(21, 24). This may reflect the small conformational
changes within the orthosteric site between active
and inactive states (21), allowing for a broader
recognition of molecules, at least in docking
screens. As important may be the insistence on
previously untested chemical entities, which
favors diverse functional properties in the newly
To optimize 9, 75 analogs from ZINC were
docked, and 15 were prioritized for testing on
the basis of docking score, EBP occupancy, interaction with predicted arrestin-biasing residue
L187ECL2 (25), and diversity (table S10). All retained the quinolone and aminergic moieties,
which engage key residues of the OBP, with most
variations in the side chain predicted to interact
with EBP residues. For example, compounds 9-6
and 9-11 (Fig. 4A and table S9) retained the
quinolone heterocycle and the bidentate amino
groups, with the hydroxymethyl replaced by simple extended side chains. While retaining major
interactions of the quinolone heterocycle with
the OBP, the increased flexibility of these analogs
facilitated better packing against the specificity-determining EBP of DRD4 (Fig. 4B and fig. S14A).
9-6 and 9-11 had inhibition constants (Ki) that
were improved 20-fold relative to that of 9 and lost
detectable binding at DRD2 or DRD3 at 10 mM
(Fig. 4, A and B; fig. S14A; and table S9). They
are potent DRD4 partial agonists with arrestin-bias factors of 20- and 7.8-fold versus quinpirole
(Fig. 4D and fig. S14B). This bias is consistent
with the docking, which selected for interactions with L187ECL2 (25) and with which the conserved quinolone interacts (Fig. 4B and fig. S14A).
EBP residue mutations disrupted the affinities of
the newly discovered agonists (table S9), consistent
with their docked poses (Fig. 4B and fig. S14A).
To further this series and increase compound
affinity and selectivity, we docked 169 analogs of
9-6 and 9-11, with a particular focus on chemically diverse moieties predicted to interact with
the EBP. Of 27 docked compounds assayed, the
most potent compound, 9-6-24, exhibited a Ki of
3 nM, with no measurable affinity for DRD2,
DRD3, or the EBP DRD4 mutant F2.61V/L3.28F,
resulting in an increased selectivity of >3300-fold
(tables S9 and S11). 9-6-24 has a phenolic ether
oxygen (Fig. 4A), which appears to form an internal hydrogen bond with the amine, thus
positioning the distal aryl ring over the nonconserved F912.61 in the EBP (Fig. 4C). With
respect to quinpirole, 9-6-24 is a partial agonist
with a 7.4-fold bias toward arrestin over Gai/o
signaling (Fig. 4E and fig. S15), which contrasts
with most existing DRD4 agonists that are either
balanced or slightly G protein–biased (fig. S16).
Given that neither the parent compound 9-6
nor 9-6-24 displayed agonist activity at 320
nonolfactory GPCRs at 1 mM (Fig. 4, F and G),
9-6-24 is among the most potent and specific DRD4 agonists characterized.
Unlike its selectivity, the agonism or bias of
9-6-24 was not initially purposely designed (al-
though we did focus on putatively biasing the
contact residue), but rather was a result of our
focus on untested chemical entities followed by
experimental selection of these desired features.
We are making 9-6-24 and a negative control
molecule with 1/2500th the affinity (9-6-16) open-
ly available to the community to use as probes for
DRD4 function, under the names UCSF924 and
UCSF924NC, respectively (table S11).
Herein we present a combination of structural,
computational, and biochemical studies that il-
luminates the structure and function of DRD4 at
atomic resolution. We further show that leverag-
ing high-resolution structural data in ligand dis-
covery campaigns guided by pharmacological
assays can rapidly uncover previously unrecog-
nized, potent, and highly selective probes with
desired functional properties.
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This work was supported by the NIH (grants R01MH112205,
U19MH82441, and HHSN-271-2013-00017-C), the Michael Hooker
Chair for Protein Therapeutics and Translational Proteomics (to
B.L.R.), and the National Institute of General Medical Sciences
(NIGMS; grant R35GM122481 to B.K.S.). We thank J. Sondek and
S. Endo-Streeter for independent structure quality-control analysis;
M. J. Miley and the University of North Carolina macromolecular
crystallization core facility supported by the NIH (grant P30CA016086);
J. W. Murphy for running FRAP (fluorescence recovery after
photobleaching) precrystallization assays; S. Sato for sharing the
original DRD4 construct; B. E. Krumm for advice on data processing;
andJ.Sm ith, R.Fi schetti, and the staff of GM/CA APS, supported by
the National Cancer Institute (ACB-12002) and NIGMS (AGM-12006).
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 Argonne National Laboratory under
contract no. DE-AC02-06CH11357. Coordinates and structure factors
have been deposited in the Protein Data Bank under accession
numbers 5WIU (sodium-free) and 5WIV (sodium-bound). UCSF924
and its deactivated negative control UCSF924NC are available as a
probe pair from Sigma Millipore (SML2022 and SML2023 for the
active and deactivated agonists, respectively).
Materials and Methods
Figs. S1 to S16
Tables S1 to S13
28 April 2017; accepted 7 September 2017
Taxon-restricted genes at the origin
of a novel trait allowing access to a
M. Emília Santos,1 Augustin Le Bouquin,1,2 Antonin J. J. Crumière,1 Abderrahman Khila1*
Taxon-restricted genes make up a considerable proportion of genomes, yet their contribution to
phenotypic evolution is poorly understood. We combined gene expression with functional and
behavioral assays to study the origin and adaptive value of an evolutionary innovation exclusive to
the water strider genus Rhagovelia: the propelling fan. We discovered that two taxon-restricted
genes, which we named geisha and mother-of-geisha, specifically control fan development.
geisha originated through a duplication event at the base of the Rhagovelia lineage, and both
duplicates acquired a novel expression in a specific cell population prefiguring fan development.
These gene duplicates played a central role in Rhagovelia’s adaptation to a new physical
environment, demonstrating that the evolution of taxon-restricted genes can contribute directly
to evolutionary novelties that allow access to unexploited ecological niches.
Morphological innovations—i.e., lineage- restricted traits that perform evolutionar- ily new functions—are important triggers of organismal diversification (1, 2). Theory predicts that the evolution of such inno-
vations allows organisms to adapt to new niches
and therefore provides access to unexploited eco-
logical opportunities (3, 4). Examples include the
evolution of plant flowers, insect wings, butter-
fly color patterns, turtle shells, lizard dewlaps
and toepads, and bird feathers, each of which is
thought to have shaped the evolutionary trajectory
386 20 OCTOBER 2017 • VOL 358 ISSUE 6361
RESEARCH | REPORTS