Design and evaluation of Trypanosoma brucei metacaspase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.099

Metacaspase (MCA) is an important enzyme in Trypanosoma brucei, absent from humans and differing significantly from the orthologous human caspases. Therefore MCA constitutes a new attractive drug target for antiparasitic chemotherapeutics, which needs further characterization to support the discovery of innovative drug candidates. A first series of inhibitors has been prepared on the basis of known substrate specificity and the predicted catalytic mechanism of the enzyme. In this Letter we present the first inhibitors of TbMCA2 with low micromolar enzymatic and antiparasitic activity in vitro combined with low cytotoxicity.

Full text of DOI:10.1016/j.bmcl.2010.01.099

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2001–2006
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and evaluation of Trypanosoma brucei metacaspase inhibitors
Maya Berg a, Pieter Van der Veken a, Jurgen Joossens a, Venkatraj Muthusamy a, Matthias Breugelmans a,
Catherine X. Moss b, Jana Rudolf b, Paul Cos c, Graham H. Coombs d, Louis Maes c, Achiel Haemers a,
Jeremy C. Mottram b, Koen Augustyns a,
*
a Laboratory of Medicinal Chemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
b Wellcome Centre for Molecular Parasitology and Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK
c Laboratory of Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Groenenborgerlaan 171,
B-2020 Antwerp, Belgium
d Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0NR, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Metacaspase (MCA) is an important enzyme in Trypanosoma brucei, absent from humans and differing
significantly from the orthologous human caspases. Therefore MCA constitutes a new attractive drug tar-
get for antiparasitic chemotherapeutics, which needs further characterization to support the discovery of
innovative drug candidates. A first series of inhibitors has been prepared on the basis of known substrate
specificity and the predicted catalytic mechanism of the enzyme. In this Letter we present the first inhib-
itors of TbMCA2 with low micromolar enzymatic and antiparasitic activity in vitro combined with low
cytotoxicity.
Received 13 November 2009
Revised 14 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Trypanosoma brucei
Metacaspase
Ó 2010 Elsevier Ltd. All rights reserved.
Inhibitor
African trypanosomiasis
Sleeping sickness
Protease inhibitor
In 2000, metacaspases (E.C. 3.4.22) were identified as a new
family of caspase-like proteins.1 Metacaspases, together with casp-
ases and paracaspases, are endopeptidases that belong to family
C14 in clan CD of the cysteine peptidases. They share the caspase
fold2 and their active site comprises a conserved His-Cys catalytic
dyad, the cysteine being the active site nucleophile. All metacasp-
ases examined to date have a basic P1 specificity towards Arg/Lys,
whereas caspases have an acidic P1 specificity towards Asp.2 This
explains why metacaspases are not able to cleave known caspase
substrates and are not inhibited by caspase inhibitors (zVAD,
DEVD-CHO).3 Metacaspases are not present in mammals and have
little overall sequence homology with human caspases, making
them good potential drug targets. In Trypanosoma brucei, five meta-
caspases have been identified denoted TbMCA1-5. Interestingly,
TbMCA1 and TbMCA4 genes encode a serine in place of a cysteine
in the catalytic dyad, thus cannot function as cysteine peptidases,
although they may still have activity through the use of the substi-
tuent serine as a nucleophile. Of the other three TbMCAs, TbMCA2
and TbMCA3 have demonstrated activity and TbMCA5 is expected
to have similar activity.4 All three have been validated as drug tar-
gets by triple RNAi.5 Triple RNAi analysis (simultaneous down-reg-
ulation) showed TbMCA2, TbMCA3 and TbMCA5 to be essential in
the bloodstream form (BSF), with parasites accumulating pre-cyto-
kinesis, meaning cell division failed. However, this effect could not
be confirmed with triple null mutants (Dmca2/3Dmca5). After an
initial slow growth phase following sequential gene deletion, they
grew well under both standard in vitro and in vivo conditions, sug-
gesting compensatory activation of alternative pathways. How-
ever, mutant strains remained highly sensitive to changes to the
standard conditions in vitro. Overall, it seems metacaspases fulfil
an important role in BSF T. brucei, but the likelihood of overlapping
functions means that therapeutic targeting would require inhibi-
tion of multiple TbMCAs and/or enzymes of the alternative
pathways.
The functions of metacaspases in protozoa remain unclear. It
has been proposed that in trypanosomatids metacaspases function
as caspase-like enzymes and are involved in Programmed Cell
Death (PCD).6–8 However, there are still doubts about the existence
of PCD in trypanosomatids and other unicellular organisms9,10 and
some authors oppose the PCD function of metacaspases in proto-
zoa by pointing out their different substrate specificity, which
makes a similar function to caspases unlikely.2 Helms et al.5 sug-
gest that MCAs have a function associated with RAB11 vesicles that
is independent of known recycling processes of RAB11-positive
endosomes. Clearly, the precise role of MCAs remains to be
* Corresponding author. Tel.: +32 38202703.
E-mail address: Koen.Augustyns@ua.ac.be (K. Augustyns).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.099

2002
M. Berg et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2001–2006
and synthesise the first specific MCA inhibitors. It is known that for
peptides the S1 subsite selectivity of TbMCA2/3 is limited to Arg/
Lys2 (Fig. 1A), leading to initial substrate-based inhibitor design
with Arg/Lys in the P1 position. Previously, potent cysteine pepti-
dase inhibitors have been obtained by replacing the scissile amide
bond by an electrophile (P10). Reversible competitive inhibitors
were designed by using a nitrile12 or
a-ketoheterocyclic war-
head.13 For the latter, the formation of a hydrogen bond between
the sp2 nitrogen of the ketoheterocycle and protonated histidine
from the His-Cys dyad was hypothesized. Irreversible inhibitors
were developed with a vinylsulfone warhead. Irreversible pepti-
dase inhibitors can be advantageous for the treatment of antipara-
sitic diseases, as these infections usually only require short courses
of treatment; thus avoiding tissue accumulation and long term
safety issues. In addition, irreversible inhibitors often show more
efficient in vivo antiparasitic activities than reversible inhibitors.11
Our strategy was to develop a small diversity set of inhibitors in
order to select a first hit from a screening assay on TbMCA2. Differ-
ent parameters were tested: a set of warheads was investigated
and peptidomimetic inhibitors were designed using a peptoid
backbone. In addition, an attempt to influence basicity at P1 posi-
tion was made. From this first screen a hit was selected which was
then subjected to a range of optimization efforts. Several Arg
mimetics were introduced in P1 and also P2 modification was ad-
dressed in order to obtain more potent inhibitors and gain insight
in the scope of the active site of MCAs.
Figure 1. (A) Schematic representation of TbMCA inhibitors based on L-Arginine or
L-Lysine. (B) First set of TbMCA inhibitors derived from
a-amino protected arginine
with nitrile P10 warhead and modifications in P1.
For a first set of inhibitors we started from the
a-amino pro-
tected arginine with a nitrile warhead 1 (Fig. 1B). In the P2 position
the protecting group benzyloxycarbonyl (Cbz) was used. Diversifi-
cation was carried out at the level of the P1 position (Fig. 1B, 2–3).
To circumvent possible membrane permeability issues which are
known for highly basic residues such as amidines and guanidines,
we selected functional groups in the P1 side chain with lower ba-
sicity. First, aromatic functions were introduced in the P1 tail (2
and 3). A second set of inhibitors was then designed with a peptoid
backbone (Fig. 2, 4–10) and a variety of functional groups in the P1
position which were intended to have a similar basicity-lowering
effect.
A third set of inhibitors (Fig. 3) was derived from
tected arginine with a -ketoheterocyclic warhead (P1’). Different
inhibitors 11–14 were screened and resulted in our first hit 11 (IC50
TbMCA2 = 0.6 M).
a-amino pro-
a
l
Figure 2. TbMCA inhibitors with peptoid backbone, nitrile P10 warhead and
modifications in P1.
elucidated. Nonetheless, the fact that T. brucei metacaspases seem
to play a vital role in the parasite, and are absent in humans makes
them attractive drug targets.11
In the absence of crystallographic data and full knowledge of
the natural substrates, we describe the application of a rational de-
sign where the substrate specificity of TbMCA2 was used to design
Figure 4. TbMCA inhibitor derived from
vinylsulfone P10 warhead.
a-amino protected arginine with a-
Figure 3. TbMCA inhibitors derived from a-amino protected arginine with a
-ketoheterocyclic P10 warhead.

M. Berg et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2001–2006
2003
Scheme 1. Reagents and conditions: (a) (1) N-(OH)-Suc, DCC, THF, rt, 1 h; (2) NH3/MeOH 7 N, rt, 1 h (68%); (b) TFAA, pyridine, THF, rt, 2 h; (c) TFA/DCM, rt, 1 h (19%).
Scheme 2. Reagents and conditions: (a) benzylchloroformate, NaOH, water/dioxane, 0 °C, 3 h (95%); (b) SnCl2, EtOH, reflux, 3 h (98%); (c) (Boc)2O, water, dioxane, Et3N, 0 °C to
rt, 12 h (17%); (d) (1) N-OH-Suc, DCC, THF, rt, 1 h; (2) NH3/MeOH 7 N, rt, 1 h (47%); (e) TFAA, pyridine, THF, rt, 3 h (8%); (f) TFA/DCM, rt, 1 h (65%).
diversity set, derived from
a-amino protected arginine with a
vinylsulfone warhead 15 (Fig. 4).
The synthesis of inhibitors 1–15 is shown in Schemes 1–6. For
compound 1, commercially available Z-Arg-Pmc-OH (21) was con-
verted to the corresponding amide (22) in the presence of N-
hydroxysuccinimide, dicyclohexyl carbodiimide (DCC) and ammo-
nia. The amide was then dehydrated with trifluoroacetic anhydride
(TFAA) to the corresponding nitrile. TFA deprotection afforded tar-
get compound 1 (Scheme 1).
Scheme 3. Reagents and conditions: (a) bromoacetonitrile, K2CO3, 0 °C to rt, 12 h
(95%); (b) benzylchloroformate, NaOH, dioxane, 0 °C, 3 h (43%); (c) TFA/DCM, rt, 1 h
(26%).
4-Nitro-L-phenylalanine 23 was first protected with a Cbz
group. Then the nitro group was reduced with SnCl2 and the ob-
tained amine function was protected with a tert-butyloxycarbonyl
One inhibitor with a warhead known to have irreversible bind-
ing properties towards cysteine peptidases was included in our
Scheme 4. Reagents and conditions: (a) bromoacetonitrile, K2CO3, 0 °C to rt, 12 h (95%); (b) benzylchloroformate, DCM, Et3N, 0 °C, 3 h (45%); (c) MsCl, Et3N, rt, 2 h (96%); (d)
NaI, dry acetone, 60 °C, 12 h (59%); (e) R = pyrrolidine (6), imidazole (7), piperidine (8), 1-(4-fluorophenyl)piperazine (9), 1-(bis(4-fluorophenyl)methyl)piperazine (10), DCM,
rt, 1–12 h (6, 43%; 7, 99%; 8, 79%; 9, 17%; 10, 26%).
O
O
O
CbzHN
HN
CbzHN
HN
CbzHN
HN
N
O
R
OH
a
b
PmcN
NH2
HN
NH2
PmcN
NH2
30
11
12
13
R = benzothiazole
R = pyridine
21
R = thiazole
14 R = 1-methyl-1H
benzo[d]imidazole
Scheme 5. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, TBTU, Et3N, DMF, 0 °C, 12 h (96%); (b) (1) THF, n-BuLi, heterocycle, ꢀ78 °C, 2 h; (2) TFA/
DCM, rt, 1 h (11, 17%; 12, 29%; 13, 17%; 14, 26%).

2004
M. Berg et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2001–2006
O
O
O
CbzHN
CbzHN
O
CbzHN
H
N
OH
d
a, b, c
NH
NH
NHBoc
NHBoc
BocN
NHBoc
BocN
32
31
33
CbzHN
SO2Ph
e, f
OEt
P
O
NH
NH2
EtO
SO2Ph
HN
34
15
Scheme 6. Reagents and conditions: (a) N,O-dimethylhydroxylamine hydrochloride, TBTU, Et3N, DMF, 0 °C, 3 h (95%); (b) TFA/DCM, rt, 1 h (99%); (c) tert-butyl (1H-pyrazol-1-
yl)methylenedicarbamate, rt, 20 h (15%); (d) LiAlH4, THF, 0 °C, 30 min (81%); (e) NaH, THF, 0 °C, 1.5 h (14%); (f) TFA/DCM, rt, 1 h (70%).
Table 1
Inhibition of TbMCA2 and T. b. brucei, T. cruzi, L. infantum and P. falciparum (IC50) for 1–15
Compound
IC50 Tb MCA2 (
l
M)
IC50 T. b. brucei (
l
M)
IC50 T. cruzi (lM)
IC50 L. infantum (
lM)
IC50 P. falciparum (
l
M)
IC50 MRC-5 (lM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
1.9
>64
30.3
>64
32.5
>64
52.1
>64
>64
22.6
2.1
32.9
>64
>64
32.5
45.3
>64
28.6
27.2
34.6
>64
>64
35.7
>64
7.0
>64
>64
>64
>64
>64
>64
>64
>64
>64
5.9
>64
>64
>64
>64
>64
50.2
16.6
25.4
28.0
>64
20.5
17.2
6.9
>64
7.6
8.0
>64
24.7
7.1
>64
>64
>64
>64
>64
>64
>64
>64
>64
11.3
>64
>64
>64
>64
>64
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.6
2.6
20.9
>64
38.7
28.3
19.0
62
2.2
38.4
3.9
3.4
Cytotoxicity was measured against human fibroblasts (MRC-5).
For target compound 3, intermediate 25 was first Boc-deprotec-
ted in order to introduce a Boc-protected guanidine function, then
a similar synthetic strategy was followed.
For target compound 4, synthesis was started from the amino-
acetonitrile derivative 27 which was synthesised from tert-butyl
4-aminopiperidine-1-carboxylate 26 and bromoacetonitrile. After
Cbz-protection of 27 followed by a final TFA deprotection, the de-
sired compound was obtained (Scheme 3).
Target compound 5 was synthesised from commercially avail-
able 5-aminobutanol (28) and bromoacetonitrile, followed by a
Cbz-protection of the free amine (Scheme 4). Compound 5 was
then converted via a mesylate to intermediate 29 using a Finkel-
stein reaction.14 A nucleophilic substitution with different amino
moieties afforded target compounds 6–10 (Scheme 4).
For compounds 11, 12 and 14 the synthesis described by Cost-
anzo et al. was followed.13 For compound 13,25 the protocol re-
ported by Lin et al. was used.15 The synthesis is shown for 11–14
starting from Z-Arg-Pmc-OH (21) (Scheme 5).
To obtain compound 15, commercially available Z-Orn-Boc-OH
(31) was converted to the corresponding Weinreb amide. The amine
function was deprotected and converted to Boc-protected guani-
dine (32). Then the protected arginine Weinreb amide was reduced
to the aldehyde (33) in which a Horner–Emmons reaction was exe-
cuted in the presence of diethyl phenylsulfonylmethylphosphonate
Figure 5. TbMCA inhibitors derived from hit 11 with modifications at P1 and P2
level.
(Boc) group (24). This intermediate was converted to the final com-
pound 2 following the same synthetic steps as described for 1
(Scheme 2).

M. Berg et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2001–2006
2005
NHCbz
O
NHCbz
O
O
O
n
O
n
HN
BocHN
HN
HN
S
S
S
N
N
N
c
a, b
HN
HN
18
HN
PbfN NH2
NH2
Leu n=1
PbfN
NH2
36
35
19 Val n=0
Scheme 7. Reagents and conditions: (a) TFA/DCM (10%, 4 equiv), rt, 3–5 h (85%); (b) (1) Z-Leu/Z-Val, HOBt, EDC, DCM, rt, 2 h; (2) Et3N, rt, 12 h; (n = 1, 19%; n = 0, 13%); (c)
TFA/DCM, rt, 1 h (18, 76%; 19, 63%).
Table 2
Inhibition of TbMCA2 and T. b. brucei, T. cruzi, L. infantum and P. falciparum (IC50) for 16–19
Compound
IC50 Tb MCA2 (
l
M)
IC50 T. b. brucei (
lM)
IC50 T. cruzi (lM)
IC50 L. infantum (
lM)
IC50 P. falciparum (
lM)
IC50 MRC-5 (lM)
16
17
18
19
1.6
4.0
1.4
1.1
10.6
11.4
5.7
10.8
5.5
5.6
22.8
45.4
36.8
>64
3.00
5.6
2.6
3.3
20.0
19.0
29.0
32.6
11.2
7.3
Cytotoxicity was measured against human fibroblasts (MRC-5).
34 to give the desired vinylogous arginine.16 A final deprotection in
acidic medium provided target compound 15 (Scheme 6).
All target compounds were biochemically evaluated by in vitro
testing as competitive inhibitors of TbMCA2 (Table 1). Six com-
Many arginine mimetics are able to interact with carboxylic groups
in the active site21 such as homo-arginine 17 and less basic lysine
16 and ornithine. At the P2 level, the Cbz protecting group was
substituted for a mimic of the P2 amino acid, which is known to
be a hydrophobic uncharged aliphatic amino acid.22 This resulted
in dipeptide analogues 18 and 19.
pounds showed significant inhibition (IC50 <2.2
lM), compound
11 was the most potent inhibitor with an IC50 of 0.6
lM. In addi-
tion, compounds 1 and 11–14 were tested against TbMCA3 and
the overall inhibition profile was similar to that for TbMCA2 (data
not shown). TbMCA2 inhibitors would be expected to show inhibi-
tion of TbMCA3 as they share 89% sequence identity.17 As simulta-
neous inhibition of all catalytically active TbMCAs is probably
necessary, inhibition of multiple TbMCAs by an inhibitor would
be advantageous. The specificity of inhibitors 1 and 11–14 was as-
sessed using human caspase-3, a member of the same peptidase
family as the MCAs. Caspase-3 activity was unaffected by any of
these inhibitors (data not shown), demonstrating their selectivity
for TbMCAs.
Next, a biological evaluation was performed in vitro against the
following protozoa (Table 1): T. b. brucei (BSF), Trypanosoma cruzi
and Leishmania infantum intracellular amastigotes, and Plasmodium
falciparum (ring stage and schizont). Leishmania parasites have a
single metacaspase gene,18 whilst P. falciparum19 and T. cruzi20
both have 2 MCAs. Cytotoxicity was assessed by testing the com-
pounds on human lung fibroblasts (MRC-5). Compound 1 has an
Compound 16 was synthesised as per the reported procedure by
McGrath et al.23 Compound 17 was synthesised in the same way as
11–14 (Scheme 5). For this compound, the amine function of N-
e-
Boc-N- -Z- -Lysine was first deprotected and converted to a Boc-
a
L
protected guanidine. The ornithine analogue could not be obtained
due to formation of the inactive enamine after cyclisation. For
compounds 18 and 19 the first synthetic steps are comparable to
Scheme 5: commercially available Boc-Arg-Pbf-OH was trans-
formed to intermediate 35 (Scheme 7). After a selective Boc depro-
tection and activation of Z-Leu/Z-Val in the presence of 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxyben-
zotriazole (HOBt), a coupling reaction took place (36).24 A final
TFA deprotection yielded compounds 18 and 19 (Scheme 7).
Modification of P1 caused inhibition in the same range as the hit
(16, TbMCA2 IC50 = 1.6 lM and 17, TbMCA2 IC50 = 4.0 lM) with
modest antiparasitic activities. P2 modification (18 and 19) scored
comparable enzymatic and antiparasitic activities (Table 2).
We report the first MCA inhibitors with low micromolar activ-
ity. These compounds inhibit TbMCA2 and TbMCA3, possess mod-
est antiparasitic activity, but have excellent selectivity when
compared to mammalian caspases. Optimization efforts are
ongoing.
IC50 value of 1.9 lM for TbMCA2 but showed no antiparasitic activ-
ity. Modifications of the P1 tail (2 and 3) in order to lower the ba-
sicity combined with the introduction of a peptoid backbone (4–
10) resulted in inactive compounds. Introduction of a-ketohetero-
cyclic warheads with retention of the Arg P1 tail increased enzy-
matic inhibition, especially for benzothiazole (11, TbMCA2
Acknowledgments
IC50 = 0.6 lM) and thiazole (13, TbMCA2 IC50 = 2.2 lM). For 11
and 14, antiparasitic activity although very modest, was observed
against P. falciparum. Finally, irreversible inhibitor 15 also was
found to be a low micromolar inhibitor (TbMCA2 IC50 = 3.9 lM)
with modest antiparasitic activity towards P. falciparum.
Compound 11 was considered as a valuable hit, the first to be
developed towards TbMCA2, with low micromolar activity and
without cytotoxicity. To obtain more potent compounds, optimisa-
tions were carried out at both P1 and P2 level (Fig. 5). At the P1 le-
vel, the arginine side chain was substituted for arginine mimetics.
This work was funded by a research project from the Research
Foundation Flanders (FWO-Vlaanderen), the Medical Research
Council (Grant numbers G9722968 and G0700127) and an SRDG
grant (HR04013) from the Scottish Funding Council. M. Berg and
M. Breugelmans have a Ph.D. grant from the Research Foundation
Flanders (FWO-Vlaanderen). P. Van der Veken has a postdoctoral
grant from the Research Foundation Flanders (FWO-Vlaanderen).
The laboratory of Medicinal Chemistry and LMPH are partners of
the Antwerp Drug Discovery Network (ADDN, www.addn.be).

2006
M. Berg et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2001–2006
J. M.; de Garavilla, L.; Haertlein, B. J.; Maryanoff, B. E. J. Med. Chem. 2005, 48,
Supplementary data
1984.
14. Enders, D.; Schusseler, T. Tetrahedron Lett. 2002, 43, 3467.
15. Lin, J.; Deng, H.; Jin, L.; Pandey, P.; Quinn, J.; Cantin, S.; Rynkiewicz, M. J.; Gorga,
J. C.; Bibbins, F.; Celatka, C. A.; Nagafuji, P.; Bannister, T. D.; Meyers, H. V.;
Babine, R. E.; Hayward, N. J.; Weaver, D.; Benjamin, H.; Stassen, F.; Abdel-
Meguid, S. S.; Strickler, J. E. J. Med. Chem. 2006, 49, 7781.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.01.099.
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