A cysteine protease inhibitor cures Chagas' disease in an immunodeficient-mouse model of infection

Abstract

Chagas' disease, caused by the parasite Trypanosoma cruzi, remains the leading cause of cardiopathy in Latin America with about 12 million people infected. Classic clinical manifestations derive from infection of muscle cells leading to progressive cardiomyopathy, while some patients develop megacolon or megaesophagus. A very aggressive clinical course including fulminant meningoencephalitis has been reported in patients who contract Chagas' disease in the background of immunodeficiency. This includes patients with human immunodeficiency virus infection as well as patients receiving immunosuppressive therapy for organ transplant. Currently, only two drugs are approved for the treatment of Chagas' disease, nifurtimox and benznidazole. Both have significant limitations due to common and serious side effects as well as limited availability. A promising group of new drug leads for Chagas' disease is cysteine protease inhibitors targeting cruzain, the major protease of T. cruzi. The inhibitor N-methyl-Pip-F-homoF-vinyl sulfonyl phenyl (N-methyl-Pip-F-hF-VS phi) is in late-stage preclinical development. Therefore, the question arose as to whether protease inhibitors targeting cruzain would have efficacy in Chagas' disease occurring in the background of immunodeficiency. To address this question, we studied the course of infection in recombinase-deficient (Rag1(-/-)) and normal mice infected with T. cruzi. Infections localized to heart and skeletal muscle in untreated normal animals, while untreated Rag1(-/-) mice showed severe infection in all organs and predominantly in liver and spleen. Treatment with the dipeptide N-methyl-Pip-F-hF-VS phi rescued immunodeficient animals from lethal Chagas' infection. The majority (60 to 100%) of inhibitor-treated Rag1(-/-) mice had increased survival, negative PCR, and normal tissues by histopathological examination.

FIG. 1.

Acute lethal Chagas' infection in immunocompetent mice. Large T. cruzi nests (arrow) and an intense inflammatory response (arrowhead) are observed in heart (A) and skeletal muscle (B) of immunocompetent animals infected with CA-I/72 T. cruzi. Other tissues remain free of parasites. (C and D) Normal heart (C) and skeletal muscle (D) in uninfected controls. (Hematoxylin and eosin staining; magnification, ×100 to 400).

FIG. 2.

Acute lethal Chagas' infection in Rag1 knockout mice. Massive infection occurs predominantly in liver (C) and spleen (D), though all tissues harbor T. cruzi amastigotes (arrows). A, heart; B, skeletal muscle; C, liver; D, spleen; and E, colon. (Hematoxylin and eosin staining; ×400 magnification.)

FIG. 3.

Treatment of T. cruzi-infected Rag1 knockout mice with a cysteine protease inhibitor. Untreated controls died 30 to 40 days postinfection from acute Chagas' disease in three independent experiments (1C, 2C, and 3C). One hundred percent (3/3) of animals treated with N-methyl-Pip-F-hF-VSφ for 27 days survived in the first experiment until euthanized 16 weeks (112 days) postinfection (1T). In the second experiment, 60% (3/5) of treated animals survived for at least 20 weeks (140 days) (2T). One of five mice presented with Chagas' symptoms and was sacrificed (day 40), and one of five mice was incorrectly euthanized (day 50). In the third independent experiment, 75% (3/4) of N-methyl-Pip-F-hF-VSφ-treated mice survived until sacrificed 24 weeks (162 days) postinfection (3T).

FIG. 4.

Cure of T. cruzi infection in Rag1^−/− mice. No infection was observed in N-methyl-Pip-F-hF-VSφ-treated Rag1^−/− mice. A, heart; B, skeletal muscle; C, liver; D, spleen; and E, colon. (Hematoxylin and eosin staining; magnification, ×400.)

FIG. 5.

(A) T. cruzi-specific PCR in Rag1^−/− mice at 27 days postinfection. Representative results shown are from tests 2C and 2T (Table 1; Fig. 3). Blood DNA specimens collected 27 days postinfection were subjected to PCR with a specific T. cruzi probe. With the exception of one animal (lower gel, lane 6), all untreated controls (upper gel, lanes 4 to 6) and most inhibitor-treated mice (lower gel, lanes 2 to 5) had positive PCRs 27 days postinfection (arrow; 330-bp band). Upper gel: lane 1, molecular weight ladder; lane 2, negative control (no DNA); lane 3, noninfected mouse blood; lanes 4 to 6, blood from untreated, T. cruzi-infected Rag1 mice; lane 7, not applicable; lane 8, positive control, T. cruzi DNA. Lower gel: lane 1, DNA ladder; lanes 2 to 6, blood from N-methyl-Pip-F-hF-VSφ-treated and T. cruzi-infected Rag1 mice no. 1 through 5, respectively. (B) Negative PCR at 5 months for Rag1^−/− animals treated with N-methyl-Pip-F-hF-VSφ. Five months after treatment with the inhibitor, PCRs with blood collected from the same surviving animals were negative (330-bp T. cruzi DNA band absent; arrow). Lane 1, DNA ladder; lanes 2 to 3, N-methyl-Pip-F-hF-VSφ-treated infected Rag1^−/− mouse no. 4; lane 4, not applicable; lanes 5 to 6, N-methyl-Pip-F-hF-VSφ-treated infected Rag1^−/− mouse no. 5; lane 6, not applicable; lane 7, positive control, T. cruzi DNA.

FIG. 6.

Negative PCR with tissues of Rag1^−/− animals treated with N-methyl-Pip-F-hF-VSφ. Upper gel: lane 1, DNA ladder; lane 2, no DNA (negative control); lane 3, macrophage DNA (negative control); lane 4, liver of K11777-treated mouse 1 from experiment 1T; lane 5, spleen of K11777-treated mouse 1 from experiment 1T; lane 6, heart of K11777-treated mouse 1 from experiment 1T; lane 7, liver of K11777-treated mouse 2 from experiment 1T; lane 8, spleen of K11777-treated mouse 2 from experiment 1T; lane 9, heart of K11777-treated mouse 2 from experiment 1T; lane 10, T. cruzi DNA (positive control). Lower gel: Lane 1, DNA ladder; lane 2, no DNA (negative control); lane 3, macrophage DNA (negative control); lane 4, liver of untreated control mouse 1C; lane 5, spleen of untreated control mouse 1C; lane 6, heart of untreated control mouse 1C; lane 7, empty; lane 8, T. cruzi DNA (positive control); lanes 9 and 10, empty. Amplified T. cruzi kDNA is indicated with an arrow; lower molecular weight bands are primers.