Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain

Article abstract of DOI:10.1016/j.bmcl.2015.08.074

Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

Full text of DOI:10.1016/j.bmcl.2015.08.074

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4509–4512
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of non-peptidic cysteine reactive fragments
as inhibitors of cysteine protease rhodesain
Danielle McShan a, Stefan Kathman b, Brittiney Lowe a, Ziyang Xu b, Jennifer Zhan b, Alexander Statsyuk b,
Ifedayo Victor Ogungbe a,
⇑
a Department of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217, United States
b Department of Chemistry, Center for Molecular Innovation and Drug Discovery, Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense,
the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a
fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of
T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent
fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic
assays will further expand the use of the covalent tethering method, a simple fragment-based drug
discovery technique to discover covalent drug leads.
Received 29 June 2015
Revised 24 August 2015
Accepted 27 August 2015
Available online 2 September 2015
Keywords:
Rhodesain
Trypanosomes
Covalent fragments
Cysteine protease
Ó 2015 Elsevier Ltd. All rights reserved.
Fragment-based drug discovery (FBDD) is a powerful method to
discover drug leads and has been widely adopted in both academia
and industry.1 FBDD can be used to explore chemical diversity
space with libraries which are smaller in size, producing drug leads
with high ligand-binding efficiency.2 It also provides a rational
path to high-affinity lead compounds that possess drug–like prop-
erties.3 The fragment-based approach is particularly well-suited
for target-based drug discovery because the structural require-
ments for inhibition or inactivation can be used to guide the choice
of pharmacophore and other structural motifs during drug design.4
The human African trypanosomiasis (sleeping sickness), a dis-
ease caused by the kinetoplastid protozoans Trypanosoma brucei
rhodeseinse and Trypanosoma brucei gambiense, is one of the
neglected tropical diseases (NTDs). The disease is fatal if untreated,
and current treatment options available for the disease are ineffec-
tive and have well-documented adverse effects. It is important to
note that the incidence of sleeping sickness has decreased in the
last decade, although the need to develop new and effective drugs
remains a key objective in controlling and eradicating the disease.5
A promising drug candidate in clinical trials for human African try-
panosomiasis is the nitroimidazole fexinidazole. Fexinidazole is
also being developed as a potential treatment for Chagas Disease.6,7
The major cathepsin L-like cysteine protease in T. brucei rhode-
siense, rhodesain, is a validated drug target. The cysteine protease
is essential for the survival and infectivity of the parasite. Its
important role in the ability of the parasite to proliferate has been
investigated by several groups.8–10
Steverding et al. have also shown that pharmacological inhibi-
tion of rhodesain is lethal to T. brucei.11 A number of potent inhibi-
tors of rhodesain that also have antitrypanosomal activity have
been reported.8 Among them are peptide-based covalent inhibitors
with Michael acceptors such as 1 and 2 and thiosemicarbazone
based inhibitors for example 3 and 4 (Fig. 1). Furthermore, a pep-
tide derived covalent inhibitor of its homologue in Trypanosoma
cruzi, K777, was recently a promising pre-clinical drug candidate
for the treatment of Chagas disease (American trypanosomiasis),
which highlights the importance of rhodesain as a drug target.12,13
This provides the rationale that covalent or non-covalent inhibi-
tors of rhodesain may be advanced into the drug development
pipeline against African sleeping sickness. To discover rhodesain
inhibitors, we employed the irreversible tethering method to dis-
cover covalent inhibitors of cysteine proteases.14 In this method,
a mixture of cysteine reactive electrophilic fragments were incu-
bated with the cysteine protease, allowing the best binding frag-
ments to covalently and irreversibly modify the catalytic cysteine
of the protease, and the covalent cysteine protease–inhibitor com-
plexes were subsequently detected using mass spectrometry
methods. The fragments can subsequently be elaborated into drug
leads while retaining the original Michael acceptor electrophile.
The originally developed method requires mass spectrometry to
identify fragment hits, and this requirement limits the widespread
use of this technology. We thought to expand the method and
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2015.08.074
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.

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D. McShan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4509–4512
Table 1
follow-up assay using 200 lL reaction mixtures in 96-well plates
to confirm the activity of the active compounds led to the identifi-
cation of compounds 5 and 7 as hits, the other five compounds
caused <40% inhibition of rhodesain at 10 lM (Table 1, Fig. 2).
Fragments 5 and 7 were recently reported as inhibitors of papain,
a cysteine protease structurally and biochemically related to
rhodesain.14 Fragments 5 and 7 seem to be better inhibitors of
rhodesain than papain judging by the kinact/KI values, however, it
is important to note that the optimal assay conditions for the
two cysteine proteases are different, and the assay conditions
may influence the observed inactivation constants. Since K777
Antitrypanosomal activity, cytotoxicity and protease inhibitory data of active
acrylates and their vinyl sulfone analogues
Compound Rhodesain Kinact/KI
T. brucei IC50
M)
Hep G2 IC50
M)
(MÀ1 sÀ1
)
(l
(l
5
6
7
8
9
10
18.32
3.47
13.22
Inactive
Inactive
Inactive
30.31 1.92
30.16 0.86
43.36 2.50
29.52 2.83
>50
>150
69 13.68
>150
76 13.17
>150
>150
>50
has
a vinyl sulfone electrophile as a Michael acceptor, we
investigated if vinyl sulfone analogues of 5 and 7 were capable of
inhibiting rhodesain.16 We have found that compound 6, which
is the vinyl sulfone analogue of 5, was also active in our assays,
but it was less reactive towards the cysteine protease than the
acrylate (5) (Table 1). However, compound 8, the vinyl sulfone ana-
log of 7, was inactive in the enzymatic assay, suggesting that
unique structural features of 7 drive its activity against rhodesain.
In addition, we investigated if the previously reported14 papain
inhibitor 9 can inhibit rhodesain, and found that it was not active
at inhibiting rhodesain. The vinyl sulfone analogue of 9 (10) was
then synthesized and tested, and it was also inactive towards
rhodesain. We then determined the time dependent inhibition con-
stant kinact/KI of compounds 5, 7 and 6, which were 18.32, 13.22 and
3.47 MÀ1 sÀ1, respectively (Table 1, Fig. 3). Taken together, our pre-
liminary SAR analysis indicates that a combination of specific struc-
tural features of the fragment and the electrophile make inhibitors
of rhodesain. Crystal structure analysis of the fragment-rhodesain
complexes is underway, and it should provide a structural ratonale
for extensive SAR studies. Although the identified covalent inhibi-
tors of rhodesain are fragments, and therefore need to be optimized
further to increase their potency and selectivity, we asked if any of
those compounds have antitrypanosomal activity. We rationalized
that those fragments that display antitrypanosomal activity, yet are
not toxic to human hepatocellular carcinoma (Hep G2), could be
further optimized into inhibitors that display selective toxicity to
T. brucei but not to human cells.
H
H
BnO2C
N
N
CO2Bn
O
O
O
EtO2C
N
H
CO2Bn
2
1
H
H
Cl
Cl
N
NH2
Cl
Cl
N
NH2
N
N
S
S
3
4
Figure 1. Inhibitors of rhodesain that have antitrypanosomal activity.
asked if the electrophilic fragments can be screened individually in
enzymatic assays to identify weak and irreversible fragment inhi-
bitors. Notably, such an approach would contradict current prac-
tices in academia and industry, in which reactive compounds are
removed from compound screening collections. We envisioned
that if successful, this strategy would substantially expand the
use of irreversible tethering in laboratory settings in which mass
spectrometry services are not available. Thus, a previously made
library of 200 electrophilic fragments was screened for inhibitor
activity against rhodesain in in vitro enzymatic assays.14,15 The
active hits were then investigated for their antitrypanosomal activ-
ity and cytotoxicity to human Hep G2 cells. The initial screen of the
Therefore, compounds 5–10 were tested for growth inhibitory
activity on T. brucei as well as their cytotoxicity on Hep G2 cells.
Compounds 5 and 7 have emerged as promising lead structures,
since both compounds were potent at inhibiting rhodesain
in vitro (kinact/KI values 18.32 and 13.22, respectively), and
fragment library (10 lM) against rhodesain in 384-well assay
plates led to the identification of seven positive hits (SI Fig. 1).
These seven compounds caused P85% inhibition of rhodesain. A
O
O
F
F
O
N
H
S
S
N
H
S
O
O
O
N
N
5
6
O
O
O
N
H
S
N
H
O
O
N
O
N
O
O
8
7
O
O
O
N
H
S
N
O
H
N
O
N
O
O
O
10
9
Figure 2. Inhibitors of rhodesain from this study.

D. McShan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4509–4512
4511
Figure 3. Pseudo-first order and second-order inhibition plots for compounds 5, 6 and 7.
Scheme 1. Overview of rhodesain-fragment conjugation.
displayed selective toxicity to T. brucei without being toxic to Hep
G2 cells (Table 1). Importantly, compound 8 was inactive in our
in vitro assays, despite the fact that it was active in the antitry-
panosomal assay. This indicates that 8 may be reactive towards
one or more other catalytic cysteines in T. brucei, although the
weak selectivity index of 8 makes it a less desired lead compound.
In conclusion, an electrophilic fragment library was evaluated
for inhibitory activity against the cathepsin-L like cysteine pro-
tease rhodesain. The unique feature of this approach is that reac-
tive compounds were screened in an enzymatic assay in a 384
well plate format to identify specific hits, which stands in sharp
contrast to the currently accepted dogma in the pharmaceutical
industry that reactive compounds must be excluded from all HTS
screens, because reactive compounds can display promiscuous
reactivity toward their protein targets. Our results show that in
fact it is possible to screen a library of cysteine reactive fragments
in enzymatic assays in a 384 well plate format if the library of the
cysteine reactive fragments is properly designed.14 Furthermore,
the non-peptidic nature of the identified inhibitors of rhodesain
could result in better pharmacokinetic properties of the covalent
rhodesain inhibitor drug leads. Furthermore, current known cova-
lent inhibitors of rhodesain have two electron withdrawing groups

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D. McShan et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4509–4512
present at the Michael acceptor site, which can increase the num-
ber of off-target effects for such inhibitors. In contrast, our frag-
ment libraries have only one electron-withdrawing group at the
Michael acceptor site, which should reduce the electrophilicity
and non-specific reactivity of these fragments (Scheme 1). We
envision that fragments that contain other electrophiles can be
assembled and tested against other cysteine proteases either using
mass spectrometry or enzymatic assays in the 96 or 384 well plate
format, which will significantly expand the use of the irreversible
tethering technology. Further optimization of the identified rhode-
sain inhibitor fragments into potent and selective lead compounds
will be reported in the near future. Although compounds 5 and 7
were also previously identified as papain hits, we believe that we
can achieve reasonable selectivity for rhodesain amongst other
papain-family cysteine proteases upon growth of the fragment into
a drug lead, similar to how selectivity amongst ATP competitive
kinase inhibitors is achieved.
fragments) with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmcl.2015.08.074.
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Acknowledgments
This work was supported in part by the US National Institutes of
Health (SC2GM109782 to I.V.O., T32GM105538 to S.K), American
Cancer Society Institutional Research Grant (93-037-18 to A.S.),
Chemistry of Life Processes Institute Lambert Fellowship (Z.X.),
the ACS Medicinal Chemistry Fellowship (S.K.) and Northwestern
University (A.S.) A.S. is a Pew Scholar in the Biomedical Sciences,
supported by the Pew Charitable Trusts. We thank Rama Mishra
and the Center for Molecular Innovation and Drug Discovery for
assisting with the initial design of the library of electrophilic
fragments.
14. Kathman, S.; Xu, Z.; Statsyuk, A. A. J. Med. Chem. 2014, 57, 4970.
15. The fragment library includes compounds that satisfy the following criteria:
molecular weight (MW) 6 350 Da, AlogP 6 3, hydrogen-bond acceptors 63,
hydrogen-bond donors 63, rotatable bonds 63, and polar surface area 680.
The library has a diversity index of 0.75.
Supplementary data
16. Supplementary material: chemical synthesis, bioassay, compound
characterization data, activity data and structures of fragments are provided
as Supporting material.
Supplementary data associated (chemical synthesis, bioassay,
compound characterization data, activity data and structures of