 |
Introduction |
Chagas' disease, the result of infection with the protozoan parasite Trypanosoma cruzi, is the leading cause of
heart disease in Latin America (1). Over 1.6 × 107 people
are infected, up to 80% in endemic areas, and over 9 × 107
are at risk (2). By definition, the acute phase of Chagas' disease lasts up to 60 d and parasites are easily detected by direct examination of peripheral blood (3). Acute Chagas'
disease results in myocarditis in ~60% of patients with an
estimated 9% mortality occurring in endemic areas (4).
Chagasic encephalitis, not uncommon in children, is also
associated with immunosuppression and AIDS (5). Gastrointestinal megasyndromes are common especially in Brazilian patients. Most chagasic patients die from heart failure associated with cardiomyopathy during the chronic phase
of the disease (3, 8).
Therapy for Chagas' disease is unsatisfactory. Because of
significant toxicity, chemotherapy with nifurtimox or benznidazole must be carried out under close medical supervision. In addition to dermatotoxicity and digestive disorders,
benznidazole induces chromosomal damage in chagasic
children (9). Long-term use of nifurtimox and benznidazole
in humans has not been documented, but its association
with malignant lymphomas in experimental animals precludes prolonged treatment of patients including immunocompromised patients (10). Both compounds may shorten
the acute phase and decrease mortality, but they achieve
parasitologic cures in only ~60% of acute patients and are
not used during the chronic phase of the disease (10). Finally, a large gradient of susceptibility to nifurtimox and
benznidazole treatments ranging from 0-100% correlates
with geographic region and may delineate distribution of
drug-resistant T. cruzi (8, 11, 12).
One approach to novel chemotherapy for Chagas' disease has focused on the development of specific inhibitors
of cruzain (a.k.a. cruzipain, gp 57/51), the major cysteine
protease of T. cruzi (13). Diazomethane or fluoromethyl ketone (FMK)1 cysteine protease inhibitors (CPI)
that effectively blocked cruzain activity prevented growth
and differentiation of T. cruzi in cell culture models of infection (18). A new generation of CPI has been synthesized with chemical modifications aimed at enhancing specificity and in vivo stability and minimizing toxicity.
We now report that CPI treatment rescued mice from the
acute phase of a lethal experimental T. cruzi infection and
cleared parasitemia in chronically infected mice without
toxicity to the mammalian host. The inhibitors induced
major ultrastructural alterations leading to death of the intracellular amastigote stage that were similar to those previously observed in the extracellular insect stage epimastigotes after exposure to the same protease inhibitors (21).
 |
Materials and Methods |
Growth Inhibition of T. cruzi Amastigotes by CPI.
J774 macrophages were cultured in RPMI-1640 medium with 5% heat-
inactivated FCS (RPMI medium). For growth inhibition assays, J774 macrophages were irradiated (3,000 rad) to arrest cell growth and cultured on coverglasses within six-well plates for 24 h at 37°C. After infection with T. cruzi trypomastigotes of the Y strain for 3 h, monolayers were washed with RPMI medium and
treated with inhibitors at 20 µM in RPMI medium. Inhibitor
stocks were made at 20 mM in DMSO and all assays included
DMSO (0.01-0.02%, vol/vol) controls. FMK inhibitors (Mu-F-hF-VS
, Mu-F-K-VS
, Mu-F-V-VS
, Mu-F-S(OBzl)-VS
,
Mu-L-hF-VS
, Mu-Yii-hF-VS
, Boc-tic-hF-VS, Mu-tic-hF-VS, Mu-Y-hF-VS, Mu-F-hF-FMK, Mu-F-hF-VAmBzl, N-Pip-F-hF-VS
, Z-F-A-FMK, Mu-bsu-hF-FMK, and Mu-F-hF-FMK) were
provided by Prototek (Dublin, CA) and vinyl sulfones were provided by Axys Pharmaceuticals (South San Francisco, CA). CPI
were evaluated in T. cruzi-infected macrophage cultures for 21-
30 d. Trypomastigote output, indicative of the completion of the
intracellular cycle, was then assayed in treated and untreated cultures to determine growth inhibition of intracellular T. cruzi
amastigotes (20).
After this initial inhibitor screen, T. cruzi-infected macrophages
were treated with 20 µM Mu-F-hF-VS
and Mu-F-V-VS
for up to 76 h. Monolayers were washed, fixed with 4% paraformaldehyde, and then Giemsa stained at determined intervals. To
evaluate treatment, the percentage of infected macrophages and
the total number of intracellular amastigotes in 100 infected
macrophages were quantified. A decrease in the number of intracellular generations indicated inhibition of intracellular growth of
T. cruzi amastigotes and was calculated from the total number of
intracellular amastigotes per 100 infected macrophages (n = 3).
Effect of CPI on the Survival of T. cruzi-infected Mice.
3-wk-old female C3H mice weighing initially between 17-19 g were used in
all experiments. In the first experiment (see Fig. 1), mice (five animals per lot) were infected with 105 trypomastigotes of the Y
strain and treated with a 1-mg i.p. injection of FMK inhibitors
(Mu-F-hF-FMK, Mu-bsu-hF-FMK, and Z-F-A-FMK) twice
per day. Controls included intraperitoneal injection with equal
volume of DMSO. Treatment was initiated 24 h after infection and continued until death of the animals or the end of the experiment, as appropriate. Parasitemias were determined every 48 h
for each animal on alternating days from 5 µl of blood extracted
from the tail and diluted 1/4 (vol/vol) in RPMI medium. The
numbers of parasites per milliliter, calculated in a Neubauer
chamber, were expressed as a mean of two or three animals per
day. The experiment was terminated 18 d after infection.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 1.
Levels of parasitemia and survival of mice treated with peptidomimetic fluoromethyl ketones. CH3 mice were infected with T. cruzi
and treated twice daily with 1 mg i.p. of Z-F-Ala-FMK ( ); Mu-bsu-hF-FMK ( ); Mu-F-hF-FMK ( ); and controls with and without DMSO i.p.
( , ). Parasitemias were determined every 48 h in each animal on alternating days. Results are mean of two to three animals per day.
|
|
Approximate dosing regimes and different inhibitor chemistries were then analyzed (see Table 3, Experiment 1). Mice (five animals per lot) were infected (intraperitoneally) with 4 × 106 tissue culture-derived trypomastigotes. 24 h after infection, mice
were treated with two daily doses of 1 mg i.p. Mu-F-hF-FMK and Mu-F-hF-VS
for up to 16 d. In a third experiment (see Table 3, Experiment 2), animals were infected with 106 trypomastigotes and treated with the inhibitors with the same regimen for 12 d.
Finally (see Table 3, Experiment 3), mice were infected with
105 T. cruzi trypomastigotes and treated with three daily intraperitoneal doses of N-Pip-F-hF-VS
(2.1 mg/d) for 24 d. Blood
(hemo) cultures of untreated controls (n = 6) and N-Pip-F-hF-VS
-treated animals (n = 10) were performed 16, 22, and 46 d
after infection using arrested macrophages as host cells. In brief,
6-µl aliquots of blood were resuspended in RPMI medium with
5% FCS and antibiotics. Irradiated J774 macrophages were infected with blood dilutions, and incubated for up to 30 d at 37°C
in a 5% CO2 atmosphere. Blood cultures were considered positive if T. cruzi infected macrophages and/or free trypomastigotes
were observed. Hemocultures were considered negative if no infected host cells and no free trypomastigotes were observed for up
to 30 d, after which macrophages died.
Six animals from Table 3, Experiment 3, survived the acute
phase of the infection. Three out of six mice had consistently negative blood cultures while the remaining three out of six were positive. After being allowed to establish chronic infection for 3 mo,
the latter three mice were retreated with three daily doses (2.1 mg/d i.p.) of N-Pip-F-hF-VS
for 21 d (see Table 4, Experiment 4). Hemocultures from the six animals were performed repeatedly as described above but with a larger volume of blood sampled (6-50 µl).
T. cruzi-infected, CPI-treated mice, noninfected CPI-treated
mice, and untreated T. cruzi-infected animals were necropsied to
evaluate toxicity of the inhibitor regimen. Mice were weighed, examined grossly, and all major organs also examined by routine hematoxylin-eosin histopathology. The toxicity of Mu-F-hF-VS
and N-Pip-F-hF-VS
was further evaluated in uninfected
C3H mice treated for 45 d with 4 mg/d i.p., and 12 mg/d per
oral (p.o.), respectively.
Fluorescence Microscopy to Determine the Effect of CPI in the Parasite.
Irradiated macrophages were infected with T. cruzi trypomastigotes for 2 h at 37°C. Monolayers were washed 24 h after
infection and reincubated with or without the addition of the
CPI Mu-F-hF-VS
(10 µM). After 48 h, culture medium containing Bodipy FL ceramide (Molecular Probes, OR) was substituted for 15 min at 37°C. Monolayers were subsequently processed according to manufacturer's instructions and observed by
fluorescence microscopy (21).
Ultrastructure and Immunocytochemistry of T. cruzi Intracellular
Amastigotes.
T. cruzi-infected, irradiated J774 macrophages were
treated or not with Mu-F-hF-VS
(10 µM) for 48 h. Monolayers
were trypsinized, washed, fixed, and processed for electronmicroscopy. Cells were fixed with 1.5% glutaraldehyde in 0.66 M sodium
cacodylate buffer, pH 7.4, at room temperature for 2 h, embedded
in EPONATE 12 (Ted Pella, Inc., Redding, CA), sectioned,
stained, and then observed with a Zeiss 10C electronmicroscope
(Carl Zeiss Inc., Thornwood, NY; reference 22). The techniques
(23) and reagents used for immunocytochemistry have been described previously for T. cruzi epimastigotes (21). Heart muscle
from a mouse treated 6 d after infection with Mu-F-hF-VS
(2 mg/d i.p. for 3 d) was fixed and processed as described above.
Confirmation of Inhibitor Targets by Labeled Inhibitor.
Irradiated J774
macrophages were infected with Y strain trypomastigotes and incubated at 37°C for 48 h to allow intracellular development of
amastigotes. T. cruzi-infected macrophages, uninfected macrophage controls, and T. cruzi epimastigotes were radiolabeled with 20 µM [14C]Mu-F-hF-VS
for 2 h at 37°C or 26°C, as appropriate.
For competition experiments, duplicate cultures were treated
with 20 µM Mu-F-hF-VS
for 3 h before addition of the radiolabeled inhibitor. Infected macrophages were scraped and centrifuged at 1,700 g for 12 min at 4°C. Pellets were resuspended and
lysed in a tissue grinder to release intracellular amastigotes, centrifuged at 260 g for 5 min, and the amastigote-containing supernatant centrifuged at 2,000 g for 12 min. Amastigote pellets were
resuspended, transferred to Eppendorf tubes, and washed three
times with PBS before sonication. Radiolabeled, noninfected
macrophages and T. cruzi epimastigotes were sonicated as above.
Purified recombinant cruzain was also radiolabeled as a control.
Samples were boiled in sample buffer (24), electrophoresed in
10% acrylamide gels, and autoradiographed (21). Western blots
were developed with anti-cruzain antibody (21).
 |
Results |
Effect of CPI on the Intracellular Development of T. cruzi.
The efficacy of a number of CPI on the mammalian stages
of the life cycle of T. cruzi was evaluated as the percentage
of growth inhibition of intracellular amastigotes in the
presence of the inhibitors for up to 21 d (Table 1). The first
intracellular cycle of untreated controls was completed
within 6 d after infection. Mu-F-hF-VS
was the most effective compound and inhibited growth 100%. Mu-F-V-VS
and BOC-tic-hF-VS, which are less effective inhibitors of the protease itself, produced 30% and 60% growth
inhibition, respectively.
Based on this initial screening of inhibitors, Mu-F-hF-VS
with Mu-F-V-VS
for comparison were further
tested (Table 2). 20 µM concentrations of Mu-F-hF-VS
cured T. cruzi-infected macrophages. Although intracellular
amastigotes were still visible in Giemsa-stained cultures 24-
78 h after infection, their morphology was extremely abnormal 50 h after treatment. Cultures were amastigote free
after 12 d. No intracellular amastigotes or release of trypomastigotes were observed in cultures treated for 12 d and
maintained without CPI for up to 30 d in numerous independent experiments, indicating that Mu-F-hF-VS
not
only blocked the intracellular development of T. cruzi, but
eventually eliminated all parasites. In contrast, amastigotes
divided at normal rate in the presence of the chemically related, but less effective inhibitor of cruzain, Mu-F-V-VS
.
Resolution of Acute Experimental Chagas' Disease.
Levels of
parasitemia in T. cruzi-infected C3H mice were first analyzed after treatment with Z-F-A-FMK, Mu-bsu-hF-FMK, and Mu-F-hF-FMK (Fig. 1). These FMK inhibitors
were chosen based on results of tissue culture screens (20, Table 1) and included a natural amino acid dipeptide (Z-F-A-FMK) and pseudopeptides designed to increase in vivo
half-life (Mu-bsu-hF-FMK and Mu-F-hF-FMK). Control
animals died at day 12 after infection while all animals treated with Mu-bsu-hF-FMK and Mu-F-hF-FMK survived throughout the experiment (18 d). The levels of parasitemia were lowest in mice treated with Mu-F-hF-FMK
and ranged from 10-500 trypomastigotes per milliliter of
blood. This corresponded to a reduction of 3 log units
from untreated controls. Mice treated with the related but
less effective (in protease substrate assays) dipeptide inhibitor Mu-bsu-hF-FMK had persistent high parasitemia in the
range of 4 × 104-5 trypomastigotes per milliliter of blood.
Not only was Z-F-A-FMK ineffective, but mice had
higher parasitemia and died earlier than controls.
From tissue culture screens (Tables 1 and 2), vinyl sulfone derivatized pseudopeptides with high efficacy but less
toxicity than FMK inhibitors were selected for further
evaluation in a mouse model of acute Chagas' disease. The
effect of CPI-treatment on the survival of T. cruzi infected C3H mice is shown in Table 3. Untreated controls
infected with a very high dose (4 × 106) of tissue culture-
derived trypomastigotes all died by 4-5 d after infection.
All five mice treated with Mu-F-hF-VS
survived for 14-
16 d at which time the experiment was terminated and animals killed (Table 3, Experiment 1). When the infectious
dose was reduced to 1 × 106 T. cruzi trypomastigotes and
mice treated with one 24-d regimen of Mu-F-hF-VS
,
two out of six mice survived up to 180 d after infection at
which time the experiment was terminated (Table 3, Experiment 2).
From these pilot studies, the most promising lead compound was then evaluated in a new dosing regimen based
on a pharmacokinetics analysis of Mu-F-hF-VS
(25).
Mice were treated three times daily with 2.1 mg/d i.p. of
N-Pip-F-hF-VS
(Table 3, Experiment 3). As indicated in
Table 3, all 10 mice survived at least 7 d longer than untreated mice and 6 out of 10 mice survived over 270 d after
infection. Three of the six long-term surviving mice had
consistently negative blood-parasite cultures over the entire treatment period while the remaining three out of six were
still positive, although well below controls. After being allowed to establish chronic infection for 3 mo, this latter
group of 3 mice were retreated with 2.1 mg/d i.p. of
N-Pip-F-hF-VS
for 21 d (Table 4, Experiment 4). Blood
cultures (10-50 µl) were negative for the retreated animals.
In summary, 5 of 10 treated mice from this study have now
survived for 9 mo without symptomatology and with negative parasitemia and hemocultures (one mouse died of accidental trauma after 100 d). Similar results were obtained
with 3-wk-old outbred Swiss mice weighing initially 19-
21 g (data not shown).
In the studies described above, CPI treatment did not induce gross or microscopic abnormalities in either infected
or uninfected mice. To further assess toxicity, doses of 4 mg/d i.p. of Mu-F-hF-VS
and 12 mg p.o. of N-Pip-F-hF-VS
for 4 d were also evaluated. No gross or microscopic abnormalities were observed in necropsied animals.
Confirmation that CPI Enters Amastigotes and Targets
Cruzain.
CPI have been shown to produce a characteristic Golgi abnormality in the extracellular epimastigote stage
of T. cruzi (21).
To confirm that the same effect was produced in the
treated amastigotes, amastigotes within host macrophages
that had been treated with Mu-F-hF-VS
(10 µM) for 48 h
were examined after labeling with Bodipy FL ceramide that
accumulates in the Golgi compartment. Cells were observed by contrast phase (Fig. 2, A and B) and fluorescence
microscopy (Fig. 2, C and D). No Bodipy FL labeling was
apparent in untreated intracellular amastigotes (Fig. 2 C),
whereas the larger Golgi complex (G) of untreated host
cells was visible. In contrast, the Golgi apparatus (g) of Mu-F-hF-VS
-treated amastigotes had abnormally large, fluorescent vesicles consistent with ultrastructural alterations in
the Golgi complex previously observed in treated epimastigotes (Fig. 2 D). To confirm these results, the normal
morphology of control intracellular amastigotes (Fig. 3 A)
was compared with the ultrastructure of CPI-treated tissue
culture amastigotes (Fig. 3 B). Golgi complex and cytoplasmic vesicle alterations found in amastigotes (Fig. 3 B) resembled those described for the epimastigote stage, and consisted of significant dilation of cisternae (21). Similar ultrastructural alterations were evident in amastigotes isolated from heart muscle of T. cruzi-infected mice treated with
Mu-F-hF-VS
(Fig. 3 C). The amounts of cruzain expressed on the surface of Mu-F-hF-VS
-treated and untreated amastigotes were quantified by immunoelectronmicroscopy (Fig. 4). A marked decrease in cruzain expressed
on the cell surface of CPI-treated cells (Fig. 4 B) was evident compared with untreated amastigotes (Fig. 4 A).

View larger version (164K):
[in this window]
[in a new window]
|
Fig. 2.
Treatment of T. cruzi-infected macrophages with a cysteine protease inhibitor and a fluorescent probe specific for the Golgi complex. Phase
contrast (A) and fluorescence (C) microphotograph of an untreated T. cruzi-infected macrophage. Several intracellular amastigotes are visible within the
cytoplasm of the host cell. The Golgi complex (G) of the host cell is labeled. Bodipy FL does not induce visible fluorescence in the Golgi complex of untreated amastigotes. Phase contrast (B) and fluorescence microphotograph (D) of an infected macrophage treated with 20 µM Mu-F-hF-VS for 48 h.
Large, fluorescent Golgi vesicles (g) are evident in CPI-treated amastigotes indicative of vesicle dilation abnormality (21). a, amastigote; G, macrophage-
Golgi complex; g, amastigote-Golgi complex; N, macrophage nucleus.
|
|

View larger version (221K):
[in this window]
[in a new window]
|
Fig. 3.
Electronmicroscopy of T. cruzi intracellular amastigotes. T. cruzi amastigotes observed within culture macrophages (A and B) or heart muscle
of Mu-F-hF-VS -treated mice (C). The normal ultrastructure of untreated intracellular amastigotes within irradiated macrophages (A) contrasts with the
altered morphology of intracellular amastigotes treated with 20 µM of Mu-F-hF-VS for 48 h (B). More dilated Golgi vesicles and perinuclear membrane similar to that reported in treated epimastigotes (21). Similar ultrastructural alterations were observed in amastigotes isolated from heart muscle (C)
of experimentally infected mice treated with 2 mg/d i.p. Mu-F-hF-VS for 4 d before necropsy and isolation of heart muscle. A, Untreated amastigotes;
B, CPI-treated cell culture amastigote; C, amastigote infecting the heart muscle of a CPI-treated animal. Nuclear membrane (large arrows); Golgi complex
(small arrow); vesicle ( ). No abnormalities were noted in Golgi complex or other organelles of host cells. Bar, 1 µm.
|
|

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 4.
Immunoelectronmicroscopy of cell surface membranes of T.
cruzi amastigotes. Cell surface membranes of untreated (A) and Mu-F-hF-VS -treated (B) amastigotes were immunocytochemically labeled with a
specific anti-cruzain antibody. Note markedly diminished gold label on
surface of treated parasite consistent with retention of unprocessed cruzain
in Golgi (21). PM, parasite cell surface membrane; IGL, immunogold label. Bar, 0.2 µm.
|
|
T. cruzi intracellular amastigotes were incubated with or
without cold inhibitor before incubation labeling with
[14C]Mu-F-hF-VS
. T. cruzi epimastigotes and recombinant cruzain were also radiolabeled and autoradiographed
as standards (Fig. 5 A). 14C-inhibitor labeling of amastigote
cruzain (lane 1) was abolished by preincubation with unlabeled Mu-F-hF-VS
(lane 2). In amastigotes isolated from
infested host cells two protease species were labeled. One
comigrated with the epimastigote cruzain species (~50 kD) known to contain both the catalytic and COOH-terminal
domains (13) while the second comigrated with recombinant cruzain containing only the catalytic domain and with a
macrophage protease (~30 kD).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 5.
Autoradiogram of extract from radiolabeled [14C]Mu-F-hF-VS intracellular amastigotes. Binding of the radiolabeled
inhibitor to amastigote cruzain
was abolished by preincubation
with unlabeled Mu-F-hF-VS
followed by [14C]CPI. Epimastigote cruzain and recombinant
cruzain controls are shown as
standards. Note two species labeled in amastigotes that comigrate with either epimastigote
cruzain (50 kD) which has both
catalytic and COOH-terminal
domains (13) or with recombinant cruzain (30 kD) that only has
catalytic domain. Lane 1, T. cruzi
intracellular amastigotes labeled
with [14C]CPI; lane 2, T. cruzi intracellular amastigotes preincubated with unlabeled inhibitor;
lane 3, sample buffer; lane 4, recombinant cruzain labeled with
[14C]CPI; lane 5, epimastigotes radiolabeled with [14C]Mu-F-hF-VS .
Note 57/51-kD doublet characteristic of native cruzain which retains
COOH-terminal domain (13). Recombinant cruzain lacks the COOH-terminal domain (14).
|
|
 |
Discussion |
High toxicity and low efficacy make current chemotherapy for Chagas' disease highly unsatisfactory. Moreover,
commercial nifurtimox production has been discontinued
and benznidazole is at present the only treatment available.
Until recently, it was still controversial as to whether drug
treatment would have any effect on the more common
chronic stage of Chagas' disease because it was unclear whether parasites were still present. However, there is now
a clear association between parasitic burden and degree of
myocardial damage (26). A recent follow up study in Argentina of benznidazole-treated versus untreated chronic
chagasic patients showed less cardiomyopathy in the first
group (27). Electrocardiogram patterns of benznidazole-treated patients confirmed an important reduction in disease progression (27). Andrade et al. (28) reported a correlation between heart disease and persistent parasitemia in
mice. These studies emphasize the importance of the development of more effective and less toxic chemotherapy
for both the acute and the chronic phase of Chagas' disease.
We have targeted cruzain (a.k.a. cruzipain, gp57/51),
the major cysteine protease of T. cruzi (reviewed in 13, 29,
30), for the development of new chemotherapy. Previously
tested dipeptide-based cruzain inhibitors that mimic substrate (20) were further modified to increase affinity for the
catalytic site of cruzain, increase half-life in vivo, provide
oral bioavailability, and reduce toxicity. In a previous report, we showed that these CPI induced death in the extracellular epimastigote stage of T. cruzi as a consequence of
blocking the autocatalytic processing of cruzain precursor protein. This resulted in Golgi complex and endoplasmic
reticulum abnormalities secondary to accumulation of unprocessed cruzain precursor molecules in the vesicle compartments (21). We now present evidence that these CPI
also inhibit T. cruzi amastigote growth within macrophages
by the same mechanism. Among the inhibitors tested, Mu-F-hF-VS
most effectively blocked the intracellular cycle
of the parasite (Table 2) and resulted in amastigote death in
vitro. None of the inhibitors produced abnormalities in the host cells at the concentrations necessary to interrupt the
parasite life cycle.
Subsequent testing of a number of CPI in an experimental mouse model of acute Chagas' disease showed that the
inhibitors also disrupted the life cycle of T. cruzi in vivo.
Initially, T. cruzi-infected animals were treated with the
fluoromethyl ketone-derivatized peptidomimetics Mu-F-hF-FMK, Mu-bsu-hF-FMK, and Z-F-A-FMK (Fig. 1).
Treatment with Mu-F-hF-FMK reduced parasitemia by 3 log units which resulted in the survival of infected mice
throughout the experiment, terminated 18 d after infection.
In contrast, animals treated with Mu-bsu-hF-FMK, a less
effective inhibitor of the protease itself, and Z-F-A-FMK, a
peptide with natural amino acids, had ~100- and 10,000-fold
higher parasitemias, respectively. Survival in the Z-F-A-FMK group (8 d) was significantly lower than in controls (12 d). The increased mortality and parasitemia probably
result from a toxic metabolite of the natural amino acid inhibitor. Z-F-A-FMK is cleaved between the phenylalanine
and alanine in vivo by an as yet unknown mammalian protease releasing alanine-FMK which, in turn, enters and inhibits the Krebs cycle (31). This fluoride-dependent toxicity results in hypothermia in mice, and T. cruzi has a higher growth rate at 35°C than at 37°C (32, 33). The peptidomimetics containing at least one nonnatural amino acid analogue (e.g., hF) do not undergo this cleavage and metabolism (25). To completely avoid the potential toxicity of
FMK derivatives, vinylsulfone analogues were tested and
found to extend survival to 120-180 d after infection with
106 trypomastigotes. We further evaluated the more aqueous soluble derivative N-Pip-F-hF-VS
that is absorbed
after oral dosing. 5 out of 10 treated mice have now survived for over 9 mo, 4 of them with repeatedly negative
hemocultures indicative of parasitological cure. No toxicity
was observed at doses two- to sixfold higher than the therapeutic doses administered p.o. or intraperitoneally for 20 d
or when mice were treated for 45 d with the therapeutic dose of 2 mg/d inhibitor.
To elucidate the mechanism of action of CPI versus intracellular amastigotes, the pathogenic stage of T. cruzi, we
infected host cells whose cell cycles were arrested. Intracellular amastigotes treated with Mu-F-hF-VS
showed major
morphological alterations as early as 24-48 h after treatment, and macrophage cultures were amastigote free within
12 d. Mu-F-hF-VS
produced abnormalities in the protein
trafficking pathway (nuclear membrane, ER, Golgi complex) and induced the appearance of double-membrane
vacuoles with similar morphology to autophagosome vacuoles (Fig. 3). Cruzain normally localizes to the cell surface
and lysosome membrane of T. cruzi amastigotes (34, 35).
CPI treatment significantly reduced the amounts of cruzain
appearing both on the cell membrane (Fig. 4) and in the lysosome consistent with an arrest of cruzain transport. An
increase of fluorescence with Bodipi FL of the Golgi complex in treated amastigotes was similar to that described for epimastigotes and correlated with enlargement of the Golgi
complex secondary to retention of unprocessed cruzain
(Fig. 2; reference 21). Radiolabeled inhibitor was used to
confirm entry into amastigotes and specific labeling of the
target protease cruzain (Fig. 5). The absence of any host
cell or animal toxicity at therapeutic doses suggests that the
parasites are more susceptible to inhibitor perhaps because
of the redundancy of cysteine proteases in mammalian cells
versus the parasite.
We have identified peptidomimetic cysteine protease inhibitors that consistently rescued mice from acute lethal infections of T. cruzi, reduced parasitemia by up to 3 log
units, and cured mice in the chronic stage of disease treated
by a 21-d regimen. These results provide an important
"proof of concept" for the development of cysteine protease inhibitors as chemotherapy for a number of disease
entities including cancer cell invasion, inflammation, osteoporosis, and microbial infections where cysteine proteases are thought to play a key role in pathogenesis (36).
Although further improvements on vinyl sulfone inhibitor
leads should be forthcoming, they clearly demonstrate that
animals can tolerate CPI at concentrations and dosing
schedules that eliminate an intracellular parasite. The pharmacokinetics of these inhibitors were adequate to sustain
therapeutic levels and the N-methyl piperazine derivative demonstrated oral bioavailability. The efficacy and lack of
toxicity of CPI in treating both acute and chronic T. cruzi
infections supports a call for further development of these
leads.
Address correspondence to James H. McKerrow or Juan C. Engel, Tropical Disease Research Unit, 4150 Clement St., 113B, San Francisco, California 94121. Phone: 415-221-4810, ext. 2625; Fax: 415-750-6947;
E-mail: jmck{at}socrates.ucsf.edu or jcengel{at}itsa.ucsf.edu
The authors thank Drs. M. Zimmerman and R. Smith (Prototek, Dublin, CA), and J. Palmer (Axys Pharmaceuticals, San Francisco, CA) for cysteine protease inhibitors; and G. Gan (Anatomic Pathology Service,
VAMC San Francisco) for processing of histological samples.
1.
|
Libow, L.F.,
V.P. Beltranni,
D.N. Silvers, and
M.E. Grossman.
1991.
Post-cardiac transplant reactivation of Chagas'
disease diagnosed by skin biopsy.
Cutis.
48:
37-40
[Medline].
|
2.
| Godal, T., and J. Nagera. 1990. Tropical diseases. In WHO
Division of Control in Tropical Diseases, 12-13. World
Health Organization; Geneva, Switzerland.
|
3.
|
The National Foundation of Brazil.
1996.
Etiological treatment for Chagas' disease.
Parasitol. Today
13:
127-128
.
|
4.
|
Parada, H.,
H.A. Carrasco,
N. Anez, and
I. Inglessis.
1997.
Cardiac involvement is a constant finding in acute Chagas'
disease: a clinical, parasitological and histopathological study.
Int. J. Cardiology.
60:
49-54
[Medline].
|
5.
| Chagas, C. 1981. Carlos Chagas: Coletanea de trabalhos cientificos. Editora Universidade de Brasilia. 6:247-258.
|
6.
|
Di Lorenzo, G.A.,
M.A. Pagano,
A.L. Taratuto,
M.L. Garau,
F.J. Meli, and
M.D. Pomsztein.
1996.
Chagasic granulomatous encephalitis in immunosuppressed patients. Computed
tomography and magnetic resonance imaging findings.
J.
Neuroimaging.
6:
94-97
[Medline].
|
7.
|
Pimentel, P.C.,
B.W. Handfas, and
M. Carmignani.
1996.
Trypanosoma cruzi meningoencephalitis in AIDS mimicking
cerebral metastasis: case report.
Arq. Neuro-Psiquiatr.
54:
102-106
[Medline].
|
8.
|
Filardi, L.S., and
Z. Brener.
1987.
Susceptibility and natural
resistance of Trypanosoma cruzi strains to drugs used clinically
in Chagas' disease.
Trans. Royal Soc. Trop. Med. Hyg.
81:
755-759
[Medline].
|
9.
|
Gorla, N.B.,
O.S. Ledesma,
G.P. Barbieri, and
I.B. Larripa.
1988.
Assessment of cytogenetic damage in chagasic children
treated with benznidazole.
Mutat. Res.
206:
217-220
[Medline].
|
10.
|
Kirchhoff, L.V..
1993.
American Trypanosomiasis (Chagas'
disease) a tropical disease now in the United States.
New
Engl. J. Med
329:
639-644
[Free Full Text].
|
11.
|
Cerisola, J.A.,
M. Alvarez, and
A.M. De Rissio.
1970.
Immunodiagnostico da doenca de Chagas. Evolucao serologica de
pacientes com doenca de Chagas.
Rev. Inst. Med. Trop. Sao
Paulo.
18:
357-364
.
|
12.
|
Andrade, S.G.,
J.B. Magalhaes, and
A.L. Pontes.
1985.
Evaluation of chemotherapy with benznidazole and nifurtimox in
mice infected with Trypanosoma cruzi strains of different types.
Bull. W H O
63:
721-726
[Medline].
|
13.
|
Cazzulo, J.J.,
V. Stoka, and
V. Turk.
1997.
Cruzipain, the
major cysteine proteinase from the protozoan parasite Trypanosoma cruzi.
Biol. Chem.
378:
1-10
.
[Medline] |
14.
|
Eakin, A.E.,
M.E. McGrath,
J.H. McKerrow,
R.J. Fletterick, and
C.S. Craik.
1993.
Production of crystallizable cruzain,
the major cysteine protease from Trypanosoma cruzi.
J. Biol.
Chem
9:
6115-6118
.
|
15.
|
Ring, C.S.,
E. Sun,
J.H. McKerrow,
G.K. Lee,
P.J. Rosenthal,
I.D. Kuntz, and
F.E. Cohen.
1993.
Structure-based inhibitor design by using protein models for the development of antiparasitic agents.
Proc. Natl. Acad. Sci. USA.
90:
3583-3587
[Abstract].
|
16.
|
Eakin, A.E.,
J.H. McKerrow, and
C.S. Craik.
1995.
A cysteine protease is a target for the enzyme structure-based design of antiparasitic drugs.
Drug Inf. J.
92:
1501S-1517S
.
|
17.
|
McGrath, M.E.,
A.E. Eakin,
J.C. Engel,
J.H. McKerrow,
C.S. Craik, and
R.J. Fletterick.
1995.
The crystal structure of
cruzain: a therapeutic target for Chagas' disease.
J. Mol. Biol.
247:
251-259
[Medline].
|
18.
|
Ashall, F.,
H. Angliker, and
E. Shaw.
1990.
Lysis of trypanosomes by peptidyl fluoromethyl ketones.
Biochem. Biophys.
Res. Commun.
170:
923-929
[Medline].
|
19.
|
Meirelles, M.N.,
L. Juliano,
E. Carmona,
S.G. Silva,
E.M. Costa,
A.C.M. Murta, and
J. Scharfstein.
1992.
Inhibitors of
the major cysteinyl proteinase (GP57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro.
Mol. Biochem. Parasitol.
52:
175-184
[Medline].
|
20.
|
Harth, G.,
N. Andrews,
A.A. Mills,
J.C. Engel,
R. Smith, and
J.H. McKerrow.
1993.
Peptide-fluoromethyl ketones arrest intracellular replication and intercellular transmission of
Trypanosoma cruzi.
Mol. Biochem. Parasitol.
58:
17-24
[Medline].
|
21.
|
Engel, J.C.,
P.S. Doyle,
J. Palmer,
I. Hsieh,
D.F. Bainton, and
J.H. McKerrow.
1998.
Cysteine protease inhibitors alter
Golgi complex ultrastructure and function in Trypanosoma
cruzi.
J. Cell Sci.
111:
597-606
[Abstract/Free Full Text].
|
22.
|
Stenberg, P.E.,
M.A. Schuman,
S.P. Levine, and
D.F. Bainton.
1984.
Redistribution of alpha-granules and their contents
in thrombin-stimulated platelets.
J. Biol. Chem.
98:
748-760
.
|
23.
|
Kjeldsen, L.,
D.F. Bainton,
H. Sengelov, and
N. Borregaard.
1993.
Structural and functional heterogeneity among peroxidase-negative granules in human neutrophils: identification of
a distinct gelatinase-containing granule subset by combined
immunocytochemistry and subcellular fractionation.
Blood.
82:
3183-3191
[Abstract].
|
24.
|
Laemli, U.K..
1970.
Cleavage of structural proteins during the
assembly of the head of bacteriophage T4.
Nature.
222:
680-685
.
|
25.
| Zhang, D. 1998. The roles of cytochrome P450 3A and P-glycoprotein in the absorption, metabolism and elimination
of a novel cysteine protease inhibitor. Ph.D. thesis. University of California, San Francisco.
|
26.
|
Tarleton, R.L.,
L. Zhang, and
M.O. Downs.
1997.
Autoimmune rejection of neonatal heart transplants in experimental
Chaga's disease is a parasite-specific response to infected host-tissue.
Proc. Natl. Acad. Sci. USA.
94:
3932-3937
[Abstract/Free Full Text].
|
27.
|
Viotti, R.,
C. Vigliano,
H. Armenti, and
E.L. Segura.
1994.
Treatment of chronic Chagas' disease with benznidazole:
clinical and serological evolution of patients with long-term
follow up.
Am. Heart J.
127:
151-162
[Medline].
|
28.
|
Andrade, S.G.,
S. Stocker-Guerret,
A.S. Pimentel, and
J.A. Grimaud.
1991.
Reversibility of cardiac fibrosis in mice
chronically infected with Trypanosoma cruzi, under specific
chemotherapy.
Mem. Inst. Oswaldo Cruz
86:
187-200
[Medline].
|
29.
|
Robertson, C.D.,
G.H. Coombs,
M.J. North, and
J.C. Mottram.
1996.
Parasite cysteine proteinases.
Perspect. Drug Discov. Des.
6:
1-20
.
|
30.
|
McKerrow, J.H.,
M.E. McGrath, and
J.C. Engel.
1995.
The
cysteine protease of Trypanosoma cruzi as a model for antiparasite drug design.
Parasitol. Today
11:
279-282
.
|
31.
|
Eichhold, T.H.,
E.B. Hookfin,
Y.O. Taiwo,
B. De, and
K.R. Wehmeyer.
1997.
Isolation and quantification of fluoroacetate in rat tissues following dosing of Z-Phe-Ala-CH2-F, a peptidyl fluoromethyl ketone protease inhibitor.
J.
Pharm. Biochem. Anal.
16:
459-467
.
|
32.
|
Marinkelle, C.J., and
E. Rodriguez.
1968.
The influence of
environmental temperature on the pathogenicity of Trypanosoma cruzi in mice.
Exp. Parasitol.
23:
260-263
[Medline].
|
33.
|
Bertelli, M.S.,
R.R. Golgher, and
Z. Brener.
1977.
Intraspecific variation in Trypanosoma cruzi: effect of temperature on
the intracellular differentiation in tissue culture.
J. Parasitol.
63:
434-437
[Medline].
|
34.
|
Fresno, M.,
C. Hernandez-Munain,
J. de Diego,
L. Rivas,
J. Scharfstein, and
P. Bonay.
1994.
Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a
GPI anchor.
Braz. J. Med. Biol. Res.
27:
431-437
[Medline].
|
35.
|
Nascimento, A.E., and
W. de Souza.
1996.
High resolution
localization of cruzipain and Ssp4 in Trypanosoma cruzi by
replica staining label fracture.
Biol. Cell.
86:
53-58
[Medline].
|
36.
|
McKerrow, J.H., and
M.N.G. James.
1996.
Cysteine proteases: evolution, function, and inhibitor design.
Perspect.
Drug Discov. Des.
6:
1-125
.
|