Structure-guided development of selective TbcatB inhibitors

Article abstract of DOI:10.1021/jm900908p

The trypanosomal cathepsin TbcatB is essential for parasite survival and is an attractive therapeutic target. Herein we report the structure-guided development of TbcatB inhibitors with specificity relative to rhodesain and human cathepsins B and L. Inhib

Full text of DOI:10.1021/jm900908p

J. Med. Chem. 2009, 52, 6489–6493 6489
DOI: 10.1021/jm900908p
Structure-Guided Development of Selective TbcatB Inhibitors
Jeremy P. Mallari,†,‡ Anang A. Shelat,‡ Aaron Kosinski,‡ Conor R. Caffrey,§ Michele Connelly,‡ Fangyi Zhu,‡
James H. McKerrow,§ and R. Kiplin Guy*,†,‡
†Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, California 94143-2280, ‡Department of
Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and Department of Cellular and
Molecular Pharmacology, University of California, San Francisco, California 94143-2280
§
Received June 19, 2009
The trypanosomal cathepsin TbcatB is essential for parasite survival and is an attractive therapeutic target.
Herein we report the structure-guided development of TbcatB inhibitors with specificity relative to rhodesain
and human cathepsins B and L. Inhibitors were tested for enzymatic activity, trypanocidal activity, and
general cytotoxicity. These data chemically validate TbcatB as a drug target and demonstrate that it is
possible to potently and selectively inhibit TbcatB relative to trypanosomal and human homologues.
Introduction
Inhibitor Design
The burden of human African trypanosomiasis (HATa) on
thehealthandeconomies ofsub-Saharan Africa, coupled with
the well-known shortcomings of the currently available drugs,
clearly illustrates the need for new drug development.1 RNAi
studies have demonstrated the essentiality of the trypanoso-
mal cathepsin TbcatB, both in vitro and in vivo.2,3
We have previously reported that the purine nitrile scaffold
produced several potent inhibitors of TbcatB and that all of
those compounds were also fairly nonselective, being inhibi-
tors of human cathepsin L.4 Furthermore, the lead compound
from that series, 4, showed moderate activity against human
cathepsin B. Cathepsin L has been implicated by conditional
knockout studies in regulating pro-opiomelanocortin hor-
mone production.5,6 While the role of cathepsin B alone
remains unclear,5 the coordinate action of cathepsin B and
cathepsin L has been implicated in maintaining normal brain
physiology in adults.6 For these reasons, this suboptimal
selectivity profile raised concerns as to the feasibility of devel-
oping nontoxic TbcatB inhibitors.
In addition, the first generation compounds were found to
be potent inhibitors of the trypanosomal cathepsin L-like
protease rhodesain (unpublished data), indicating that rho-
desain inhibition may be a determinant of trypanocidal acti-
vity. Although TbcatB is known to be essential,2,3,4 the inter-
play between TbcatB and rhodesain inhibition in killing the
parasite is not well understood.
We sought to address these questions by developing specific
TbcatB inhibitors, relative to cathepsin L, cathepsin B, and
rhodesain using a structure-guided approach. Herein, we report
the development of a series of TbcatB inhibitors with greatly
enhanced selectivity against rhodesain and human cathepsins B
and L. This series includes the most potent TbcatB inhibitors to
date, and these compounds were shown to kill Trypanosoma
brucei with a high degree of selectivity relative to a panel of four
mammalian cell lines (BJ, HEK 293, HEP G2, and Raji).
The previously reported purine nitrile inhibitors of TbcatB
also inhibit rhodesain and human cathepsin L.4 To explore
possible structural explanations for the overlap between the
inhibition profiles of these proteases, lead TbcatB inhibitor 4
was modeled as a covalent adduct with cysteine and docked to
the previously reported homology model of TbcatB4 and crystal
structures of human cathepsin L and rhodesain (PDB codes
1mhw and 2p86, respectively) (Figure 1, see Supporting Infor-
mation for modeling details).
Compound 4 was predicted to make similar interactions
with each protease, consistent with the lack of selectivity
observed for this inhibitor. In each model, the 3-hydroxypro-
pyl side chain of the ligand projects into solvent on the prime
side of the protease binding pocket. The N9 amine forms a
hydrogen bond to the carbonyl of either Gly72 (TbcatB), Gly68
(cathepsin L), or Gly66 (rhodesain). Finally, the 3,4-dichloro-
phenyl ring makes strong van der Waals contact with the well-
defined, hydrophobic S2 pockets of each protease.
In TbcatB, residues His179 to Gly188 form a loop oriented
towardthe primeside of the active site cleft. Consequently, the
entrance to the S2 pocket near Asp165 is much wider in
comparison to those of cathepsin L and rhodesain. In con-
trast, the homologous loop region in cathepsin L points away
from the prime side, in part because of a disulfide bridge
between Cys156 and Cys204. As a result, Met161 truncates
the S2 pocket in cathepsin L. At the other side of the active site
cleft, Asp73 projects toward solvent and acts to constrict the
S3 pocket in TbcatB, while in cathepsin L, the orientation of
Tyr72 results in a much wider S3 pocket that bridges the S2
site via Leu69. Rhodesain shares structural traits from both
the TbcatB and cathepsin models: Leu160 plays a similar role
to Met161 in cathepsin L, but Leu67 and Phe61 occlude the S3
pocket just as Asp73 does in TbcatB.
In summary, the S2 pocket of TbcatB is expected to be
much larger and more negatively charged than the S2 pockets
of rhodesain and cathepsin L, whereas the S3 pocket is most
accessible incathepsin L. Itwas envisioned that the differences
between the proteases’ S2 binding sites could be exploited by
increasing steric bulk at the 6-amino substituent in order to
improve inhibitor potency and selectivity for TbcatB.
*To whom correspondence should be addressed. Phone: (901) 495-
5714. Fax: (901) 495-5715. E-mail: kip.guy@stjude.org.
a Abbreviations: HAT, human African trypanosomiasis; DMF, di-
methylformamide; DMSO, dimethyl sulfoxide; ACN, acetonitrile; PAM-
PA, parallel artificial membrane permeability assay.
r
2009 American Chemical Society
Published on Web 09/21/2009
pubs.acs.org/jmc

6490 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Mallari et al.
Figure 1. Targeting the S2 pocket to increase TbcatB selectivity. Compound 4 (space-filling representation) docked to Connolly surface depictions
of (a) TbcatB, (b) cathepsin L, and (c) rhodesain. Polar pockets are magenta, hydrophobic pockets are green, and exposed surfaces are red.
Scheme 1. Synthesis of Purine-Derived Nitrilesa
Table 1. Aryl Substitutents
a Conditions: (i) 3-Bromobenzylamine, 2-butanol, 60 °C, 3 to 12 h;
(ii) 3-bromopropanol, K2CO3, DMF, 60°C, 10 to 16 h; (iii) NaCN, DMSO,
μW, 180 °C, 160 W, 5-20 min; (iv) Ar-B(OH)2, Na2CO3, Pd(PPh3)4,
1,4-dioxane, μW, 150 °C, 300 W, 5-40 min.
Chemistry
Intermediate 2was synthesized by the general route (Scheme 1)
previously described.4,7 Briefly, 1 was reacted with 3-bromoben-
zylamine in 2-butanol to install the 6-amino substituent. The
crude reaction was concentrated in vacuo and resuspended in
dimethylformamide (DMF) with K2CO3. Alkylation at N9
was accomplished by heating the crude reaction product with
3-bromopropanol. Purification was accomplished by flash chro-
matography, and overall yield for the two reactions was 60%.
Subsequent reaction with sodium cyanide in dimethyl sulfoxide
(DMSO) with microwave acceleration afforded intermediate 2,
which was purified by preparative C18 chromatography with a
resulting yield of 70%. Installation of the distal aryl ring was
performed by Suzuki cross coupling. The desired aryl boronic
acid, intermediate 2, Na2CO3, and Pd(PPh3)4 were reacted in 1,4
dioxane with microwave acceleration. The target inhibitors
3(a-ae) were purified by preparative C18 chromatography with
yields of 10-60%. Purity of target compounds of g95% was
confirmed by LCMS on both C4 and C18 columns.
Determining the Activity of Purine Inhibitors 3(a-ae).
Inhibitors 3(a-ae) were tested in vitro to determine inhibitory
activity against TbcatB, rhodesain, cathepsin L, and cathepsin B
(Table 2, 3). Compounds were also assayed to determine
antiproliferative activity against the cultured bloodstream form
of T. brucei (Table 4). Finally, cytotoxicity was evaluated in BJ,
HEK 293, Raji, and HEP G2 cell lines to determine a thera-
peutic window.
The initial inhibitor set focused on introducing sterically and
electronically varied aryl groups at the 30 position of the
6-amino benzyl ring (Table 2). This design strategy was quite
successful, producing several inhibitors with greater than
5-fold enhanced potency against TbcatB relative to parent
compound 4 (EC50 =1.4 μM). It was found that a wide range
of aryl moieties were suitable, including small heterocycles,
substituted phenyl groups, and larger fused ring systems.
Activity against cathepsin B in this series is almost nonexistent,
especially among the most potent TbcatB inhibitors. In addi-
tion, many compounds in this series (3c, 3h, 3o, 3p, 3v) showed
modest (2-3 fold) selectivity for TbcatB over cathepsin L and
rhodesain, a marked improvement over the parent compound,
which preferentially inhibits both proteases (approximately
10-and 25-fold, respectively) over TbcatB. Compound 3b was
the most selective TbcatB inhibitor relative to both cathepsin L
(∼5-fold) and rhodesain (∼25-fold). This inhibitor was one of
only two compounds (3b and 3n) to incorporate an ortho-
substituted biphenyl group, and it was hypothesized that this
substitution pattern constrains the biphenyl moiety in a con-
formation that gives rise to TbcatB selectivity.
To test this hypothesis, a second series of compounds was
synthesized to further optimize the ortho-substituent at the distal
aryl ring (Table 3). This round of optimization yielded a com-
pound with exquisite selectivity for TbcatB relative to rhodesain
(3ab, >100-fold selective) as well as the compound most sele-
ctive for TbcatB relative to cathepsin L (3aa, >10-fold selective).
Compounds 3ac-3ae all displayed good selectivity relative to
rhodesain and modest selectivity relative to cathepsin L.
Each inhibitor was screened against a panel of four mam-
malian cell lines (BJ, Raji, Hep G2, and HEK 293). Almost
every inhibitor displayed EC50 values weaker than 10-20 μM

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6491
Table 2. Protease Inhibition of Initial Inhibitor Series
Table 4. Trypanocidal Activity and Potency against Trypanosomal
Proteases
IC50
vs Tbcat B vs rhodesain vs T. brucei
IC50
EC50
compd
Ar
(μM)
(μM)
(μM)
3
phenyl
2-Cl-phenyl-
2-Me-phenyl-
1.8 ( 0.2
1.3 ( 0.1
0.39 ( 0.03
0.7 ( 0.2
20 ( 15
>50
1.4 ( 0.2
4.6 ( 0.3
1.05 ( 0.09
0.63 ( 0.08
0.63 ( 0.09
0.7 ( 0.1
3aa
3ab
3ac
3ad
3ae
IC50 vs
ID cat B rhodesain
(μM) (μM)
IC50 vs
IC50 vs
cat L
IC50 vs
cat B
2, 6 dimethyl phenyl- 0.27 ( 0.03
>15
>15
2-ipr-phenyl-
2-methoxycarbonyl-
phenyl-
0.31 ( 0.03
10 ( 0.2
compd
3a phenyl
3b 2-CF3-phenyl-
3c 3-vinyl-phenyl-
3d 3-Cl-phenyl-
Ar
(μM)
(μM)
>10
1.8 ( 0.2 0.7 ( 0.2 0.8 ( 0.3 8.4 ( 0.8
0.28 ( 0.03 8 ( 3 1.5 ( 0.6 >15
0.8 ( 0.2 1.5 ( 0.1 1.5 ( 0.2 >50
0.8 ( 0.1 1.2 ( 0.4 0.7 ( 0.2 >50
3b
3c
3d
3e
2-CF3-phenyl-
3-vinyl-phenyl-
3-Cl-phenyl-
0.28 ( 0.03
0.8 ( 0.2
0.8 ( 0.1
0.8 ( 0.1
8 ( 3
0.46 ( 0.08
2.6 ( 0.3
3.7 ( 0.7
2.7 ( 0.4
1.5 ( 0.1
1.2 ( 0.4
0.5 ( 0.2
3e 3-methoxycarbonyl- 0.8 ( 0.1 0.5 ( 0.2 0.8 ( 0.2 >15
phenyl-
3-methoxycarbonyl-
phenyl-
3f
3-acetamidophenyl- 1.6 ( 0.2 0.40 ( 0.07 0.4 ( 0.1 >15
3f
3g
3-acetamidophenyl-
3-dimethylamino-
phenyl-
1.6 ( 0.2
1.2 ( 0.2
0.40 ( 0.07
0.9 ( 0.3
4.1 ( 0.5
18 ( 0.2
3g 3-dimethylamino-
phenyl-
3h 4-vinyl-phenyl-
3i
3j
1.2 ( 0.2 0.9 ( 0.3 1.4 ( 0.4 >15
0.9 ( 0.4 1.8 ( 0.2 1.1 ( 0.1 >50
1.7 ( 0.2 1.4 ( 0.1 1.9 ( 0.7 >15
3h
3i
3j
4-vinyl-phenyl-
4-Cl-phenyl-
0.9 ( 0.4
1.7 ( 0.2
1.6 ( 0.3
1.8 ( 0.2
1.4 ( 0.1
1.2 ( 0.3
2.2 ( 0.2
1.9 ( 0.4
4.0 ( 0.6
4-Cl-phenyl-
4-methoxycarbonyl- 1.6 ( 0.3 1.2 ( 0.3 1.8 ( 0.3 >50
phenyl-
4-methoxycarbonyl-
phenyl-
3k 4-methoxy-phenyl- 1.2 ( 0.9 0.8 ( 0.1 1.1 ( 0.3 >50
3l
3m 3,5-Cl2-phenyl-
3n 3-bromo-2-isopro-
poxy-phenyl-
3o pentaflourophenyl- 0.9 ( 0.3 5.2 ( 0.9 2.7 ( 0.5 >15
3p 2-napthyl-
3q furan-2-yl-
3r
3s
3t
3u pyridin-3-yl-
3v 2,4-dimethoxypyri- 2.9 ( 0.7
midin-5-yl-
3w indol-5-yl-
3x indol-6-yl-
3y 1-phenylsulfonyl-
indol-2-yl
3z 1-phenylsulfonyl-
indol-3-yl
3k
3l
4-methoxy-phenyl-
3,4-Cl2-phenyl-
3,5-Cl2-phenyl-
3-bromo-2-isopro-
poxy-phenyl-
pentaflourophenyl-
2-napthyl-
1.2 ( 0.9
1.2 ( 0.2
1.2 ( 0.1
0.9 ( 0.2
0.8 ( 0.1
0.9 ( 0.2
1.1 ( 0.2
1.5 ( 0.3
2.9 ( 0.3
2.3 ( 0.2
3.3 ( 0.4
1.1 ( 0.1
3,4-Cl2-phenyl-
1.2 ( 0.2 0.9 ( 0.2 1.6 ( 0.3 >50
1.2 ( 0.1 1.1 ( 0.2 0.9 ( 0.2 >50
0.9 ( 0.2 1.5 ( 0.3 1.3 ( 0.2 >50
3m
3n
3o
3p
3q
3r
3s
3t
0.9 ( 0.3
0.23 ( 0.03
1.0 ( 0.3
0.46 ( 0.1
0.6 ( 0.3
0.37 ( 0.09 0.24 ( 0.04 0.43 ( 0.06
0.6 ( 0.2
2.9 ( 0.7
5.2 ( 0.9
0.7 ( 0.1
31 ( 0.07
0.03 ( 0.02 0.56 ( 0.08
0.8 ( 0.3 1.0 ( 0.1
4.7 ( 0.5
1.1 ( 0.2
1.6 ( 0.3
0.23 ( 0.03 0.7 ( 0.1 0.8 ( 0.1 >50
1.0 ( 0.3 0.31 ( 0.07 0.84 ( 0.09 4.7 ( 0.4
0.46 ( 0.1 0.03 ( 0.02 0.6 ( 0.1 8.6 ( 0.5
0.6 ( 0.3 0.8 ( 0.3 0.9 ( 0.1 2.5 ( 0.2
0.37 ( 0.09 0.24 ( 0.04 0.48 ( 0.07 4.0 ( 0.1
0.6 ( 0.2 0.26 ( 0.07 1.6 ( 0.3 3.9 ( 0.3
furan-2-yl-
furan-3-yl-
furan-3-yl-
thiophen-2-yl-
thiophen-3-yl-
thiophen-2-yl-
thiophen-3-yl-
pyridin-3-yl-
4 ( 2
6 ( 1
>15
3u
3v
0.26 ( 0.07
4 ( 2
1.4 ( 0.3
5.9 ( 0.8
2,4-dimethoxy-
pyrimidin-5-yl-
indol-5-yl-
0.25 ( 0.03 0.5 ( 0.2 0.19 ( 0.04 13 ( 3
0.24 ( 0.09 0.26 ( 0.05 0.46 ( 0.09 13 ( 2
3w
3x
3y
0.25 ( 0.03
0.24 ( 0.09 0.26 ( 0.05
0.5 ( 0.2
0.38 ( 0.06
0.8 ( 0.1
42 ( 0.05
>15
9 ( 3
1.8 ( 0.8 >50
indol-6-yl-
1 -phenylsulfonyl-
indol-2-yl
>15
9 ( 3
>15
2.0 ( 0.4 1.0 ( 0.2 >50
3z
1 -phenylsulfonyl-
indol-3-yl
>15
2.0 ( 0.4
2.0 ( 0.4
Table 3. Optimization of Ortho-Substituted Phenyl Moiety
IC50 vs IC50 vs IC50 vs IC50vs
TbcatB rhodesain Cat L Cat B
a subset of inhibitors, many compounds in this study were far
more promiscuous than expected. It was believed that the
smaller S2 pockets in cathepsin L and rhodesain could not
accommodate aryl substitution of the 6-amino benzyl ring. To
gain insight into this unexpected activity, compounds 3a, 3aa,
3ab, 3i, and 3x were docked against TbcatB, cathepsin L, and
rhodesain. This inhibitor set exhibits a wide range of selectivity:
3a, 3i, and 3x are active against all three proteases, 3aa is
TbcatB selective, and 3ab is inactive against rhodesain.
As expected, the wider S2 pocket in TbcatB easily accom-
modates the distal aryl ring of the 6-amino side chains. In
contrast, most cathepsin L ligands occupy the S3 pocket and
most rhodesain compounds spill out of the S2 pocket by
packing against Leu160 (Figure 2). The two exceptions are
the 4-chlorophenyl compound, 3i, which adopts a similar pose
in both cathepsin L and rhodesain by pointing into a hydro-
phobic pocket beyond S2, and the 2-chlorophenyl compound,
3aa, which displaces Phe61 and occupies the S3 pocket in the
rhodesain model (Figure S1, Supporting Information).
compd
Ar
(μM)
(μM)
(μM) (μM)
3b
2-CF3-phenyl-
0.28 ( 0.03
1.3 ( 0.1
0.39 ( 0.03 >50
8 ( 3 1.5 ( 0.6 >15
3aa 2-Cl-phenyl-
3ab 2-Me-phenyl-
20 ( 15 16 ( 7
>15
1.1 ( 0.5 >15
1.0 ( 0.4 >15
0.9 ( 0.3 >15
1.4 ( 0.6 >15
3ac 2, 6-dimethyl-phenyl- 0.27 ( 0.03 >15
3ad 2-ipr-phenyl-
3ae 2-methoxycarbonyl-
phenyl-
0.31 ( 0.03 >15
1.0 ( 0.2 >10
across all four cell lines. Overall, HEK 293 was the most
sensitive cell line, while Hep G2 and BJ were the least sensitive.
Several inhibitors display a therapeutic index between
25- and 50-fold across four different cell lines, further
supporting the proposition that targeting TbcatB is a
promising therapeutic strategy. It is interesting to note that
potency against TbcatB correlates with therapeutic index,
although selectivity for TbcatB relative to cathepsin L does
not necessarily improve the therapeutic index.
These molecular modeling studies offer no clear explana-
tion for the observed selectivity of the ortho-substituted
compounds. Nevertheless, it is apparent that the aryl
Additional Docking Studies to Rationalize Cathepsin L and
Rhodesain Activity. Although good selectivity was achieved in

6492 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20
Mallari et al.
Figure 2. Second generation purine nitriles exploit different binding pockets in (a) TbcatB, (b) cathepsin L, and (c) rhodesain. The phenyl
substituted compound, 3a, adopts the most common binding mode observed for each protease (see Supporting Information for additional models).
PA-based (parallel artificial membrane permeability assay)
percent membrane retention and log-normalized permeability,
and two calculated descriptors (SlogP_VSA9 and a_count),
which appeared in the regression model describing the first
generation series.4 An iterative model building process yielded
an equation with three terms (standard errors in parentheses,
see Supporting Information for regression details):
T: brucei log10ðEC50Þ ¼ ½0:85ð0:13Þꢀ½TbcatB log10ðEC50Þꢀ
þ ½0:12ð0:06Þꢀ½rhodesain log10ðEC50Þꢀ
- ½0:11ð0:06Þꢀ½log10ðpermeabilityÞꢀ þ ½0:54ð0:13Þꢀ
Increasing potency (decreasing EC50) against TbcatB and
rhodesain and increasing PAMPA permeability results in
greater trypanocidal activity. In this model, the TbcatB term
is dominant, whereas permeability and rhodesain inhibition
contribute similarly.
Figure 3. Heatmap of EC50 values for the second generation purine
nitriles. Compounds are hierarchically clustered by Tanimoto che-
mical similarity along the x-axis, whereas the assays are hierarchi-
cally clustered along the y-axis according to activity profile.
Compounds 3y and 3z were excluded from analysis because they
incorporate an unusually large phenylsulfonyl indole substitution at
the Ar position.
The regression model fits the training set data with an
adjusted r2 = 0.667 (Figure 4) and demonstrates reasonable
predictive accuracy as assessed by cross-validation: the aver-
age predictive squared correlation coefficient, q2,13 is 0.578,
and the average root-mean squared error (RMSE) is 0.208 (in
log10(EC50) units). The TbcatB term is highly significant by
ANOVA (p<10-5), whereas the rhodesain and permeability
covariates are marginally significant (p=0.063 and p=0.091,
respectively). Importantly, rhodesain inhibition and PAMPA
permeability are not statistically significant by themselves.
The addition of both variables together significantly improves
model quality relative to TbcatB alone (Δq2 is [0.035-0.054]
and ΔRMSE is [-0.012 to -0.008] at the 95% confidence
interval) to justify inclusion.
It is clear from the plot of experimental versus predicted
T. brucei EC50 that the refined model does a poor job of
predicting the activity of the first generation compounds.
However, the TbcatB term was not the dominant term in the
earlier study. Moreover, the two calculated descriptors
(SlogP_VSA9 and a_count) are no longer relevant to the
current model. It is likely that the first generation series,
which had greater chemical diversity at both the N6 and N9
positions, exerted some of its trypanocidal activity via off-
target effects or was more prone to differences in membrane
permeability or intracellular localization.
substituted 6-amino benzyl side chain is much more flexible
than previously imagined. Additional optimization of cathe-
psin L selectivity might be achieved by exploiting the differ-
ent electronic environments of the S2 pocket in TbcatB and
the S3 pocket of cathepsin L. It may also be productive to
adjust the placement of the distal aryl ring by replacing the
6-amino benzyl linker.
Protease Inhibition and Trypanocidal Activity. Although
the purine nitrile scaffold is thought to kill T. brucei by
inhibition of TbcatB, the contribution of rhodesain inhibition
to trypanocidal activity remains poorly understood. With a
set of structurally similar inhibitors that exhibit a range of
selectivity profiles, it is now possible to examine the individual
importance of these two putative protease targets in determi-
ning trypanocidal activity.
The heatmap in Figure 3 summarizes the protease and
trypanocidal activity of the compounds in this study.
All ortho-substituted analogues partition into the right
cluster (3f-3n) formed from the first bifurcation of the
dendrogram along the x-axis. Two conclusions can be drawn
from this analysis. First, it is clear that ortho-substitution is
generally favorable for TbcatB, but it is not well tolerated in
either rhodesain or cathepsin B. Second, when comparing
activity profiles along the y-axis, the TbcatB protease assay
behaves more similarly to the trypanocidal assay, whereas
the rhodesain assay clusters more closely to that of
cathepsin L.
In contrast, data from the current study strongly suggests
that TbcatB inhibition is primarily responsible for the
antiparasitic activity of the aryl substituted 6-aminophenyl
purine nitriles. The importance of rhodesain inhibition
appears to be minimal. This conclusion is clearly illustrated
by the TbcatB selective ortho-substituted series (com-
pounds 3b and 3aa-3ae), which maintains potent trypano-
cidal activity despite poor potency against rhodesain
(Table 4).
To further investigate how trypanocidal activity relates to
TbcatB inhibition, linear regression analysis was performed to
model T. brucei EC50 as a function of the following independent
variables: log-normalized TbcatB and rhodesain EC50, PAM-

Brief Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6493
preparation, and building the modified cysteine residues. Protein
models were charged at physiological pH using the PDB2PQR8
and PropKa9 programs. All molecular dynamic simulations were
performed using AMBER10 in conjunction with AMBER Tools
version 1.010 and visualized in VMD 1.8.6 (http://www.ks.uiuc.
edu/Research/vmd/).11 Diagnostics plots and statistics were calcu-
lated using custom PERL and R scripts.12 Default parameters and
settings were used unless otherwise noted.
Acknowledgment. This work was supported by the American
Lebanese Syrian Associated Charities (ALSAC) of St. Jude
Children’s Research Hospital, National Institute of Allergy
and Infectious Diseases grant AI35707, Drugs for Neglected
Diseases initiative, and the Sandler Family Supporting Foun-
dation. We also thank Cindy Choy and David Smithson for
their advice and feedback.
Figure 4. Experimental trypanocidal activity vs trypanocidal acti-
vity predicted from the three-term linear model. The red line has
unity slope and represents perfect predictions.
Supporting Information Available: Spectroscopic data and
LCMS data for all listed compounds. Experimental details for
molecular docking, heatmap, and regression modeling studies.
Experimental details for inhibitor synthesis, protease purification,
and cellular and biochemical assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
Conclusion
This paper describes the structure-guided optimization of
selectivity in a series of purine nitrile inhibitors. By exploiting
predicted differences at the S2 pockets of TbcatB, rhodesain,
and cathepsin L, a series of selective inhibitors with enhanced
potency for TbcatB was developed. The activity profiles of
these compounds across a panel of proteases strongly support
the proposed binding model for TbcatB and provide a struc-
tural rationale for the further development of selective pro-
tease inhibitors. In addition, these results suggest that it is
possible to effectively target TbcatB with selectivity relative to
the human cathepsins, reaffirming the potential of TbcatB as a
therapeutic target. Finally, it was found that inhibition of
TbcatB was a key determinant of trypanocidal activity for this
compound series while inhibition of rhodesain was relatively
unimportant.
References
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Experimental Section
Synthesis. Amines and alkyl bromides were purchased from
Sigma Aldrich. Sodium cyanide, potassium carbonate, sodium
carbonate, and 2,6-dichloropurine 1 were purchased from Sig-
ma Aldrich. Boronic acids were purchased from Sigma Aldrich,
TCI America, Maybridge, Alfa Aesar, and Fluka. All reaction
solvents were anhydrous. LCMS data were collected on a
Waters system using XTerra C18 and YMC Pro C4 columns
(MeOH:H2O, 1% HCOOH). Intermediate nitrile 2 was synthe-
sized as previously described.4
Synthesis of 3a-3ae. Compound 2(23 mg, 0.06 mmol), Na2CO3
(25 mg, 0.24 mmol, 4.0 equiv), Pd(PPh3)4 (14 mg, 0.012 mmol,
0.2 equiv), and the appropriate aryl boronic acid (0.12 mmol, 2.0
equiv) were dissolved in anhydrous 1,4-dioxane (0.5 mL, 0.12 M) in
a 0.5 mL sealed microwave reaction vial (Biotage). The vial was
heated to 150 °C for increments of 5-15 min in a Biotage Initiator
microwave until the reaction was complete as shown by LC/MS, a
total of 20-90 min. The crude product was purified by reverse-
phase HPLC (Waters XTerra preparative C18 column, ACN:H2O,
1% HCOOH, linear gradient from 40 to 70% ACN) to afford target
inhibitor 3a-3ae with a resulting yields of 10-70%.
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Wang, J.; Duke, R. E.; Luo, R.; Crowley, M; Walker,R. C.; Zhang,
W.; Merz, K. M.; Wang, B.; Hayik, S.; Roitberg, A.; Seabra, G.;
ꢀ
Kolossvary, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wu, X.;
Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Yang, L.; Tan, C.;
Mongan, J.; Hornak, V.; Cui, G.; Mathews, D. H.; Seetin, M. G.;
Sagui, C.; Babin, V.; Kollman, P. A. AMBER 10; University of
California: San Francisco, 2008.
Cloning, Expression, and Purification of TbcatB. TbcatB was
cloned and prepared as previously described.4
Cellular and Biochemical Assays. Inhibitors were screened for
protease inhibition, trypanocidal activity, and toxicity against
mammalian cell lines as previously described.4
Molecular Docking Studies. The previously reported TbcatB
homology model,4 the crystal structure of human cathepsin L
(PDB code 1mhw), and the crystal structure of T. brucei rhodesain
(PDB code 2p86) served as the starting point for the molecular
docking studies. The MOE program (Chemical Computing
Group, version 2008.10) was used for visualization, initial protein
(11) Humphrey, W.; Dalke, A.; Schulten, K., VMD: visual molecular
dynamics. J. Mol. Graph. 1996, 14, (1), 27-28; 33-38.
(12) R Development Core Team R: A Language and Environment
for Statistical Computing; R Foundation for Statistical Computing:
Vienna, 2005; http://www.R-project.org.
(13) Schuurmann, G.; Ebert, R. U.; Chen, J.; Wang, B.; Kuhne, R.
External validation and prediction employing the predictive
squared correlation coefficient test set activity mean vs training
set activity mean. J. Chem. Inf. Model. 2008, 48 (11), 2140–2145.