Journal of Agricultural and Food Chemistry
Article
inhibitory activity of these compounds is identical in trend. The
specific method is as follows: according to the known coupled enzyme
reaction,38,39 the inhibitory activity of the tested compounds against
AtHPPD was determined by spectrophotometrically monitoring the
production of maleylacetoacetate using a Molecular Devices Synergy
H1 microplate reader (Biotek, Winooski, VT) with excitation at 318
nm at 30 °C.40,41 All experiments were carried out in triplicate. By
fitting the data of absorbance change over time, the IC50 values
(Tables 1 and 2) of compounds 9−60 and the positive controls MBQ
and mesotrione were processed and calculated using OriginPro 7.5
software (Origin Lab, Northampton, MA).
Determination of Ki Value. Using the experimental methods as
previously described,31,32,41,42 the kinetics of time-dependent enzyme
inhibition of representative compounds were studied on a Lambda 45
ultraviolet spectrophotometer (PerkinElmer, Waltham, MA). In short,
we first determined the parameters vs, v0, and kobs (expressed as
steady-state velocity, initial velocity, and the observed first-order rate
constant, respectively) in the presence of representative compounds
according to the process curves by fitting the equations described in
(association and dissociation rate constants) according to the
relationship between kobs and substrate concentrations by fitting the
(Ki) is equal to k−0/k+0.
Crystallization and Structure Determination. The crystal
structures of AtHPPD-MBQ (PDB code: 5YWK) and AtHPPD-60
(PDB code: 6M6D, unreleased) were obtained by crystallization
experiments under the conditions listed in Table S2. All of the
diffraction data were collected at the Shanghai Synchrotron Radiation
Facility 17 U1 and 19U1 beamlines and then processed using
HKL2000 software.43 Subsequently, these structures were solved by
molecular replacement using Phaser,44 taking AtHPPD complexed
with BQ (PDB code: 5YY6)32 as the search model. The standard
refinement was carried out with programs PHENIX45 and Coot.46
Data Collection and Refinement Statistics are presented in Tables S3
and S4. All complex structures were visualized in Pymol software
visually after 30 days of treatment. To minimize experimental errors,
we performed three sets of parallel experiments for all of the tested
compounds. All of the inhibition rates of the compounds are listed in
Tables 1 and 2. The herbicidal spectrum and crop selectivity data of
inhibitors MBQ, 12, 28, 29, and 60 are summarized in Table 4. These
statistical inhibition rate data of the tested compounds against crops
or weeds are determined by the severity of the bleaching symptoms
produced by the plants.
Toxicity Experiment. Nontarget organisms including the bee,
earthworm, silkworm, and fish were selected to investigate the acute
toxicity of the representative compound. All of the bioassays of acute
toxicity were set up according to the criterion for chemical pesticide
environmental safety assessment released from the General
Administration of Quality Supervision, Inspection and Quarantine
of the Peopleʼs Republic of China. The detailed description of toxicity
tests for the above-mentioned organisms is presented in the
Xishan Zhongke Drugs Research and Development Co., Ltd. (Xishan,
Suzhou, China).
RESULTS AND DISCUSSION
■
Crystal Structure of AtHPPD-MBQ Complex and
Structure-Based Design. Previously, we discovered the
lead compound, BQ, that potently inhibits AtHPPD activity
via virtual screening, suggesting that triketone-quinazoline-2,4-
dione represents a novel scaffold of HPPD inhibitors.36 As this
work progressed, we carefully probed the SARs of the Ra to Rd
positions in compound BQ. The methyl group was regarded to
be the optimum insertion at the Ra and Rb positions of the
quinazoline-2,4-dione moiety due to its contribution to the
enhancement of both HPPD inhibition and herbicidal efficacy.
Incorporating structural fine-tuning at the Rc and Rd sites led
to the discovery of a new lead compound, MBQ, showing an
excellent IC50 value of 270 nM against AtHPPD (Figure 1A).
Based on the above information, an optimization study aimed
at improving the binding affinity of MBQ with AtHPPD was
begun.
Binding Free Energy Calculations. Three-step energy mini-
mization was carried out for the AtHPPD-inhibitor complex before
binding free energy calculation using the sander module in AMBER
16 package (Amber, San Francisco, CA) first to minimize the ions and
hydrogens and others were fixed. Second, only backbone atoms of
AtHPPD were fixed and other atoms were allowed to move. Third, all
atoms were free to move. In the three steps, we all used a 2000-step
steepest descent method and the conjugated gradient method for the
next 200 steps. Then, the binding free energies (ΔGcal) of
representative inhibitors with AtHPPD were calculated using the
MM-PBSA strategy47 in AMBER 16 program. The detailed
Greenhouse Herbicidal Activity and Crop Selectivity. As
described previously,35,36 the stock solutions of title compounds 9−60
were prepared with predefined application dosages of 30, 60, 120, and
150 g ai/ha. Echinochloa crus-galli (E.c.), Setaria viridis (S.v.), Digitaria
sanguinalis (D.s.), Amaranthus retroflexus (A.r.), Chenopodium
serotinum (C.s.), and Abutilon theophrasti (A.t.) were selected as
representative test weeds. When these weeds had grown to the four-
leaf stage, they were treated by the solutions containing inhibitor at
the corresponding concentrations. The postemergence herbicidal
responses of the prepared samples against six representative species of
weeds were evaluated using a visual measurement method previously
published.33−36 In the same way, some important gramineous weeds
such as Aegilops squarrosa (A.s.), Avena fatua (A.f.), Bromus japonicus
(B.j.), Alopecurus aequalis (A.a.), and Alopecurus japonicus (A.j.) were
selected to explore the weed-controlling spectrum of the representa-
tive compounds. Besides, four-leaf crops such as wheat, rice, cotton,
soybean, rape, peanut, and maize, which were cultivated under similar
conditions to the aforementioned weeds, were used to evaluate crop
safety in the presence of the selected compounds at the dosages of
30−120 g ai/ha. Herbicidal activity and crop safety were evaluated
In an attempt to perform this project more rationally, a
cocrystal structure of AtHPPD complexed with MBQ was
determined (PDB code: 5YWK). Careful inspection of the
crystal structure of the AtHPPD-MBQ complex (Figure 1B)
suggested that MBQ engaged in two sets of apparent
interactions in the active pocket of AtHPPD. One interaction
was that of the quinazoline-2,4-dione fragment parallelly
stacking between the aromatic side chains of residues
Phe381 and Phe424, forming a close π−π interaction. The
other interaction was that of tight chelating, which was
visualized between the two carbonyl groups of the triketone
ring of MBQ and the metal ion of the HPPD catalytic center.
It is also noteworthy that these two sets of interactions in this
region are shown in the binding modes of almost all
commercial HPPD inhibitors with the target protein.
Accordingly, we retained the quinazoline-2,4-dione ring and
triketone ring as vital elements for the subsequent optimization
of the binding affinity of new analogues with AtHPPD.
Furthermore, the N-substituted benzene of compound MBQ
could insert into the hydrophobic cavity at the entrance of the
active cavity and form weak hydrophobic contacts with
Met335, Phe392, and Pro384 residues therein. Inspired by
the above structural information, we speculated that establish-
ing stronger interactions with these hydrophobic residues
located in the highlighted region of Figure 1 could be
instrumental in improving quinazoline-2,4-dione inhibitor
binding with AtHPPD. Moreover, this region possessed a
vast space that could be capable of accommodating different
464
J. Agric. Food Chem. 2021, 69, 459−473