Communication
wards the side chains of Thr335 and Ile339, with additional
close CÀH···p interactions with His461’ (Figures S5 and S6 in
the Supporting Information). In the structure of 5, similar inter-
actions with Tyr110 (d(N···OTyr110)=3.1 ꢁ) and the hydrophobic
sub-pocket (Val53, Val58, and Ile106) are observed (Figure 2c).
Overall, the observed binding mode of (+)-2 in active site B of
the X-ray co-crystal structure corresponds to the one predicted
in our design (see Section S2 in the Supporting Information).
The origin of the significantly weaker TR binding of 5 (Ki
value: 1.5 mm) compared to (+)-2 (73 nm) remains unclear
after structural analysis. The most probably protonated prop-
argyl amine of (+)-2 forms a longer, much weaker H bond
(4.1 ꢁ) to the phenolic O-atom of Tyr110 than the neutral
amine in 5 (3.1 ꢁ). Also, the CF3 group does not engage in re-
pulsive electrostatic interactions[24] but rather accommodates
well in a hydrophobic surrounding shaped by Ile106, Leu339’,
Val58, Val53, and His461’.[24]
In previously published structures,[5c,7] a large unexplained
density had been observed close to the entrance to the Z-
site[25] in active site B. The higher resolution of our present co-
crystal structures allows to assign this density to a HEPES (2-[4-
(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer mole-
cule (Figure 2 and Figures S7–9 in the Supporting Information).
This observation highlights that further opportunities for
ligand-protein interactions are available in the wide TR active
site. It provides inspiration for the structure-based develop-
ment of even stronger inhibitors combining the interactions
established by current ligands and the HEPES molecule, for ex-
ample, by extending the propargylic vector to also displace
the buffer molecule (see Figure S7, Supporting Information, for
a detailed representation of its binding mode). In the struc-
tures of (+)-2 and 5, the closest distances between the HEPES
molecule and the propargylic vector of the ligands in active
site B are only 4.5 and 3.9 ꢁ, respectively. In the complex with
(+)-4 an additional ligand molecule is found stacking to the
main binding ligand in active site B (Figure S8 in the Support-
ing Information), as already observed in other co-crystal struc-
tures of TR inhibitors.[26]
future in vivo studies. We propose a further extension of the
propargylic vector to replace the buffer molecule.
Acknowledgements
The authors thank the ETH Research Council (ETH-01 13-2) for
support. Research at the University of Heidelberg was support-
ed by the Deutsche Forschungsgemeinschaft (DFG KR 1242/8-
1) and at the University of Toronto by the Natural Sciences and
Engineering Research Council of Canada (RGPIN-2015-04877)
and the Canadian Research Chairs Program. We thank Dr. Mas-
similiano Donzelli and Bjçrn Wagner from F. Hoffmann-La
Roche Ltd for PAMPA and microsomal stability measurements.
We are grateful to Dr. Bruno Bernet (ETH) for proofreading the
Experimental Section, and Dr. Cꢂdric Schaack (ETH) for proof-
reading the entire manuscript and helpful discussions. Monica
Cal (Swiss TPH), Romina Rocchetti (Swiss TPH) and Sonja Keller-
Mꢃrki (Swiss TPH) helped with the parasite assays. We thank
Natalie Dirdjaja (BZH) for the preparation of trypanothione di-
sulfide and TR enzyme, and G. Prive (UHN) for use of diffraction
equipment. Use of the IMCA-CAT beamline 17-ID at the Ad-
vanced Photon Source was supported by the companies of the
Industrial Macromolecular Crystallography Association through
a contract with Hauptman-Woodward Medical Research Insti-
tute. This research used resources of the Advanced Photon
Source, a U.S. Department of Energy (DOE) Office of Science
User Facility operated for the DOE Office of Science by Ar-
gonne National Laboratory under Contract No. DE-AC02-
06CH11357.
Conflict of interest
The authors declare no conflict of interest.
Keywords: antiprotozoal agents
· co-crystals · molecular
recognition · neglected diseases · X-ray diffraction
In conclusion, we investigated two main structural modifica-
tions of lead compound 1, namely the improvement of the
indole N-substituent and the introduction of a lean propargylic
substituent in position 4 of the thiazole moiety. These modifi-
cations provided a remarkable enhancement of the binding af-
finity towards T. brucei TR with five inhibitors showing Kic
values in the sub-micromolar range. The most potent ligand of
the series (+)-2 resulted in a Kic value of 73 nm. To the best of
our knowledge, it is the strongest competitive inhibitor of this
enzyme reported to date. Only a noncompetitive polyamine-
based ligand reported by Chitkul and Bradley had a similar po-
tency, with a Ki value for T. cruzi TR of 76 nm; this ligand has
not been tested on cells.[27] Our new ligands showed strong in
vitro activities with IC50 values for (+)-2 and 5 of 120 nm and
50 nm, respectively. Their binding modes were elucidated from
co-crystal structures which also revealed the complexation of a
HEPES buffer molecule in close proximity. This new structural
insight paves the way for the structure-based design of the
next-generation inhibitors with low nanomolar activities for
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