10.1002/anie.201810470
Angewandte Chemie International Edition
COMMUNICATION
Inhibitor-induced dimerization of an essential oxidoreductase
from African Trypanosomes
Annika Wagner,[a][b] Thien Anh Le,[c] Martha Brennich,[d] Philipp Klein,[e] Nicole Bader,[f] Erika Diehl,[a][b]
Daniel Paszek,[a] A. Katharina Weickhmann,[b] Natalie Dirdjaja,[g] R. Luise Krauth-Siegel,[g] Bernd
Engels,[c] Till Opatz,[e] Hermann Schindelin*[f] and Ute A. Hellmich*[a][b]
presenting a serious health risk as well as an economical threat.
The essential oxidoreductase tryparedoxin (Tpx) in the T. brucei
Abstract: Trypanosomal and leishmanial infections claim tens of
thousands of lives each year. The metabolism of these single cell
eukaryotic parasites differs from the human host and their enzymes
thus constitute promising drug targets. Tryparedoxin (Tpx) from
Trypanosoma brucei is the essential oxidoreductase in the parasite’s
hydroperoxide clearance cascade. Functional in vitro and in vivo
assays show that a small, selective inhibitor efficiently inhibits Tpx.
With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD
simulations, ITC, and NMR spectroscopy, we show how covalent
binding of this monofunctional inhibitor leads to Tpx dimerization.
Intra- and intermolecular inhibitor-inhibitor, protein-protein and
inhibitor-protein interactions stabilize the dimer. The behavior of this
efficient antitrypanosomal molecule thus constitutes an exquisite
example of chemically induced dimerization with a small, monovalent
ligand that can be exploited for future drug design.
hydroperoxide clearance cascade,
a
distant relative of
thioredoxins,[1] presents a promising drug target.[2] It undergoes
thiol exchange reactions in its WC40PPC43 active site motif leading
to peroxidase reduction and hydroperoxide clearance, protecting
the parasite from oxidative damage (Scheme 1). The cascade
also delivers reducing equivalents for DNA synthesis and protein
repair, which are vital for pathogen metabolism and proliferation.[3]
Scheme 1. (a) The T. brucei hydroperoxide clearance cascade consists of
NADPH as the electron donor, trypanothione reductase (TR), trypanothione
(TS2/T(SH)2), the oxidoreductase tryparedoxin (Tpx) and peroxidases (Px). CFT
inhibits Tpx in vitro and in vivo (Fig. 1, Fig. S1). (b) Structure of the Tpx inhibitor
CFT.[4]
Kinetoplastids, comprising African and South American
Trypanosoma and Leishmania species, are the causative agents
of devastating diseases. Trypanosoma brucei species cause
human African Trypanosomiasis (African sleeping sickness), a
neglected tropical disease, and Nagana cattle disease thus
A
selective
inhibitor
(2-(chloromethyl)-5-(4-
fluorophenyl)thieno[2,3-d]pyrimidin-4(3H)-one, CFT) for Tpx was
previously described to covalently interact with C40 in the Tpx
active site in vitro as well as in vivo (Fig. 1, S1).[4] In parasite cell
culture, our EC50 data for CFT agree with previously published
values (Fig. S1a).[4] Tpx activity and CFT inhibition efficiency were
quantified in vitro via the rate of NADPH consumption in the
reconstituted hydroperoxide clearance cascade (Scheme 1, Fig.
S1b-d). Removing the chloride leaving group renders the inhibitor
ineffective in vivo and in vitro (Fig. S1), thus highlighting the
importance of the covalent interaction of CFT with Tpx.
To elucidate the structural basis of T. brucei Tpx inhibition, we
studied the Tpx-CFT complex by X-ray crystallography, SAXS,
MD simulations, analytical SEC, SEC-MALS, NMR and ITC and
strikingly, found that CFT dimerizes Tpx.
“Small” molecules that promote chemically induced dimerization
(CID), so called dimerizers, have been used, e.g. to control
cellular signaling pathways.[5,6] Certain drugs, such as rapamycin,
[a]
A. Wagner, E. Diehl, D. Paszek, Prof. Dr. U. A. Hellmich
Institute of Pharmacy and Biochemistry
Johannes Gutenberg-University Mainz
Johann-Joachim-Becherweg 31, D-55128 Mainz, Germany
E-mail: u.hellmich@uni-mainz.de
[b]
[c]
[d]
[e]
[f]
A. Wagner, A. K. Weickhmann, Prof. Dr. U. A. Hellmich
Biomolekulares Magnetresonanz Zentrum (BMRZ)
Goethe-Universität Frankfurt
Max-von-Laue Str. 9, D-60438 Frankfurt/M, Germany
T.A. Le, Prof. Dr. B. Engels
Institute of Physical and Theoretical Chemistry
Julius-Maximilians-University Würzburg
Emil-Fischer-Straße 42, D-97074 Würzburg, Germany
Dr. M. Brennich
Synchrotron Crystallography Team
EMBL Grenoble Outstation
71 Avenue des Martyrs, 38042 Grenoble, France
P. Klein, Prof. Dr. T. Opatz
Institute of Organic Chemistry
linking
mTOR
and
FKBP,
are naturally occurring
heterodimerizers.[6] Covalently linked dimers of e. g. FK506 were
used as artificial symmetric homodimerizers.[5] Most dimerizers
tend to be relatively large and are almost always bifunctional.[5,6]
In contrast, CFT presents a small, monofunctional dimerizer that
covalently binds to Tpx.
Johannes Gutenberg-University Mainz
Duesbergweg 10-14, D-55128 Mainz, Germany
N. Bader, Prof. Dr. H. Schindelin
Rudolf Virchow Center for Experimental Biomedicine
Julius-Maximilians-University Würzburg
Josef-Schneider-Straße 2, Haus D15, D-97080 Würzburg, Germany
E-mail: hermann.schindelin@virchow.uni-wuerzburg.de
N. Dirdjaja, Prof. Dr. R. L. Krauth-Siegel
Biochemistry Center (BZH)
Crystal structures exist of oxidized T. brucei Tpx as well as photo-
reduced and glutathionylspermidine (a trypanothione mimic)
bound C. fasciculata Tpx.[7] However, to date, no structure of a
Tpx in complex with a drug molecule to serve as a starting point
for structure-based drug design is available.
We thus determined the X-ray crystal structures of the T. brucei
Tpx-CFT complex at 1.6 and 1.8 Å resolution (Fig. 1, Table S1).
Each unit cell contains three chains, A. B and C, of the thioredoxin
fold protein, each in complex with CFT (see SI for a more detailed
description).
[g]
Ruprecht-Karls-University Heidelberg
Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.