Leishmania major: Molecular Modeling of Cysteine Proteases and
Prediction of New Nonpeptide Inhibitors
Paul M. Selzer,*,2Xiaowu Chen,†,‡ Victor J. Chan,* Maosheng Cheng,‡ George L. Kenyon,†
I. D. Kuntz,†,‡ Judy A. Sakanari,* Fred E. Cohen,†,‡,§,? and James H. McKerrow*,†,‡,1
Departments of Pathology,* Cellular and Molecular Pharmacology,† Pharmaceutical Chemistry,‡ Biochemistry and
Biophysics,§ and Medicine,? University of California, San Francisco, California 94143, U.S.A.
Selzer, P. M., Chen, X., Chan, V. J., Cheng, M., Kenyon, G. L., Kuntz, I. D., Sakanari, J. A.,
Cohen, F. E., and McKerrow, J. H. 1997. Leishmania major: Molecular modeling of cysteine pro-
teases and prediction of new nonpeptide inhibitors. Experimental Parasitology 87, 212–221. The
crystal structures of papain, cruzain, and human liver cathepsin B were used to build homology-based
enzyme models of a cathepsin L-like cysteine protease (cpL) and a cathepsin B-like cysteine protease
(cpB) from the protozoan parasite Leishmania major. Although structurally a member of the cathep-
sin B subfamily, the L. major cpB is not able to cleave synthetic substrates having an arginine in
position P2. This biochemical property correlates with the prediction of a glycine instead of a
glutamic acid at position 205 (papain numbering). The modeled active sites of the L. major cpB and
cpL were used to screen the Available Chemicals Directory (a database of about 150,000 commer-
cially available compounds) for potential cysteine protease inhibitors, using DOCK3.5. Based on both
steric and force field considerations, 69 compounds were selected. Of these, 18 showed IC50’s
between 50 and 100 ?M and 3 had IC50’s below 50 ?M. A secondary library of compounds,
originally derived from a structural screen against the homologous protease of Plasmodium falcipa-
rum (falcipain), and subsequently expanded by combinatorial chemistry, was also screened. Three
inhibitors were identified which were not only effective against the L. major protease but also
inhibited parasite growth at 5–50 ?M.
© 1997 Academic Press
Index Descriptors and Abbreviations: Leishmania major; cysteine proteases; homology modeling;
drug design; docking; reversible inhibitors; cpL, cathepsin L-like cysteine protease; cpB, cathepsin
B-like cysteine protease; Z, benzoyloxycarbonyl; AMC, 7-amino-4-methyl coumarin.
Parasitic diseases are major worldwide health
problems now exacerbated by the emergence of
drug-resistant organisms (Moran and Bernard
1989). Leishmaniasis, a spectrum of diseases
produced by protozoan parasites of the genus
Leishmania, affects more than 12 million
people. Current therapy for leishmaniasis is
suboptimal due to toxicity of available thera-
peutic agents and the emergence of drug resis-
tance (Grogl et al. 1992). Compounding these
problems is the fact that many countries and
regions where the disease is endemic are eco-
nomically poor. As a result, major pharmaceu-
tical companies have historically had little in-
terest in anti-leishmanial drug development.
To facilitate the cost-effective development
of new antiparasitic chemotherapy, we have
been exploring the application of structure-
based drug design, utilizing computational
screens of available chemical databases as an
inexpensive shortcut to identify potential che-
motherapeutic leads. One appealing target in the
Leishmania parasites is a family of cysteine pro-
teases required for parasite replication and viru-
lence (Coombs and Baxter 1984; McKerrow et
1To whom correspondence should be addressed at: Vet-
erans Affairs Medical Center, 4150 Clement Street 113B,
San Francisco, CA 94121. Phone: 415-476-2940. Fax: 415-
750-6947. E-mail: email@example.com.
2To whom correspondence should be addressed at pres-
ent addresses: I. Frauenklinik, Ludwig Maximilians Univer-
sita ¨t Mu ¨nchen, Labor fu ¨r Biochemie, Maistraße 11, D-
80337 Mu ¨nchen, Germany. Phone: +49-89-5160-4256. Fax:
+49-89-5160-4916. E-mail: Paul.Selzer@med.uni-
muenchen.de; and Boehringer/Mannheim GmbH, Nonnen-
wald 2, D-82377 Penzberg, Germany. Fax: +49-8856-60-
2003. E-mail: Paul Selzer@bmg.boehringer-mannheim.
EXPERIMENTAL PARASITOLOGY 87, 212–221 (1997)
ARTICLE NO. PR974220
Copyright © 1997 by Academic Press
All rights of reproduction in any form reserved.
al. 1993; Sakanari et al. 1995; Mottram et al.
1996). Because of the relationship of these tar-
get enzymes to cysteine proteases of known
structure and catalytic mechanism, it is feasible
to utilize molecular modeling techniques to vi-
sualize the active site of the enzyme and com-
putationally design or screen for inhibitors.
While molecular modeling is not a substitute for
crystallographic structure determination, it has
proven useful in identifying or designing inhibi-
tors for two other parasitic infections, schisto-
somiasis and malaria (Ring et al. 1993). The
Leishmania cysteine proteases present a similar
opportunity in that there are sufficient data from
structural analysis of closely related enzymes to
allow a reasonable and useful model to be built.
In this initial work, we have produced struc-
tural models of the two major cysteine proteases
of L. major, a cathepsin L-like cysteine protease
(cpL) and a cathepsin B-like cysteine protease
(cpB). These models were used to develop hy-
potheses on the structural basis of substrate
specificity. The predicted active site binding re-
gions of both the L. major cpL and cpB were
then used to screen a public domain database of
small molecular weight compounds for poten-
tial chemotherapeutic leads. In addition a li-
brary of compounds derived from a lead found
by a similar approach using the homologous
malaria cysteine protease as a target (Ring et al.
1993; Li et al. 1994, 1995, 1996) was also
screened. Because reagent quantities of the L.
major cpB were available it was used for con-
firmatory screens of DOCK3.5-derived leads.
The most promising compounds were investi-
gated both for their ability to inhibit parasite
growth and for any toxicity against host cells.
MATERIALS AND METHODS
Homology Modeling and Docking of Inhibitors
The mature protein sequences of L. major cpL (216 aa,
GenBank locus U43706) and L. major cpB (243 aa, Gen-
Bank locus 43705) (Sakanari et al. 1997) were used to
search the Brookhaven Protein Databank of three-
dimensional structures (Bernstein et al. 1978). Both se-
quences show high homology with the papain family pro-
teases. Based on the crystal structures of papain (Kamphuis
et al. 1984), cruzain (McGrath et al. 1995), and human liver
cathepsin B (Musil et al. 1991) homology-based enzyme
models were built (Fig. 1) using the programs InsightII
(Biosym Technologies, San Diego, CA) and Midas Plus
(Computer Graphics Laboratory, University of California
San Francisco) (Ferrin et al. 1988; Huang et al. 1991)
(http://www.cgl.ucsf.edu/midasplus.html). For L. major cpL
the crystal structures of cruzain and papain (59 and 41%
sequence identity, respectively) were used, whereas for L.
major cpB, the three-dimensional structure of human liver
cathepsin B served as reference protein (54% sequence
identity). Identities in the structural conserved regions
(SCR) and especially within the active site cleft of the en-
zymes reached values of up to 80%. Comparing known
crystal structures with models built by homology, modeling
revealed that sequence identities higher than 70% lead to
highly accurate structural predictions (Mosimann et al.
In order to find structurally conserved regions, sequences
of the reference proteins were manually aligned based on
their secondary structure. The sequence of the correspond-
ing parasite protease was then aligned to the reference pro-
teases and the coordinates were assigned within the SCR
regions. Loops or variable regions (VR), which are located
between SCRs, were found exclusively at the surface of the
proteases, not interacting with the active site. Coordinates
for the VRs were either directly generated or assigned from
known crystal structures. The conformation of side chains
were retained in conserved positions and the statistically
most likely rotamer (rotational position of side chain based
on analysis of all known protein structures containing that
amino acid) was chosen when no conformational informa-
tion was available. The final structures were refined by
energy minimization using the AMBER potential function.
The quality of the models was validated with QPACK (Gre-
goret and Cohen 1990), VADAR (University of Alberta,
Protein Engineering Network of Centres of Excellence), and
the 3D profile method (Luthy et al. 1992).
All color figures shown are generated using Midas Plus.
Primary sequence alignments were performed using the
software package GCG (Genetics Computer Group Inc.,
Madison, WI). Three-dimensional structures of inhibitors
were generated with Sybyl and the CONCORD algorithm
(Tripos Inc., St. Louis, MO). Partial charges were calculated
using the Gasteiger–Masili method within Sybyl. Searching
the Available Chemicals Directory (ACD) for potential pro-
tease inhibitor leads was carried out using DOCK3.5 in
SEARCH mode (contact and force field scores). Com-
pounds of high interest were ‘‘redocked’’ using the
SINGLE mode function. (For further details of the DOCK
approach and the program see Kuntz 1992; Kuntz et al.
1994; and http://www.cmpharm.ucsf.edu/kuntz/dock.html).
All computer-assisted modeling and docking was performed
on Silicon Graphics Workstation (IRIS4D/35 or Indigo2).
The native L. major cpB was a generous gift of Dr.
Jacques Bouvier (Ciba–Geigy, CH-1566 St. Aubin, Swit-
zerland). Its purity was confirmed by silver-stained SDS-
L. major: MOLECULAR MODELING AND DRUG DESIGN
PAGE. Papain [EC 188.8.131.52] and mammalian cathepsin B
(bovine spleen) [EC 184.108.40.206] were purchased from Sigma.
Recombinant cruzain was produced as previously described
(Eakin et al. 1992, 1993).
All proteases were assayed at 25°C using an automated
microtiter plate spectrofluorometer (Labsystem Fluoroscan
II). Activity was detected by the liberation of 7-amino-4-
methyl coumarin (AMC) (Knight 1995a) (excitation wave-
length ? 355 nm and emission wavelength ? 460 nm)
from the synthetic peptide substrate Z-Phe-Arg-AMC or
Z-Arg-Arg-AMC (Z ? benzoyloxycarbonyl; Phe ? phe-
nylalanine; Arg ? arginine) (Enzyme Systems Products,
Livermore, CA). The enzyme concentrations were deter-
mined by active site titration (Barrett et al. 1982; Knight
1995b). Inhibitors (20-mM stock solutions, dissolved in di-
methyl sulfoxide (DMSO), stored at −20°C) at various con-
centrations were preincubated with the respective enzyme
for 5 min before the reaction was started by adding the
substrate. Enzyme activities were expressed as a percentage
of residual activity compared to an uninhibited control and
plotted versus increasing inhibitor concentrations in order to
calculate the IC50values.
L. major cpB. 100 mM Na acetate, pH 5.5, 10 mM di-
thiothreitol (DTT), 1 mM EDTA, 0.1% Triton X-100, 50
?M Z-Phe-Arg-AMC final concentration (from a 10-mM
stock solution in DMSO) Km? 7 ?M.
Papain and mammalian Cathepsin B. 100 mM Na ac-
etate, pH 5.5, 10 mM DTT, 100 ?M Z-Phe-Arg-AMC final
concentration, Km? 50 and 110 ?M, respectively.
Cruzain. The assay conditions were the same as for pa-
pain except the substrate concentration was 20 ?M, Km?
1 ?M. Kmvalues were determined by nonlinear regression
using the software Ultrafit (Biosoft Inc., Ferguson, MO).
L. major promastigotes LV39(MRHO/SU/59/P) were
grown at 27°C in RPMI-1640 containing 10% (v/v) heat-
inactivated fetal bovine serum and 20% Brain Heart Infu-
sion Tryptose. Cell growth was determined by counting the
parasites with a neubauer hemocytometer. Inhibitors dis-
solved in DMSO were added from a 20-mM stock solution.
DMSO concentrations up to 0.5% showed no effect on the
RESULTS AND DISCUSSION
The structure of the active site clefts of cys-
teine proteases from the papain family are
highly conserved. The cleavage site or catalytic
triad of all cysteine proteases consists of a nu-
cleophilic cysteine, a histidine, and an aspara-
gine. However, the S-subsites show striking dif-
ferences (Table I). These sites are responsible
for the substrate specificity (Schechter and
Berger 1967; Barrett and Kirschke 1981; Storer
and Menard 1994, 1996). The S?-subsites, while
conserved, show an obvious difference in cpLs
and cpBs. Cathepsin B-like proteases have an
occluding loop which contains the two histi-
dines (His110 and His111 in cathepsin B human
liver; His101 and His 102 in L. major cpB,
shown in Table I) that are required for the exo-
peptidase activity (dipeptidyl carboxypeptidase
activity) of cathepsin B (Aronson and Barrett
1978; Turk et al. 1995; Illy et al. 1997). The
polypeptide chain of cysteine proteases from
the papain family is folded into two domains,
between which the V-shaped active-site cleft is
formed (Musil et al. 1991). Although the inter-
domain interface has a similar shape in papain
and in cathepsin B, the amino acid residues in-
volved are different. Specific residues of the
Structural Alignment of Active Site Residues
A77E245 V247Y75 P76A173A200 C29 H199N219 Q23 W221H110 H111
L. major cpL
L. major cpB
Note. Key residues of the active site clefts of papain family cysteine proteases are shown. The top three proteases served
as reference proteins to build the homology models of the two Leishmania proteases. The catalytic triad and the S? subsites
are highly conserved whereas the S subsites show differences responsible for differences in substrate specificity. Note the
two histidines in the S2? subsite, appearing only in cathepsin B and L. major cpB.
SELZER ET AL.
domain interface of human liver cathepsin B are
Glu36, Ser39, Glu171, Arg202, and Ala218
(Musil et al. 1991). The occluding loop and the
interface residues are structural features of
members of the cathepsin B subfamily. An oc-
cluding loop with the two histidines appears in
L. major cpB and the residues within the inter-
face are identical to human liver cathepsin B
with only one minor exchange of Arg202 to
Lys. With these structural features the L. major
cpB is clearly a member of the cathepsin B
subfamily. Consistent with this conclusion is
the fact that the primary sequence of L. major
cpB has high overall identity (54%) to human
liver cathepsin B but a much lower overall iden-
tity to papain (30%) or other cathepsin L-like
proteases. The sequence identity of the L. major
cpB (Sakanari et al. 1997) to the L. mexicana
cpB, (Bart et al. 1995) is 80% consistent with
the close relationship of these two species.
A biochemical property of cathepsin B-like
proteases is their ability to cleave synthetic sub-
strates with Arg in position P2(e.g., Z-Arg-Arg-
AMC). Cathepsin L-like proteases have a
stricter preference for substrates with a Phe in
position P2(e.g., Z-Phe-Arg-AMC) (Khouri et
al. 1991; Storer and Menard 1996). In the assay
system used in this study bovine spleen cathep-
sin B cleaves both substrates at about the same
rate, whereas papain has about 10% activity to-
ward Z-Arg-Arg-AMC compared to 100% to-
ward Z-Phe-Arg-AMC (data not shown). This is
consistent with previous data indicating that ca-
thepsin B prefers Phe over Arg in position P2
only 3.6-fold while papain favors it by a factor
of 904 (Storer and Menard 1996). Glutamic acid
205 (papain numbering, Table I) is responsible
for the ability to cleave substrates with an Arg
in position P2. In a double mutant of papain
(Val133→ Ala/Ser205→ Glu), exchanging ser-
ine 205 for glutamic acid by site-directed mu-
tagenesis enhanced the ability of papain to
cleave Z-Arg-Arg-AMC substrates by about
100-fold (Khouri et al. 1991). Recently the
crystal structure of cathepsin B and a peptide
inhibitor with an Arg in position P2finally
proved this theory (Jia et al. 1995). Position 205
is also a glutamic acid in cruzain which has the
ability to cleave Z-Arg-Arg-AMC but still pre-
fers Z-Phe-Arg-AMC at acidic pH (data not
shown). L. major cpB shows no activity toward
Z-Arg-Arg-AMC but is active against Z-Phe-
Arg-AMC. Its counterpart from L. mexicana
prefers Phe over Arg in Position P2but still
shows 10% activity toward Z-Arg-Arg-AMC
(Robertson and Coombs 1993). This difference
in the substrate specificity is reflected in posi-
tion 205. The L. mexicana cpB has a serine, like
papain, at this position, whereas L. major cpB
has a glycine (Table I). Replacement of glu-
tamic acid 205 by a glycine in L. major cpB
results in the S2 site having a much larger and
more hydrophobic pocket (Fig. 1, Fig. 2). Thus,
while L. major cpB belongs to the cathepsin B
subfamily by virtue of its structural homology,
its activity is more ‘‘cathepsin L-like’’. This
observation confirms that there is a need for
analyzing sequence, structure, and enzymatic
data before concluding an enzyme is a member
of a specific protease subfamily. It also under-
scores the potential functional diversity of the
cathepsin B proteases as evidenced by the abil-
ity to significantly alter substrate specificity by
a single residue change in S2.
The structural models of the L. major cpB
and cpL were used to search the ACD for po-
tential protease inhibitors. ACD is a database of
about 150,000 commercially available com-
pounds formerly known as the Fine Chemicals
Directory distributed by Molecular Design Lim-
ited Information System (San Leandro, CA).
This approach was performed with the software
DOCK3.5 (University of California, San Fran-
cisco) (Kuntz 1992; Kuntz et al. 1994). The
computational software DOCK3.5 is a suite of
programs for locating feasible binding orienta-
tions, given the structure of a ligand molecule
and a receptor molecule. In SEARCH mode,
orientations are generated for each of the scor-
ing molecules in a database, then the best-
scoring orientation of each molecule is saved,
and the best-scoring molecules are saved to a
file. Two scoring lists were generated, a contact
score and a force field interaction energy score.
The top 3% for each scoring method was saved
and visually examined for size, packing, and
L. major: MOLECULAR MODELING AND DRUG DESIGN
interactions within the active site. In order to
find new lead inhibitors, the most promising
compounds (about 0.05%) were selected to be
tested in vitro. Out of 150,000 compounds 4500
were saved for each scoring method (force field
and contact score). Following visual examina-
tion of fit, 69 compounds were finally selected
for testing. Of these 69 compounds, 43 were
from the contact list, 26 were from the force
field list, and 15 compounds appeared on both
lists. Forty-five of the 69 cpB compounds also
appeared within the top 3% lists (contact and
force field score) from the L. major cpL screen.
Because the L. major cpB is available in re-
agent quantities it was chosen for biochemical
screens. Eighteen of the chosen compounds
showed an IC50between 50 and 100 ?M and 3
(PS44, reactive orange, 16, 2-((4-(7-acetamido-
nyl)-ethyl sulfate, contact list; PS50, succin-
imidyl 4-(p-maleimidophenyl)butyrate, both
lists; PS28, 3,5 dichlorofolic acid, L-glutamic
both lists) showed an IC50below 50 ?M (Table
II, Fig. 3). Thirteen of the 18 compounds in-
cluding PS28, PS44, and PS50 were also found
on the lists for the L. major cpL. PS50 is likely
an irreversible inactivator of the protease. Its
maleimide group binds specifically to free sul-
fur groups (Smyth et al. 1964). In L. major cpB
the highly reactive sulfur in the cysteine of the
catalytic triad is a likely target. This would lead
to a covalent bond between the maleimide
group and the L. major cpB active site. Indeed
inhibition by PS50 could not be competed by
excess amounts of substrate consistent with ir-
PS44 contains three sulfate groups which
make it a highly negatively charged compound
and difficult to derivatize. Most likely these hy-
drophilic groups are responsible for the inhibi-
tory activity. However, since the S-subsites of
the target proteases are rather hydrophobic,
PS44 was not further considered as a lead com-
The most promising lead compound is PS28.
PS28 is a derivative of folic acid and therefore
FIG. 1. Homology-based space filling protein models of the L. major cpL (a) and cpB (b). The active site cleft
is formed between the two main domains. The S subsites (S) and the S? subsites (S?) are shown in cyan, the
catalytic triad (T) in yellow. The two histidines of the occluding loop are shown in red.
SELZER ET AL.
belongs to a well-studied group of compounds,
some of which are drugs. Dihydrofolic acid is a
substrate of the dihydrofolic acid reductase
(DHFR), itself a drug target (Schweitzer et al.
1990). Methotrexate and aminopterin, used in
anticancer treatment, are highly active inhibi-
tors of DHFR (Schweitzer et al. 1990). These
latter 2 compounds were among the 18 com-
pounds showing IC50’s between 50 and 100
?M. While PS28 was a byproduct of anticancer
drug development (Martinelli and Chaykovsky
1980), until now no enzyme inhibition activity,
or biological activity, had been reported for this
The three-dimensional structure of PS28 was
modeled with Sybyl and docked into the active
site of L. major cpB by using the SINGLE mode
function of DOCK3.5. In SINGLE mode
DOCK3.5 generates many orientations of one
ligand for both contact scoring and force field
scoring. These orientations can be examined for
interactions of the ligand and its receptor to de-
termine the most likely orientation. A high per-
centage of the orientations (contact and force
field score) generated by DOCK3.5 for PS28
and L. major cpB showed the pteroic acid half
Inhibition of Cysteine Proteases
Note. Inhibitory activity of first (PS)- and second-
generation compounds (ZL) toward different cysteine pro-
teases. Note differences in activity of parasite versus non-
parasite proteases from 2- to 100-fold. IC50determined
from plot of five or more assays at specific inhibitor con-
FIG. 2. The main residues of the active site cleft of the L. major cpB protease and a putative binding
orientation generated by DOCK3.5 of the lead compound PS28. Numbers refer to the L. major cpB mature
sequence. Residues are colored: S, subsites in magenta, catalytic triad in yellow; S?, subsites in cyan and
backbone in gray. The ligand is colored: carbons in green, nitrogens in blue, oxygens in red, and chlorines in
gray. Note the position of the hydrophilic glutamic acid group of the ligand close to the hydrophobic S subsites.
L. major: MOLECULAR MODELING AND DRUG DESIGN
of the compound close to the catalytic triad and
the S?-site and at least one of the chlorides
would interact with the catalytic triad (Fig. 2).
The hydrophilic glutamic acid was predicted as
pointing into either the hydrophobic S1-site or
the wide open hydrophobic S2-site, both unfa-
vored interactions. This suggests that com-
pounds having a more hydrophobic moiety may
be better inhibitors. The synthesis of such
chemical derivatives is now a goal of our proj-
ect. The carboxylic acids of the glutamic acid
will be eliminated, and the glutamic acid will be
replaced by phenylalanine or homophenylala-
A second approach to inhibitor discovery and
design for the L. major cysteine proteases was
to test a library of compounds, synthesized by
combinatorial chemistry, and based on an origi-
nal lead compound [oxalic bis(2-hydroxy-1-
naphthylmethylene)hydrazide] found by DOCK
using the malaria cysteine protease falcipain as
a target (Ring et al. 1993). Of special note is the
fact that the hydrazide lead was also among the
compounds selected by computer search of
ACD for the L. major cpB and cpL. From this
lead a second generation of compounds had
been synthesized (ZL-compounds, Fig. 3) and
tested against falcipain as well as against the
malaria parasite in cell culture. Some of these
compounds proved to be potent inhibitors of
both the protease and parasite growth in culture
(Li et al. 1994, 1995, 1996). Although the over-
all sequence identity of falcipain and the L. ma-
jor cpB is only 31%, key regions of substrate
binding are conserved and several of these in-
hibitors had IC50’s in the nanomolar range ver-
sus the L. major cpB (Table II). The enhanced
inhibitory activity of these second-generation
compounds is the result of specific synthetic
modifications based on computer predictions
made with DOCK. For the L. major cpB, these
results again emphasize that while its overall
homology is to cathepsin B, it shares substrate-
binding similarities with cathepsin L-like en-
zymes like falcipain.
The three hydrazides (ZLIII43A, ZLIII115A,
and ZLIII133A) were also tested in L. major
cell cultures. Inhibitors were added to replicat-
ing promastigotes (106cells ml−1) as a single
dose and cell growth was monitored over 3
days. All three compounds showed very similar
effects on the replication rate of the parasite.
Concentrations of 5 ?M led to about half maxi-
mal growth whereas 20 and 50 ?M totally in-
hibited cell growth (Fig. 4). Exchanging the me-
dia every day for a total of 3 days, thereby keep-
FIG. 3. Chemical structures of first (PS28)- and second-generation (ZL’s) compounds.
SELZER ET AL.
ing inhibitor concentrations stable (20 and 50
?M), led to death of all the parasites. After the
fourth day the media was exchanged with fresh
media without inhibitor and the flasks were kept
under culture conditions. Even after 10 days no
parasites could be detected, indicating a total
cure of the Leishmania culture by the cysteine
protease inhibitors. ZLIII43A and ZLIII115A at
40 ?M had no effect on the growth or appear-
ance of J774 cells, a mammalian macrophage
cell line. Evaluation of these compounds on in-
tracellular L. major amastigotes and in mice
will be reported in another paper (Selzer, Pin-
gel, Hsieh, Chan, Engel, Sakanari, and McKer-
row, submitted for publication).
The results of these cell culture assays sug-
gest that the cysteine proteases of L. major are
crucial to the parasite. Mottram and co-workers
reported that a cpL null mutant of L. mexicana
showed an 80% decrease in virulence but was
still able to grow (Mottram et al. 1996). L. mexi-
cana has multiple cysteine proteases of both the
cpL and cpB type (Robertson et al. 1996). Since
the inhibitors we tested inhibit both cpLs and
cpBs, they may target both types of proteases
within the parasite, overcoming the redundancy
in activity suggested by the null mutant studies.
Because the inhibitors are reversible, it was not
possible to identify specific protease targets by
inhibitor labeling. The lack of toxicity to mam-
malian cells at the inhibitor concentrations
which kill parasites may reflect a greater prote-
ase redundancy in mammalian lysosomes (Ma-
son 1991; Kirschke et al. 1995) or differential
uptake of inhibitor by parasites versus host cells
(McGrath et al. 1995).
The authors thank Christopher Franklin for excellent
technical assistance. This investigation received financial
support from the UNDP/WORLD BANK/WHO Special
Programme for Research and Training in Tropical Diseases
(TDR) (Grant No. T21/181/29 to J.A.S. and No.
M20/181/232 to F.E.C.) and the National Institutes of
Health (AI35707-1) to J.H.M. The Midas Plus program
from the Computer Graphics Laboratory, University of
California, San Francisco, was supported by the National
Institutes of Health (RR-01081). James H. McKerrow is
supported by a Burroughs Wellcome Molecular Parasitol-
ogy Scholar Award. Paul M. Selzer is supported by a fel-
lowship of the Deutsche Forschungsgemeinschaft (Se
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Received 28 March 1997; accepted with revision 18 August
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