Identification of a new class of nonpeptidic inhibitors of cruzain

Article abstract of DOI:10.1021/ja710254m

Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of Chagas disease and is a promising target for the development of new chemotherapy. With the goal of developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was used to screen a library of protease substrates initially designed to target the homologous human protease cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor 38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored β-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely depend on leaving group pK_a, with 2,3,5,6- tetrafluorophenoxymethyl ketone 54 identified as one of the most potent inhibitors with a second-order inactivation constant of 147,000 s^-1 M^-1. This inhibitor completely eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead to new chemotherapeutics for Chagas disease.

Full text of DOI:10.1021/ja710254m

Published on Web 04/25/2008
Identification of a New Class of Nonpeptidic Inhibitors of
Cruzain
Katrien Brak,† Patricia S. Doyle,‡ James H. McKerrow,‡ and Jonathan A. Ellman*,†
Department of Chemistry, UniVersity of California, Berkeley, California 94720, and Department
of Pathology, UniVersity of California, San Francisco, California 94158-2330
Received November 12, 2007; E-mail: jellman@uclink.berkeley.edu; jmck@cgl.ucsf.edu
Abstract: Cruzain is the major cysteine protease of Trypanosoma cruzi, which is the causative agent of
Chagas disease and is a promising target for the development of new chemotherapy. With the goal of
developing potent nonpeptidic inhibitors of cruzain, the substrate activity screening (SAS) method was
used to screen a library of protease substrates initially designed to target the homologous human protease
cathepsin S. Structure-based design was next used to further improve substrate cleavage efficiency by
introducing additional binding interactions in the S3 pocket of cruzain. The optimized substrates were then
converted to inhibitors by the introduction of cysteine protease mechanism-based pharmacophores. Inhibitor
38 was determined to be reversible even though it incorporated the vinyl sulfone pharmacophore that is
well documented to give irreversible cruzain inhibition for peptidic inhibitors. The previously unexplored
ꢀ-chloro vinyl sulfone pharmacophore provided mechanistic insight that led to the development of potent
irreversible acyl- and aryl-oxymethyl ketone cruzain inhibitors. For these inhibitors, potency did not solely
depend on leaving group pKa, with 2,3,5,6-tetrafluorophenoxymethyl ketone 54 identified as one of the
most potent inhibitors with a second-order inactivation constant of 147,000 s-1 M-1. This inhibitor completely
eradicated the T. cruzi parasite from mammalian cell cultures and consequently has the potential to lead
to new chemotherapeutics for Chagas disease.
Introduction
Chagas disease (American trypanosomiasis), caused by the
parasitic protozoan Trypanosoma cruzi, is the leading cause of
heart disease in Latin America. Today, at least 12 million people
are infected with the parasite, resulting in more than 50,000
deaths each year.1 Chemotherapy for Chagas disease is unsat-
isfactory with current drugs, nifurtimox and benznidazole,
having significant toxic side effects.2 Due to the toxicity of
current chemotherapy and emerging drug resistance, there is
an urgent need for developing an effective therapy against
Chagas disease.
Figure 1. Most advanced inhibitor of cruzain.
Cruzain, a cysteine protease of the papain family, is the
primary cysteine protease of the T. cruzi parasite. It is involved
in intracellular replication and differentiation and is essential
at all stages of the parasite’s life cycle.3 Recently, it has been
demonstrated that T. cruzi infection can be cured in cell, mouse,
and dog models by treatment with irreversible inhibitors of
cruzain.4,5 Parasite vulnerability to cruzain inhibition results
from the lack of redundancy of this enzyme. Moreover, parasite
localization provides a means for preferential inhibition of
cruzain over the highly homologous human papain superfamily
cysteine proteases cathepsins B, L, K, S, F, and V because the
parasite resides in the host cell cytoplasm, whereas the cathe-
psins are located in the less accessible lysosomes.6 For these
reasons, cruzain is a highly attractive therapeutic target for the
treatment of Chagas disease.7
Dipeptidyl vinyl sulfone 1 is the most advanced inhibitor of
cruzain and is currently in preclinical trials (Figure 1).8 Although
this peptidic inhibitor has shown good efficacy with minimal
toxicity, nonpeptidic inhibitors with improved oral bioavail-
ability could prove even more effective. As only irreversible
inhibitors of cruzain have been successful in curing parasitic
† University of California, Berkeley.
‡ University of California, San Francisco.
(1) http://www.who.int/tdr/diseases/chagas/direction.htm.
(2) de Castro, S. L. Acta Trop. 1993, 53, 83–98.
(3) Harth, G.; Andrews, N.; Mills, A. A.; Engel, J. C.; Smith, R.;
McKerrow, J. H. Mol. Biochem. Parasitol. 1993, 58, 17–24.
(4) Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. J. Exp. Med.
1998, 188, 725–734.
(6) McKerrow, J. H.; Engel, J. C.; Caffrey, C. R. Biorg. Med. Chem. 1999,
7, 639–644.
(7) McKerrow, J. H.; McGrath, M. E.; Engel, J. C. Parasitol. Today 1995,
(5) Barr, S. C.; Warner, K. L.; Kornreic, B. G.; Piscitelli, J.; Wolfe, A.;
Benet, L.; McKerrow, J. H. Antimicrob. Agents Chemother. 2005, 49,
5160–5161.
11, 279–282.
(8) Jacobsen, W.; Christians, U.; Benet, L. Z. Drug Metab. Dispos. 2000,
28, 1343–1351.
9
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10.1021/ja710254m CCC: $40.75
2008 American Chemical Society

New Class of Nonpeptidic Inhibitors of Cruzain
A R T I C L E S
Scheme 1. Fluorogenic substrate screening
Table 1. Representative Substrates from the Initial 1,2,3-Triazole
Library
infections, we sought to develop nonpeptidic irreversible inhibi-
tors of cruzain.9
Recently, we developed substrate activity screening (SAS)
as a new method for the rapid identification of nonpeptidic
enzyme inhibitors.10–14 The SAS method consists of the
identification of nonpeptidic substrate fragments, substrate
optimization, and then conversion of optimal substrates to
inhibitors. Significantly, the SAS method has successfully been
applied to the papain superfamily protease cathepsin S,10,12,13
which has high homology to cruzain.15 Using a focused substrate
library developed for cathepsin S as a starting point, we report
herein the development of a new class of nonpeptidic 2,3,5,6-
tetrafluorophenoxymethyl ketone inhibitors that exhibit potent
inhibitory activity against cruzain as well as complete eradication
of T. cruzi parasites in cell culture. This class of compounds
represents a promising and novel inhibitor class for the treatment
of Chagas disease.
Results and Discussion
11). In contrast, meta and para substituents resulted in increases
in substrate activity (substrates 9, 10 and 12, 13) with p-
methoxy-substituted benzamide substrate 13 identified as the
most efficient substrate from the screening of the initial triazole
library.
Structure-Guided Substrate Optimization. Further optimiza-
tion of substrate binding to cruzain was accomplished using
structure-based design. Recently, a crystal structure was obtained
of chloromethyl ketone inhibitor 14 bound to cathepsin S.12 The
amino acid sequences of cathepsin S and cruzain are 38%
homologous and their active sites are nearly identical (Figure
2).15 Taking into account the high homology between these two
enzymes, molecular replacement was performed to model
inhibitor binding to cruzain (Figure 3).
Due to the similarities in the active sites, the inhibitors are
predicted to bind in a similar fashion with the n-butyl group in
the S1 pocket, the methyl and isopropyl groups in the S2 pocket,
and the benzamide moiety in the S3 pocket. The majority of
prior inhibitor development for cruzain has focused on the S1′,
S1, and S2 pockets. The S3 pocket of cruzain is largely
Initial Screening. High correlation between substrate cleav-
age efficiency and inhibitory activity was observed in the
previous development of cathepsin S inhibitors.10,12 Substrate
analogues were therefore first evaluated and optimized before
conversion to inhibitors. A triazole-based substrate library
consisting of more than 150 substrates was screened against
cruzain. Substrate activity was measured by monitoring
liberation of the 7-amino-4-methyl coumarin acetic acid
(AMCA) fluorophore, which results from protease-catalyzed
amide bond hydrolysis (Scheme 1).
Shown in Table 1 is the structure–activity relationship (SAR)
for a subset of substrates from the triazole library that exempli-
fies cruzain’s substrate specificity requirements. The weakest
substrate for which a signal could be detected was substrate 2
that incorporated a simple benzyl substituent on the triazole ring.
A variety of more active hydroxyl-substituted substrates were
screened, and the optimal aliphatic functionalities identified were
the methyl and isopropyl substituents present in substrate 4.
Replacement of the hydroxyl with a benzamide moiety in
substrate 5 resulted in an increase in cleavage efficiency. The
epimeric compounds 6 and 7 demonstrate that cruzain shows
strong chiral recognition with epimer 7 being much more active.
Substitutions on the benzamide moiety indicated that ortho
substitutions were not tolerated by cruzain (substrates 8 and
(9) For the only other report of nonpeptidic cruzain inhibitors, see: Du,
X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.; Holler, T. P.;
McKerrow, J. H.; Cohen, F. E. J. Med. Chem. 2002, 45, 2695–2707.
(10) Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain, R. K.; Ellman,
J. A. J. Am. Chem. Soc. 2005, 127, 15521–15527.
(11) Salisbury, C. M.; Ellman, J. A. ChemBioChem 2006, 7, 1034–1037.
(12) Patterson, A. W.; Wood, W. J. L.; Hornsby, M.; Lesley, S.; Spraggon,
G.; Ellman, J. A. J. Med. Chem. 2006, 49, 6298–6307.
Figure 2. Alignment of cathepsin S (PDB ID: 2H7J) and cruzain (PDB
ID: 1F2C) amino acid sequences with areas around the catalytic triad
highlighted in yellow. Identical residues are indicated with “ * ” and similar
residues are indicated with “. ”. The figure was produced using Swiss-Pdb
Viewer (http://ca.expasy.org/spdbv/).
(13) Inagaki, H.; Tsuruoka, H.; Hornsby, M.; Lesley, S. A.; Spraggon, G.;
Ellman, J. A. J. Med. Chem. 2007, 50, 2693–2699.
(14) Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman, J. A.
J. Am. Chem. Soc. 2007, 129, 9613–9615.
(15) Bromme, D.; McGrath, M. E. Protein Sci. 1996, 5, 789–791.
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A R T I C L E S
Brak et al.
bond, which could be interacting with the serine residue in the
S3 pocket (Figure 3b). When comparing naphthyl 20 and
quinoline 30, a 4-fold increase in activity was observed
presumably due to this polar interaction. Moreover, the 4-fold
increase in cleavage efficiency of indole 29 relative to benzo-
triazole 21 demonstrated that hydrophobic interactions also
contribute to binding. Substrate 18 incorporating a morpholine
moiety was prepared because this substituent has led to potent
vinyl sulfone inhibitors of cruzain.16 However, this substituent
resulted in a decrease in substrate cleavage efficiency.
A notable feature in the inhibitor binding model is the
nonessential nature of the benzamide carbonyl (Figure 3b).
Therefore, amine 33 corresponding to benzamide substrate 7
was prepared, resulting in a 3-fold increase in cleavage
efficiency (Table 3). To determine if amine substrate SAR
correlated with the SAR trends observed for the corresponding
amide substrates, additional amine analogues were synthesized
and evaluated. High correlation was observed between the SAR
for the amide and amine substrate series, resulting in the
identification of quinoline amine substrate 36 and benzothiazole
amine substrate 37 with 19-fold greater cleavage efficiency than
unsubstituted benzamide 7.
Figure 3. (a) Crystal structure of cathepsin S (PDB ID: 2H7J) and (b)
molecular replacement model of cruzain (PDB ID: 1F2C) with chloromethyl
ketone inhibitor 14. The atoms are shaded according to element: protein
carbons are green, inhibitor carbons are gray, nitrogens are blue, and oxygens
are red. The figure was produced using PyMOL (www.pymol.org).
Conversion of Substrates into Inhibitors. A key advantage
of the SAS method is that the aminocoumarin group has to be
precisely oriented in the active site for amide bond hydrolysis
to occur and can therefore be directly replaced with mechanism-
based pharmacophores. The optimal quinoline amine substrate
36 was first converted to inhibitors to evaluate the effectiveness
of different cysteine protease mechanism-based pharmacophores.
Scheme 2. General Synthesis of Focused Library 1,2,3-Triazole
Substratesa
We initially chose to investigate the vinyl sulfone pharma-
cophore because it has been incorporated in potent inhibitors
of cruzain that have proven effective at eradicating Chagas
disease in both cell culture and animal models.4,5,16,17 Vinyl
sulfone inhibitor 38 was prepared via a Horner-Wadsworth-
Emmons olefination (Scheme 3). Kinetic analysis of the vinyl
sulfone inhibitor, surprisingly, indicated no time dependence
and was consistent with competitive reversible inhibition (Figure
4a).18
To gain further insight, we decided to investigate the
reversible nature of vinyl sulfone inhibitor 38. Vinyl sulfones
are thought to irreversibly alkylate cysteine proteases via a
Michael addition followed by protonation of the R-carbon by
the active-site histidine to form a covalent thioether adduct
(Figure 5a).19 Two potential reasons for the reversibility of vinyl
sulfone 38 are therefore either that the active-site cysteine is
not adding into the vinyl sulfone or that the active-site histidine
is not properly oriented for protonating the resulting anion. We
postulated that a ꢀ-chloro vinyl sulfone could distinguish
between these possibilities because cysteine protease inactivation
could be accomplished via Michael addition followed by
ꢀ-elimination of chloride ion, thereby eliminating the need for
anion protonation (Figure 5b). Although this particular phar-
a Reagents: (a) CuI, i-Pr2EtN, THF, rt; (b) R′CO2H, triphosgene, THF,
rt; (c) R′COH, NaBH(OAc)3, AcOH, THF, rt; (d) CF3CO2H, CH2Cl2, rt.
unexplored with no previous reports of significant binding
interactions in this pocket. Upon closer inspection of our
molecular replacement model, key differences in the S3 pockets
were noted. The S3 pocket of cathepsin S is small and well-
defined, whereas that of cruzain is large and open-ended. To
take advantage of cruzain’s larger S3 pocket, a focused library
of substrate analogues incorporating planar heterocycles in place
of the phenyl ring of the benzamide moiety was designed.
Heterocycles were chosen with potential for hydrophobic
interactions with the hydrophobic side of the pocket and with
potential for hydrogen-bonding interactions with the serine
residue in the S3 pocket.
The synthesis of the 1,4-disubstituted-1,2,3-triazole substrates
containing the AMCA fluorophore was accomplished on solid
support (Scheme 2). We were pleased to find that all but one
of the substrates of the focused library were more active than
the unsubstituted benzamide substrate 7 (Table 2). The most
potent substrates identified were quinoline 30 and benzothiazole
31 with 7–9 fold increases in cleavage efficiency. These
substrates both contained a nitrogen atom para to the amide
(16) Palmer, J. T.; Rasnick, D.; Klaus, J. L.; Bromme, D. J. Med. Chem.
1995, 38, 3193–3196.
(17) Roush, W. R.; Gwaltney, S. L.; Cheng, J.; Scheidt, K. A.; McKerrow,
J. H.; Hansell, E. J. Am. Chem. Soc. 1998, 120, 10994–10995.
(18) For an example of vinyl sulfone inhibitors of cruzain with reversible
nature, see: Scheidt, K. A.; Roush, W. R.; McKerrow, J. H.; Selzer,
P. M.; Hansell, E.; Rosenthal, P. J. Biorg. Med. Chem. 1998, 6, 2477–
2494.
(19) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. ReV.
2002, 102, 4639–4750.
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New Class of Nonpeptidic Inhibitors of Cruzain
A R T I C L E S
Table 2. Cleavage Efficiencies of 1,2,3-Triazole Amide Substrates against Cruzain
Table 3. Comparison of 1,2,3-Triazole Amide and Amine
Substrate Activity against Cruzain
Scheme 3. Synthesis of Vinyl Sulfone Inhibitor 38a
a Reagents: (a) CuI, i-Pr2EtN, THF, rt; (b) diazomethane, THF, rt; (c)
DIBAL-H, Et2O, -78 °C; (d) (OEt)2P(O)CH2SO2Ph, LiBr, NEt3, CH3CN,
-40 °C.
conversion of ketosulfone 45 to vinyl chloride 46 via the vinyl
triflate. Gratifyingly, time-dependence analysis and dilution
experiments indicated that ꢀ-chloro vinyl sulfone inhibitor 43
was an irreversible inhibitor of cruzain (Figure 4b). This result
suggests that the active-site cysteine is adding into vinyl sulfone
38 and that the lack of a protonation event resulted in a
reversible inhibitor. The ꢀ-chloro sulfone inhibitor 43 had a
modest second-order rate of inactivation constant of 805 s-1
M-1 (Table 4). Encouraged by this result, we decided to explore
other pharmacophores that irreversibly inactivate cysteine
proteases according to mechanisms that do not utilize a
protonation step.
macophore has never before been investigated, it is analogous
to previously characterized ꢀ-chloro R,ꢀ-unsaturated ester
inhibitors.20
ꢀ-Chloro vinyl sulfone inhibitor 43 was prepared according
to the route depicted in Scheme 4 with the key step being
(21) Smith, R. A.; Copp, L. J.; Coles, P. J.; Pauls, H. W.; Robinson, V. J.;
Spencer, R. W.; Heard, S. B.; Krantz, A. J. Am. Chem. Soc. 1988,
110, 4429–4431.
(20) Govardhan, C. P.; Abeles, R. H. Arch. Biochem. Biophys. 1996, 330,
(22) Krantz, A.; Copp, L. J.; Coles, P. J.; Smith, R. A.; Heard, S. B.
Biochemistry 1991, 30, 4678–4687.
110–114.
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Scheme 4. Synthesis of ꢀ-Chloro Vinyl Sulfone Inhibitor 43a
a Reagents: (a) diazomethane, THF, rt; (b) methylphenylsulfone, n-BuLi,
THF, 0 to -78 °C; (c) Tf2O, i-Pr2EtN, THF, -20 °C to rt; (d) TBACl,
THF, rt; (e) Na ascorbate, CuSO4, 1:1 H2O/t-BuOH, 39, rt.
Table 4. Second-Order Inactivation Rates of Cruzain Inhibitors
with Varying Pharmacophores
Figure 4. Time-dependence of (a) vinyl sulfone 38 and (b) ꢀ-chloro vinyl
sulfone 43.
Figure 5. Mechanism of inhibition of cysteine proteases by (a) vinyl
sulfones and (b) ꢀ-chloro vinyl sulfones.
a Tests were performed in quadruplicate (SD values included). b pKa
of the carboxylic acid or phenol leaving group. c No time-dependence
observed. d Ref 19. e Ref 18.
Acyloxymethyl ketone inhibitors were designed by Krantz
as more stable halomethyl ketone analogues.21,22 This pharma-
cophore has led to potent time-dependent inhibitors of cathepsins
B, L, and S.19 Hence, we next incorporated the acyloxymethyl
ketone pharmacophore. The synthesis of the acyloxymethyl
ketone inhibitors required the preparation of azide intermediates
47a and 47b (Scheme 5). Beginning with L-norleucine azido
acid 40, the common bromomethyl ketone precursor 48 was
obtained in three steps. Displacement of the bromide followed
by cyclization with propargyl amine 39 then afforded the
acyloxymethyl ketone inhibitors 49 and 50 as mixtures of
diastereomers.
inhibitor of cruzain with a second-order rate constant of 2690
s-1 M-1 (Table 4). Acyloxymethyl ketone inhibitors of the
cathepsins have shown a strong correlation between the leaving-
group pKa and the rate of inactivation.23 Accordingly, we
prepared the 2,6-bis-trifluoromethyl acyloxymethyl ketone
inhibitor 50 and were delighted to find that inhibitor 50 was
58-fold more potent than inhibitor 49 with a second-order rate
constant of 157,000 s-1 M-1 (Table 4). Inhibitors incorporating
this pharmacophore were subsequently prepared corresponding
to both the amides and amines of the benzothiazole and
We initially investigated the 2,6-dimethyl acyloxymethyl
ketone inhibitor 49 and observed that it was an irreversible
(23) Krantz, A. Methods Enzymol. 1994, 244, 656–671.
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New Class of Nonpeptidic Inhibitors of Cruzain
A R T I C L E S
Scheme 5. Synthesis of Aryl- and Acyl-oxymethyl Ketone
Scheme 6. Synthesis of Diastereomerically Pure Aryloxymethyl
Inhibitorsa
Ketone Inhibitor 58a
a Reagents: (a) isobutyl chloroformate, N-methylmorpholine, THF, -40
°C; (b) diazomethane, THF, 0 °C; (c) HBr, THF, 0 °C; (d) KF, DMF, 0
°C; (e) Na ascorbate, CuSO4, 1:1 H2O/t-BuOH, rt.
a Reagents: (a) NaBH4, 95:5 THF/H2O, 0 °C to rt; (b) Na ascorbate,
CuSO4, 1:1 H2O/t-BuOH, rt; (c) Dess-Martin periodonane, CH2Cl2, rt.
Table 5. Second-Order Inactivation Rates of 1,2,3-Triazole
and has a lower molecular weight. This pharmacophore has
proven to be particularly effective for caspase inhibition.25,26
In particular, Idun Pharmaceuticals used 2,3,5,6-tetrafluorophe-
nol as the leaving group in an aryloxymethyl ketone pan-caspase
inhibitor that has progressed to phase II clinical trials.27 In
contrast, there has only been one report of aryloxymethyl ketone
inhibitors of a member of the papain superfamily, and only
modest inhibition was observed.21 Nevertheless, we prepared
2,3,5,6-tetrafluorophenol aryloxymethyl ketone inhibitor 54
(Scheme 5). Unexpectedly, the aryloxymethyl ketone inhibitor
54 was equipotent to the acyloxymethyl ketone inhibitors with
a second-order inactivation constant of 147,000 s-1 M-1 despite
the >105 difference in the acidity of the corresponding leaving
groups (pKa of 2,3,5,6-tetrafluorophenol g 5.5, pKa of 2,6-bis-
trifluoromethyl benzoic acid ) 0.58) (Table 4). Inhibitor potency
is clearly not solely dependent on the pKa values of the leaving
group with leaving-group binding and/or orientation also playing
a significant role.25
Cruzain Inhibitorsa
The preparation of both the acyl- and aryl-oxymethyl ketone
inhibitors resulted in epimerization alpha to the pharmacophore
carbonyl. To investigate the configurational stability of the
inhibitors in ViVo, an alternative sequence for the preparation
of diastereomerically pure aryloxymethyl ketone inhibitor 54
was developed (Scheme 6). Specifically, racemization through
enolization was prevented by reducing the acyloxymethyl ketone
47c prior to the cycloaddition step. Alcohol 57 was then oxidized
back to the acyloxymethyl ketone. The diastereomerically pure
inhibitor 58 was then subjected to the assay buffer conditions
at 37 °C for several hours (Scheme 7). This resulted in complete
racemization of the inhibitor, suggesting that in ViVo the inhibitor
will be able to funnel through the active diastereomer.
Cell Culture Assays. Acyloxymethyl ketone inhibitors
50-53 and aryloxymethyl ketone inhibitors 54-55 were
tested for their effectiveness in eliminating T. cruzi infection
in irradiated (9000 rad) J744 macrophages. Host cells died
of T. cruzi infection after 5 days without treatment (Table 6).
Notably, all of the inhibitors significantly delayed T. cruzi
intracellular replication at concentrations of 5 or 10 µM. The
T. cruzi infected cells were initially treated with all of the
inhibitors at 10 µM concentrations. The cultures treated with
b
a Tests were performed in quadruplicate (SD values included). kinact
c
/Ki (s-1 M-1). kass (s-1 M-1).24
quinoline substrates (Scheme 5). There was good correlation
between substrate activity and inhibitor potency with the amine
inhibitors 50 and 51 being more potent than the amide inhibitors
52 and 53 (Table 5).
The aryloxymethyl ketone pharmacophore has the same
mechanism of inhibition as the acyloxymethyl ketone pharma-
cophore. It is more attractive, however, because it should be
less prone to nucleophilic attack, cannot undergo hydrolysis,
(24) Bieth, J. G. Methods Enzymol. 1995, 248, 59–84.
(25) For an analysis of the rate of inactivation dependence on leaving group
orientation for caspases, see : Brady, K. D.; Giegel, D. A.; Grinnell,
C.; Lunney, E.; Talanian, R. V.; Wong, W.; Walker, N. Biorg. Med.
Chem. 1999, 7, 621–631.
(26) Brady, K. D. Biochemistry 1998, 37, 8508–8515.
(27) Linton, S. D.; et al. J. Med. Chem. 2005, 48, 6779–6782.
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Scheme 7. Configurational Lability of Aryloxymethyl Ketone
Inhibitor 54
ending the treatment). Most significantly, the quinoline aryloxy-
methyl ketone inhibitor 54 was trypanocidal at 10 µM and had
completely eradicated the T. cruzi parasite with no parasites
observed at day 40 postinfection. The performance of inhibitor 54
was comparable to vinyl sulfone 1, which is the most advanced
inhibitor of cruzain to date.
Plasma Stability. Due to the excellent cell culture activity of
inhibitor 54, mouse plasma stability studies were performed by
ADMETRx as a prelude to the inhibitor’s evaluation in animal
models of the Chagas disease. The inhibitor was incubated at
37 °C in mouse plasma for 0, 5, 10, 30, or 60 min and analyzed
by LC/MS for remaining inhibitor. Incubation of the inhibitor
with mouse plasma showed no disappearance of the inhibitor
with time, indicating it to be 100% stable to mouse plasma under
these conditions.
Table 6. Effect of Inhibitors on Survival of T. cruzi Infected J744
Macrophages
survival of
T. cruzi infected
cells (days)a
Conclusion
last day of
treatment (day)
type of
inhibition
cell
toxicitye
cmpd
A potent irreversible 2,3,5,6-tetrafluorophenoxymethyl ketone
inhibitor 54 was developed that completely eradicates T. cruzi
parasites in cell culture. A substrate library containing more
than 150 triazole-based substrates developed for cathepsin S,
was first evaluated against cruzain to define important structural
features for efficient substrate cleavage. Subsequent optimization
of the substrate scaffold in the S3 pocket was guided by
structure-based design and led to nonpeptidic substrates with
even greater cleavage efficiency.
Vinyl sulfone, ꢀ-chloro vinyl sulfone, acyl- and aryloxy-
methyl ketone pharmacophores were then explored in the
conversion of the most efficient substrates to inhibitors. The
ꢀ-chloro vinyl sulfone pharmacophore, which had not previously
been reported, led to key mechanistic insight and ultimately
resulted in the development of potent irreversible aryloxymethyl
ketone inhibitors, a pharmacophore class that had previously
been little explored against the papain superfamily.
none
1
5
>40
30
26
19
23
>40
33
-
-
-
27c
14d
14d
14d
23
trypanocidal
trypanostatic
trypanostatic
trypanostatic
trypanostatic
trypanocidal
trypanostatic
nontoxic
toxic
50b
51b
52b
53
toxic
toxic
nontoxic
nontoxic
nontoxic
54
27c
27c
55b
a Effect of inhibitors on survival of J744 macrophages infected with T.
cruzi trypomastigotes, treated daily with a solution of inhibitor (10 µM).
Survival time is defined as the time before the cell monolayer is destroyed
by the infection.4 b The concentration was lowered to 5 µM after 9 (50), 7
(51), and 12 (52, 55) days. c Treatment was ended to distinguish between
trypanostatic and trypanocidal inhibitors. d Treatment was ended due to
compound toxicity. e Determined by contrast phase microscopy comparison
to uninfected macrophage controls.
each different inhibitor were compared daily by contrast phase
microscopy to uninfected macrophage controls. Inhibitors 50,
51, 52, and 55 showed toxicity as evidenced by cells that
rounded up or detached from the wells, condensed, died, or
became granular. For this reason, the concentration of inhibitors
50, 51, 52, and 55 was lowered to 5 µM. The 2,6-bis-
trifluoromethyl acyloxymethyl ketone inhibitors 50, 51, and 52
remained toxic at 5 µM. As a result, it was necessary to stop
treatment by day 14 after which point the cells died. Acyloxy-
methyl ketone inhibitor 53 was quite effective at 10 µM in
delaying T. cruzi replication; however, by day 23 the cell
monolayer had been destroyed by the infection.
Evaluation of 2,3,5,6-tetrafluorophenoxymethyl ketone inhibi-
tor 54 in animal models of the disease is currently underway.
The nonpeptidic nature of this potent class of inhibitors, coupled
with their potent cell-based activity and plasma stability, makes
these compounds very promising starting points for the develop-
ment of chemotherapy for Chagas disease.
Acknowledgment. J.A.E. acknowledges support from the NIH
(GM54051), and J.H.M. acknowledges support from the NIH
(AI35707) and the Sandler Family Supporting Foundation.
To distinguish between trypanostatic compounds that only delay
parasite replication and trypanocidal compounds that effectively
kill T. cruzi, treatment for the remaining aryloxymethyl ketone
inhibitors was ended on day 27, and the cells were monitored for
two more weeks. Benzothiazole aryloxymethyl ketone inhibitor
55 proved to be trypanostatic at 5 µM against T. cruzi because
parasites destroyed the cell monolayer by day 33 (6 days after
Supporting Information Available: Complete experimental
details, including cell culture assay and plasma stability
procedures, spectral data for all compounds, and full author list
for ref 27. This material is available free of charge via the
Internet at http://pubs.acs.org.
JA710254M
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6410 J. AM. CHEM. SOC. VOL. 130, NO. 20, 2008