inelastic neutron scattering and ab initio theo-ries of correlated electron systems.
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The research at the Joint Institute for Nuclear Research was
supported by the Russian Foundation for Basic Research project
16-02-01086. The research at Argonne National Laboratory and
Los Alamos National Laboratory was supported by the Materials
Sciences and Engineering Division, Office of Basic Energy Sciences,
U.S. Department of Energy. The research at Oak Ridge National
Laboratory’s Spallation Neutron Source was supported by the
Scientific User Facilities Division, Office of Basic Energy Sciences,
U.S. Department of Energy. Neutron experiments were performed
at the Spallation Neutron Source, Oak Ridge National Laboratory,
and the ISIS Pulsed Neutron Source, Rutherford Appleton
Laboratory. We gratefully acknowledge the computing resources
provided on Blues, a high-performance computing cluster operated
by the Laboratory Computing Resource Center at Argonne National
Laboratory. We are also grateful for useful discussions with
P. Riseborough. Files containing the data sets used in this paper
are available for download from http://dx.doi.org/doi:10.18126/
M2C914. Four-dimensional inelastic neutron scattering data from
ARCS are stored in HDF5 files, conforming to the NeXus standard
( www.nexusformat.org), which can be viewed using the open-
source application, NeXpy ( http://nexpy.github.io/nexpy/). The
data can be compared with DFT+DMFT calculations, produced
using the Wien2K+DMFT package (31), which are also stored in
NeXus files. Inelastic neutron scattering data from MERLIN are
available as RAR archives, containing files produced by the Horace
suite of MATLAB programs ( http://horace.isis.rl.ac.uk/), which can
be used to extract cuts and slices through the 4D data.
Materials and Methods
Figs. S1 to S8
27 February 2017; accepted 1 December 2017
Mapping the malaria parasite
druggable genome by using in vitro
evolution and chemogenomics
Annie N. Cowell,1 Eva S. Istvan,2 Amanda K. Lukens,3,4 Maria G. Gomez-Lorenzo,5
Manu Vanaerschot,6 Tomoyo Sakata-Kato,3 Erika L. Flannery,1 Pamela Magistrado,3
Edward Owen,7 Matthew Abraham,1 Gregory LaMonte,1 Heather J. Painter,7
Roy M. Williams,1 Virginia Franco,5 Maria Linares,5 Ignacio Arriaga,5 Selina Bopp,3
Victoria C. Corey,1 Nina F. Gnädig,6 Olivia Coburn-Flynn,6 Christin Reimer,1
Purva Gupta,1 James M. Murithi,6 Pedro A. Moura,6 Olivia Fuchs,1 Erika Sasaki,1
Sang W. Kim,1 Christine H. Teng,1 Lawrence T. Wang,1 Aslı Akidil,8 Sophie Adjalley,8
Paul A. Willis,9 Dionicio Siegel,10 Olga Tanaseichuk,11 Yang Zhong,11 Yingyao Zhou,11
Manuel Llinás,7 Sabine Ottilie,1 Francisco-Javier Gamo,5 Marcus C. S. Lee,6,8
Daniel E. Goldberg,2 David A. Fidock,6,12 Dyann F. Wirth,3,4 Elizabeth A. Winzeler1,10*
Chemogenetic characterization through in vitro evolution combined with whole-genome
analysis can identify antimalarial drug targets and drug-resistance genes. We performed
a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse
compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes
associated with drug-resistance acquisition, where gene amplifications contributed to
one-third of resistance acquisition events. Beyond confirming previously identified
multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor
pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a
pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum
resistome and druggable genome will likely guide drug discovery and structural biology
efforts, while also advancing our understanding of resistance mechanisms available to the
Malaria has a disproportionately negative impact on human health because its causal protozoan parasites are adept at changing their genomes to evade anti- malarial drugs and the human immune
system. A single human infection may result in
upwards of 1012 asexual blood-stage parasites.
Thus, even with a relatively slow random muta-
tion rate (~10−9 per nucleotide site per mitotic
division) in the parasite, within a few cycles of
replication, each base in the P. falciparum ge-
nome can acquire a random genetic change that
may render at least one parasite resistant to the
activity of a drug or a human-encoded antibody. The
recent evolution of artemisinin-resistant parasites
in Southeast Asia now threatens both life-saving
treatments and malaria-control efforts (1).
Although this rapid evolution impedes our
ability to control the disease, in vitro evolution in
the presence of known antimalarials, followed by
whole-genome sequencing of resistant clones, can
be used to discover mediators of drug resistance
(2). Testing for the evolution of resistance can
also reveal antimalarial drug targets (3). Com-
pared with targets that are validated using genetic
knockdown methods, chemically validated drug
targets are more valuable because their activity
can be inhibited in cultured parasites by a small
molecule. Furthermore, the inhibitor provides
a tool for crystallization and chemical genetic
studies. Most studies using this method to date
have focused on single gene mutations in re-
sponse to single compounds, even though, in
many cases, additional allelic changes have been
noted in P. falciparum clones during the acqui-
sition of compound resistance.
Next-generation sequencing shows both
neutral and positive selection
We systematically studied patterns of P. falciparum
genome evolution by analyzing the sequences of
clones resistant to diverse compounds with antimalarial activity across the P. falciparum life
cycle. To investigate the genomic evolutionary
response to treatment with small molecules, we
assembled a collection of isogenic P. falciparum
clones that had acquired resistance to an array
of chemically distinct small-molecule growth