested the efficacy of Taenia protease inhibitors as chemotherapeutic agents against murine cysticercosis, using compounds that were discovered to be potent, in vitro, against a prominent T. solium cyst wall cysteine protease isolated in a prior study (1). We demonstrated that treatment with Z-LLL-FMK and Z-LLY-FMK protected mice against a T. crassiceps challenge. Furthermore, most of the cysts which did survive the treatment exhibited abnormal morphology marked by tegumental degeneration and host inflammatory attack. These initial studies tested for a prophylactic effect, since inhibitor treatment began prior to infection. Additional studies are needed to test for therapeutic effects.

While tripeptides are known to be proteasome inhibitors (6), Z-LLL-FMK and Z-LLY-FMK were found to have a 10,000-fold-greater potency of inhibition than Z-LLL-VS for the purified T. solium cysteine protease (1) (unpublished data). Since the potency of inhibition of parasite development correlates with the potency of inhibition of the cysteine proteinase but not of the proteasome, we propose that the Taenia cysteine protease rather than the proteasome is the major target of inhibition.

Our studies demonstrated that Z-LLL-FMK and Z-LLY-FMK have activities in a murine model of cysticercosis. No toxicity was identified in preliminary studies. Hence, we believe that protease inhibitor development against the T. solium cysteine protease warrants greater attention for de novo drug development.


   ACKNOWLEDGMENTS
 
We thank Mark Farmer and the Center for Advanced Ultrastructural Research for access to electron microscopy facilities. We also thank James Powers (Georgia Institute of Technology) for supplying vinylsulfone and other inhibitor compounds.

This work was supported by an NIH predoctoral training grant.


   FOOTNOTES
 
* Corresponding author. Mailing address: Infectious Disease Section, Department of Medicine, One Baylor Plaza 535EA, Houston, TX 77030. Phone: (713) 798-5246. Fax: (713) 790-0681. E-mail: arthurw{at}bcm.tmc.edu. Back


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Antimicrobial Agents and Chemotherapy, August 2006, p. 2886-2888, Vol. 50, No. 8
0066-4804/06/$08.00+0     doi:10.1128/AAC.01060-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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