Proteomic profiling and potential cellular target identification of K11777, a clinical cysteine protease inhibitor, in Trypanosoma brucei

Article abstract of DOI:10.1039/c1cc16178d

We report herein the design, synthesis and application of K11777-derived activity-based probes (ABPs) allowing in situ profiling and identification of potential cellular targets of K11777 in Trypanosoma brucei.

Full text of DOI:10.1039/c1cc16178d

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Proteomic profiling and potential cellular target identification of K11777,
a clinical cysteine protease inhibitor, in Trypanosoma bruceiw
Peng-Yu Yang,a Min Wang,b Cynthia Y. Heb and Shao Q. Yao*a
Received 5th October 2011, Accepted 15th November 2011
DOI: 10.1039/c1cc16178d
We report herein the design, synthesis and application of
K11777-derived activity-based probes (ABPs) allowing in situ
profiling and identification of potential cellular targets of
K11777 in Trypanosoma brucei.
More than a billion people suffer from neglected tropical
diseases (NTDs), including malaria (caused by Plasmodium
falciparum), sleeping sickness (aka Human African trypano-
somiasis; caused by Trypanosoma brucei), Chagas’ disease (aka
American trypanosomiasis; caused by Trypanosoma cruzi),
leishmaniasis, onchocerciasis, lymphatic filariasis and
schistosomiasis.1 Most drugs available to date however are
limited by parasite resistance and marked host toxicity. New
strategies and targets that can effectively combat these
parasitic diseases are therefore needed urgently. Equally
important, from the standpoint of drug efficacy and safety,
comprehensive cellular target profiling of a given drug candidate
is becoming an integral step in the process of drug discovery.
The knowledge of potential on- and off-targets of a drug at the
earliest stages of its development will not only shed light on its
potential success (or failure) during the clinical trials, but also
provide invaluable insight into its mode of action and further
Fig. 1 (A) Workflow for proteome profiling of potential cellular
optimizations.
targets of K11777 in living parasites using cell-permeable ABPs. (B)
One promising strategy for discovering small-molecule thera-
Structures of K11002/K11777 and corresponding cell-permeable and
peutics for parasitic diseases has been to target the cathepsin L
clickable probes (1, 2 and 3; for synthesis, see ESIw).
subfamily of the papain-like (clan CA, family C1) cysteine
trypanosomes (i.e., T. brucei, L. major, T. gondii, E. histolytica)
and schistosome bloodflukes (i.e., S. mansoni),4–6 its mechanism of
proteases such as cruzain, rhodesain (also called brucipain or
trypanopain) and falcipains (FP-2 and -3). Among various
action is not well understood except for inhibition of the cathepsin
pharmacophores, vinyl sulfones have been widely studied as
L and/or B-like cysteine proteases in vitro. Little information is
potential cysteine protease inhibitors and are entering the develop-
ment pipeline as anti-parasitics (Fig. S1 in ESIw).2 One of these,
K11777 (Fig. 1), which was developed from the predecessor
available concerning its other potential cellular targets and distri-
bution. Recently, we reported a chemical profiling approach that
makes use of drug-like probes for proteome-wide profiling of
K11002 by replacing the morpholine–urea ring in K11002 with
cellular targets in living cells.7 This method is based on activity-
an N-methylpiperazine (N-Mpip) for increased oral bioavailability
based protein profiling (ABPP),8 and large-scale LC-MS/MS
and solubility in intestinal fluids, is currently in late-stage preclinical
trials for Chagas disease.3 Although it is clear that K11777 also
analysis, allowing rapid determination of potential cellular targets
of bioactive small molecules.8d,e Herein, we report the first applica-
demonstrated efficacy against other parasites as diverse as
tion of this method in live parasites. Several cell-permeable probes
based on K11777 were designed, synthesized, and used to identify
potential cellular targets of this drug candidate in T. brucei.
The design of K11777-like probes was based on the general
structure of several trypanocidal vinyl sulfones (e.g., K11777,
K11002 and Cbz-Phe-Hph-VSCH2Ph) and our previous experience
with orlistat-like probes.7a We took advantage of key properties of
a Department of Chemistry, National University of Singapore,
3 Science Drive 3, Singapore 117543. E-mail: chmyaosq@nus.edu.sg;
Fax: +65 6770169; Tel: +65 65162925
b Department of Biological Sciences, National University of Singapore,
14 Science Drive 4, Singapore 117543
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c1cc16178d
c
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Chem. Commun., 2012, 48, 835–837 835

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vinyl sulfones in our rational design of ABPs: (1) vinyl sulfones
inhibit cysteine proteases via a Michael addition to form a covalent
bond with the active-site cysteine residue (e.g., Cys 25 in cruzain/
rhodesain and Cys 51 in FP-3);2,9 (2) recent crystal structures of
rhodesain with K11777 and K11002 have shown that the N-Mpip/
morpholine–urea ring did not form any specific polar interactions
with rhodesain and thus was assumed non-essential for inhibitor
binding.2,9 Accordingly, probes 1 and 2, in which a CRC triple
bond was introduced at the P3 position, were synthesized. We also
0
synthesized probe 3 by introducing a CRC triple bond at the P1
position. Our experience was that such extremely conservative
modifications would not compromise the native biological properties
of mother compounds, whilst allowing subsequent target identifi-
cation by downstream conjugation of reporter tags using bio-
orthogonal click chemistry. The three analogues were synthesized
by using appropriate modifications of previously described methods,
as shown in the ESIw (Schemes S1ꢀS4). The resulting probes were
fully characterized (MS and NMR) before being used in subsequent
biochemical/biological experiments.
Fig. 2 (A) ED50 curves of K11777, K11002 and three probes showing
T. brucei killing after 24 h. (B) In situ proteome profiling of probes (25 mM)
against T. brucei BSF and PCF. K11777 was added to selected lanes in
competition experiments. Putative bands of labeled rhodesain and TbCatB
are indicated (with arrows). (C) Western blotting validation of rhodesain
and TbCatB labeled with VS-1 (25 mM), following in situ labeling and
affinity pull-down/WB experiments (see ESIw for details). Null pull-down
experiments (with DMSO) were used as negative controls. (D) Venn
diagram illustrating the number of proteins identified from T. brucei after
in situ labeling and large-scale pull-down/LC-MS/MS experiments.
We first evaluated the trypanocidal activities of vinyl sulfones
1, 2 and 3, together with K11002 and K11777 in cell culture of
both the bloodstream form (BSF) and the procyclic form (PCF),
which represent two distinct stages of T. brucei during its
complex life cycle alteration between mammalian hosts and
insect vectors. In this context, it is worth noting that the BSF
and PCF parasites differ extensively in morphology and meta-
bolism. All vinyl sulfones blocked the parasite growth to a
similar extent in a dose-dependent manner with ED50 values
between 4 and 8 mM (Fig. 2A). Additionally, there was no
noticeable difference in inhibition between BSF and PCF trypano-
somes by all five compounds. These data show that the introduc-
tion of a terminal alkyne handle at the indicated positions did not
noticeably affect its trypanocidal activity. Therefore, all three
probes (1, 2 and 3) should be suitable chemical probes for
proteome profiling and cellular target identification of K11777.
Only 1 (VS-1), however, was chosen for further comprehensive
proteome profiling, LC-MS/MS and cellular bioimaging experi-
ments based on its closest resemblance to K11777.
large-scale proteomic analyses using VS-1 to identify potential
cellular targets of K11777 in both parasite forms by affinity
pull-down/LC-MS/MS experiments; all proteins were identified
with a minimum protein score of 40 as well as at least two unique
peptides, and results are summarized in Fig. 2D, Table 1 and in
ESI.w In total, 49 and 111 proteins were identified from BSF and
PCF, respectively, 20 of which were from both parasite forms (see
SI_2, ESIw, for detailed protein ID). Among these proteins, some
were inevitably non-specific protein binders caused by their
‘‘sticky’’ nature as well as their high endogenous expression level
(e.g. cytoskeletal proteins and carbohydrate-metabolism-related
proteins). We focused our attention on other candidates which
possess known nucleophilic cysteine residues, because they are
more likely true cellular targets of K11777 (Table 1); in addition to
the expected rhodesain and TbCatB, other proteins such as BS2,
MCA4, AOX and two proteasome subunits were identified. It is
worth noting that vinyl sulfones had previously been identified as
potent and irreversible inhibitors of proteasomes (through mod-
ifications of their active-site threonine).12 Thus, it is not surprising
that two proteasome subunits were detected in PCF. It is also
interesting to note that many more proteins were identified from
PCF alone (91) than from BSF (29), and many of them were
cytosolic (SI_2 in ESIw).
Next, we compared the in situ proteome reactivity profiles of
our probes against their cellular targets in live BSF and PCF
(Fig. 2B). Generally, the three probes showed comparable in situ
proteome reactivity profiles, thereby indicating that they have
similar cellular targets in the parasites. However, the reactivity
profiles between BSF and PCF for VS-1 (lanes 1 and 5 in
Fig. 2B), though similar, showed noticeable differences, suggesting
the existence of both common and unique targets in the two
forms. In addition to the expected rhodesain and TbCatB
bands (B41 kDa and B31 kDa indicated by arrows, respec-
tively), which were subsequently verified by affinity pull-down/
western blotting with the corresponding antibodies (Fig. 2C),
a number of other proteins were also covalently labeled by the
three probes. These labeled bands were K11777-sensitive, that
is, their labeling was blocked by the presence of a competing
K11777 (lanes 2 and 6 in Fig. 2B). Also evident in Fig. 2B and
C is that, VS-1 labeled only the mature active enzyme form of
rhodesain (B41 kDa), but not its proform (B48 kDa).10 In
addition, TbCatB labeled by VS-1 was mostly detected in
BSF, which is consistent with previous findings that TbCatB
was up-regulated in BSF.11 Subsequently, we performed
To visualize cellular localization of VS-1 and potential cellular
targets of K11777, live parasites were directly treated with VS-1,
fixed, and reacted in situ with the rho-azide reporter (ESIw), then
imaged. Overall, no noticeable difference was observed in the
probe uptake between BSF and PCF (Fig. S4, ESIw). As shown in
Fig. 3, VS-1 signals were detected mostly in the lysosome of BSF,
where endogenous rhodesain resides (panels b–d). In contrast, the
c
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Table 1 Representative proteins identified by 1 (VS-1) in T. bruceia
T. brucei gene
Protein name
Localization
Detection
Tb927.6.1000
Tb927.10.8230
Tb927.5.1810
Tb10.70.5250
Tb927.10.7090
Tb927.10.290
Tb927.10.6080
Tb09.160.4250
Tb927.5.3350
Tb927.10.7410
Cysteine peptidase precursor (CP), Clan CA, family C1, cathepsin L-like*
Protein disulfide isomerase, bloodstream-specific protein 2 precursor (BS2)*
Lysosomal/endosomal membrane protein p67 (p67)*
Metacaspase MCA4, cysteine peptidase, Clan CD, family C13 (MCA4)**
Alternative oxidase (AOX)**
Proteasome alpha 2 subunit*
Proteasome beta 5 subunit (PRCE)*
Tryparedoxin peroxidase (TRYP1)**
Iron superoxide dismutase**
Succinyl-CoA ligase [GDP-forming] beta-chain*
L
C
L
N
M
C
C
C
Both
BSF
BSF
BSF
BSF
PCF
PCF
PCF
PCF
PCF
M
M
a
L, C, N, M and G represent lysosome, cytoplasm, nucleus, mitochondrion and glycosome, respectively. Symbols in the protein name column: (*)
sensitive to RNA interference; (**) putative drug target.
drug in both parasites and human cells. Further studies are
underway to validate some of the potential cellular targets identified
from the present study.
Funding support was provided by Ministry of Education
(R-143-000-394-112), Agency for Science, Technology and Research
(A*Star) (R143-000-391-305), and Competitive Research Program
(NRF-G-CRP 2007-04) of the National Research Foundation
(NRF). This work was also in part supported by funding from
Singapore National Research Foundation, awarded to CYH
as a research fellow.
Fig. 3 Confocal microscopy of VS-1 localization in BSF (top) and
PCF (bottom). Live parasites were treated with VS-1 (25 mM) followed
by imaging as described in ESI.w Panels (a) and (e): bright-field images.
Panels (b) and (f): 554 nm channel (pseudocolored in red) detecting
Notes and references
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Panels (d) and (h): merged images of panels (b) and (c), (f) and (g)
together with nuclei (stained with DAPI; pseudocolored in blue). All
images were acquired under the same settings. Scale bar = 10 mm.
Cells treated with DMSO are shown in Fig. S5 (ESIw).
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probe was shown to be evenly distributed throughout PCF
parasites with no specific subcellular localization (panels f–h;
similar results were obtained with HepG2 mammalian cells as
shown in Fig. S7, ESIw). These results correlate well with our
above-described proteome profiling and affinity pull-down/
LC-MS/MS data where different cellular targets were labeled in
the two parasite forms, and many more cytosolic proteins were
identified in PCF. The partial colocalization observed between
VS-1 and rhodesain in BSF presumably also reflected the presence
of other side targets of K11777. Collectively, these results estab-
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In conclusion, we have successfully synthesized and evaluated
K11777-like probes for their trypanocidal activities against
both BSF and PCF T. brucei. Subsequent in situ proteome
profiling of VS-1 enabled us to tentatively identify previously
unknown cellular targets of K11777 in both parasite forms.
Furthermore, we demonstrated the utility of our probe for
live-parasite labeling and visualization of potential K11777-
responsive targets (e.g. rhodesain). Our probes should be useful
in assisting future investigations of K11777 as an anti-parasitic
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c
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Chem. Commun., 2012, 48, 835–837 837