Use of resistant ACCase mutants to screen for novel inhibitors against resistant and susceptible forms of ACCase from grass weeds

Article abstract of DOI:10.1021/jf034738m

The aryloxyphenoxypropionic acid (AOPP) and cyclohexanedione (CHD) herbicides inhibit the first committed enzyme in fatty acid biosynthesis, acetyl CoA carboxylase (ACCase). The frequent use of AOPP and CHD herbicides has resulted in the development of resistance to these herbicides in many grass weed species. New herbicides that inhibit both the susceptible and resistant forms of ACCase in grass weeds would have obvious commercial appeal. In the present study, an attempt was made to identify molecules that target both the herbicide-sensitive and -resistant forms of ACCase. Seven experimental compounds, either CHD-like or AOPP-CHD hybrids, were synthesized and assayed against previously characterized susceptible and resistant forms of ACCase. All seven compounds inhibited ACCase from sensitive biotypes of Setaria viridis and Eleusine indica (I₅₀ values from 6.4 to > 100 μM) but were not particularly potent compared to some commercialized herbicides (I₅₀ values of 0.08-5.6 μM). In almost all cases, the I₅₀ values for each compound assayed against the resistant ACCases were higher than those against the corresponding sensitive ACCase, indicating reduced binding to the resistant ACCases. One compound, a CHD analogue, was almost equally effective against the resistant and susceptible ACCases, although it was not a very potent ACCase inhibitor per se I₅₀ of 51 and 76 μM against susceptible ACCase from S. viridis and E. indica, respectively). The AOPP-CHD hybrid molecules also inhibited some of the resistant ACCases, with I₅₀ values ranging from 6.4 to 50 μM. These compounds may be good leads for developing ACCase inhibitors that target a wider range of ACCase isoforms, including those found in AOPP- and CHD-resistant weed biotypes.

Full text of DOI:10.1021/jf034738m

5144 J. Agric. Food Chem. 2004, 52, 5144−5150
Use of Resistant ACCase Mutants To Screen for Novel
Inhibitors against Resistant and Susceptible Forms of ACCase
from Grass Weeds
AMIT SHUKLA,*,†,§ CORWIN NYCHOLAT,†,| MANI V. SUBRAMANIAN,‡,
†,X
RICHARD J. ANDERSON,‡,# AND MALCOLM D. DEVINE
Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive,
Saskatoon, Saskatchewan, Canada S7N 5A8, and Novartis Crop Protection Inc.,
975 California Avenue, Palo Alto, California 94304
The aryloxyphenoxypropionic acid (AOPP) and cyclohexanedione (CHD) herbicides inhibit the first
committed enzyme in fatty acid biosynthesis, acetyl CoA carboxylase (ACCase). The frequent use
of AOPP and CHD herbicides has resulted in the development of resistance to these herbicides in
many grass weed species. New herbicides that inhibit both the susceptible and resistant forms of
ACCase in grass weeds would have obvious commercial appeal. In the present study, an attempt
was made to identify molecules that target both the herbicide-sensitive and -resistant forms of ACCase.
Seven experimental compounds, either CHD-like or AOPP-CHD hybrids, were synthesized and
assayed against previously characterized susceptible and resistant forms of ACCase. All seven
compounds inhibited ACCase from sensitive biotypes of Setaria viridis and Eleusine indica (I50 values
from 6.4 to >100 µM) but were not particularly potent compared to some commercialized herbicides
(I50 values of 0.08-5.6 µM). In almost all cases, the I50 values for each compound assayed against
the resistant ACCases were higher than those against the corresponding sensitive ACCase, indicating
reduced binding to the resistant ACCases. One compound, a CHD analogue, was almost equally
effective against the resistant and susceptible ACCases, although it was not a very potent ACCase
inhibitor per se (I50 of 51 and 76 µM against susceptible ACCase from S. viridis and E. indica,
respectively). The AOPP-CHD hybrid molecules also inhibited some of the resistant ACCases, with
I
50 values ranging from 6.4 to 50 µM. These compounds may be good leads for developing ACCase
inhibitors that target a wider range of ACCase isoforms, including those found in AOPP- and CHD-
resistant weed biotypes.
KEYWORDS: Structure-activity relationships; acetyl coenzyme A carboxylase; ACCase; aryloxyphe-
noxypropionic acid; cyclohexanedione; herbicide resistance
INTRODUCTION
death of the plant (1). In general, ACCase from grasses is
susceptible to inhibition by AOPP and CHD herbicides, whereas
dicot ACCase is tolerant to these herbicides (3). These herbicides
inhibit the eukaryotic form of ACCase found in the plastids of
most grass species, but not the prokaryotic form, which is the
major form present in dicots (4). This is the primary mechanism
of selectivity of these herbicides between grasses and dicots.
Tolerance of these herbicides in some grasses, including some
cereal crops, is conferred by enhanced metabolism of the
herbicides to inactive compounds (5).
The aryloxyphenoxypropionate (AOPP) and cyclohexanedi-
one (CHD) herbicides inhibit the enzyme acetyl CoA carboxy-
lase (ACCase; EC 6.4.1.2), which catalyzes the conversion of
acetyl CoA to malonyl CoA (1, 2). Inhibition of ACCase leads
to inhibition of acyl lipid biosynthesis, eventually resulting in
* To whom correspondence should be addressed. Phone: 306-966-2409.
Fax: 306-966-8894. E-mail: amit.shukla@usask.ca.
† University of Saskatchewan.
§ Present address: Genome Prairie, College of Agriculture, University
of Saskatchewan, Saskatoon S7N 5A8, Canada.
| Present address: Dept. of Chemistry, University of Alberta, Edmonton,
Alberta T6G 2G2, Canada.
It has been suggested that these two classes of herbicides
share a common binding site on the target enzyme, ACCase.
Double inhibition studies with AOPP and CHD herbicides and
malonyl CoA and CoA suggest that the two groups of herbicides
overlap in their binding to the target enzyme and that they
compete with malonyl CoA for binding at this site (6). Further
studies have shown that sethoxydim, a CHD, and haloxyfop,
‡ Novartis Crop Protection Inc.
Present address: Dow Chemical Co., 5501 Oberlin Dr., San Diego,
CA 92121.
# Present address: Cropsolution Inc., 430 Ferguson Dr., Mountain View,
CA 94043.
X Present address: Bayer Cropscience N.V., Jozef Plateaustraat 22,
B-9000 Gent, Belgium.
10.1021/jf034738m CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/08/2004

Resistant ACCase Mutants
J. Agric. Food Chem., Vol. 52, No. 16, 2004 5145
More recently, cDNA fragments encoding the carboxyltrans-
ferase domain of the multidomain plastid ACCase from
herbicide-resistant maize and from herbicide-sensitive and
-resistant annual ryegrass were cloned and sequenced (25). A
leucine residue was found in ACCases from herbicide-resistant
plants at a position occupied by isoleucine in all ACCases from
sensitive grasses studied. Leucine is also present at the
equivalent position in herbicide-resistant ACCases from other
eukaryotes. It was also shown that a single isoleucine to leucine
replacement at an equivalent position changes the wheat plastid
ACCase from sensitive to resistant. These results have revealed
an important mutation conferring herbicide-resistant ACCase.
As of yet, structural information of the complete multidomain
plastid ACCase is not available.
New herbicides that inhibit both the susceptible and re-
sistant forms of ACCase in grass weeds would have obvious
commercial appeal. In an attempt to identify molecules that
target both the herbicide-sensitive and -resistant forms of
ACCase, seven experimental compounds, either CHD-like or
AOPP-CHD hybrids (Figure 1), were synthesized and assayed
against previously characterized susceptible and resistant forms
of ACCase. In addition, one compound (IV) was also assayed
against resistant and susceptible maize ACCase.
Table 1. I50 Values and R/S I50 Ratios for ACCase from
Herbicide-Sensitive and -Resistant Biotypes of Green Foxtail (S1,
Sensitive; R1 and R2, Resistant) and Goosegrass (S3, Sensitive; R3,
Resistant), Assayed in Vitro with Known ACCase Inhibitors (17, 21,
23)
green foxtail
I50 (µM)
goosegrass
I50 (µM)
I50 (µM)
herbicide
diclofop
fenoxaprop 0.6 28
fluazifop
S1 R1 R1/S1 S1
R2
R2/S1 S3
R3
R3/S3
0.7 28
47
48
4.4
30
6.9 1.0
25
25
>90
5.6 >500
quizalofop 0.08 4.7
clethodim 0.6 18
sethoxydim 1.7 85
60
31
50
31
2.1
3.2
7.7 3260
8.9
7.8
4.2
2.4 1.5
420 3.8
6.6
77
4.4
20
tralkoxydim 0.3
9.4
an AOPP, are linear, noncompetitive inhibitors with various
substrates of ACCase and that the transcarboxylase reaction
(transfer of CO2 from the biotin prosthetic group to form
malonyl CoA) is inhibited by AOPP and CHD herbicides (3).
Double inhibition studies also suggest that the hydrophobic
oxime region of CHD herbicides overlaps with the hydrophobic
aryloxyphenoxy region of AOPP herbicides at the binding site
on the enzyme (7). Their results indicated that the CoA binding
site was distinct from the herbicide binding site and suggested
synthesis of CoA conjugates of herbicides to further study
substrate and herbicide binding sites on ACCase. The conjugates
diclofopyl-CoA, haloxyfopyl-CoA, and quizalofopyl-CoA were
prepared and found to inhibit rat liver ACCase more than the
parent acids, thus confirming that CoA and herbicide binding
sites are different (8).
The frequent use of AOPP and CHD herbicides has resulted
in the development of resistance to these herbicides in many
grass weed species in North and Central America, Europe, and
Australia (9). The species in which resistance has developed
include wild oat (AVena fatua) (10, 11), green foxtail (Setaria
Viridis) (12), giant foxtail (Setaria faberi) (13), annual ryegrass
(Lolium rigidium) (14-16), goosegrass (Eleusine indica) (17),
and maize (Zea mays) (18). In most cases, resistance is due to
alteration of the target enzyme, ACCase, making it less sensitive
to inhibition by these herbicides (11, 17-24).
The results of enzyme inhibition studies suggest several
distinct mutations in the ACCase gene, conferring different
levels of resistance to various ACCase inhibitors. For example,
ACCase from one biotype of green foxtail (referred to as R1 in
this study) was highly resistant to a broad spectrum of AOPP
and CHD herbicides, with R/S I50 ratios ranging from 31 for
clethodim to 60 for quizalofop (21). (For convenience, pub-
lished I50 values for various herbicide-ACCase combinations
are summarized in Table 1.) In contrast, a second biotype of
green foxtail (referred to as R2 in this study) was very re-
sistant to sethoxydim (R/S I50 ratio of 420) but only mar-
ginally resistant to other AOPP and CHD herbicides (23;
Table 1). This is similar to resistance in a sethoxydim-re-
sistant maize line (18), also used in this study (designated R4).
A third pattern of resistance was observed in a biotype of
goosegrass (referred to as R3 in this study); it was highly
resistant to fluazifop, moderately resistant to fenoxaprop and
sethoxydim, but only marginally resistant to clethodim (R/S I50
ratios of >90, 24, 20, and 4, respectively) (17; Table 1).
ACCase from the corresponding susceptible biotypes (S1 for
green foxtail, S3 for goosegrass, and S4 for maize) was sensitive
to inhibition by the AOPP and CHD herbicides tested (Table
1; 17, 18, 21, 23).
MATERIALS AND METHODS
Synthesis of Compounds. Compound I. Wittig reaction of 3-eth-
ylthiobutyraldehyde (26) and 1-triphenylphosphoranylidene-2-pro-
panone in refluxing dichloromethane gave 7-ethylthio-3-hepten-2-one
(Z/E, 1:14), which upon treatment with dimethyl 2-methoxymalonate
and sodium methoxide in methanol yielded 5-(2-ethylthiopropyl)-4-
methoxy-4-methylcarboxy-1,3-cyclohexanedione. Decarbomethoxyla-
tion (KOH/LiOH, then acidification), O-acylation with propionyl
chloride and triethylamine, and rearrangement (acetone cyanohydrin,
triethylamine) gave 5-(2-ethylthiopropyl)-4-methoxy-2-propanoyl-1,3-
cyclohexanedione, which on treatment with O-allylhydroxylamine
hydrochloride (triethylamine in ethanol) yielded I.
Compounds II and III. In an analogous manner, condensation of
dimethyl 2-fluoromalonate with 6-(5-chloro-2-pyridylthio)-3-hexen-2-
one (sodium methoxide in xylene) followed by decarbomethoxylation,
O-propanoylation, acetone cyanohydrin-catalyzed rearrangement, and
reaction with either O-allylhydroxylamine or O-ethylhydroxylamine
gave II and III, respectively.
Compound IV. Reaction of 2,4,6-trimethylphenylacetyl chloride and
N-methylhydroxylamine (triethylamine in THF/H2O) gave the corre-
sponding N-methylhydroxamic acid, which was O-alkylated with ethyl
2-bromo-2-methylpropionate (potassium carbonate in DMF). Treatment
of the product with potassium t-butoxide in toluene and acidification
gave IV.
Compounds V and VI. The syntheses of these oxazinediones were
achieved by methodology previously described (27). Thus, O-acylation
of 2,6,6-trimethyl-2H-1,2-oxazine-3,5(4H, 6H)-dione with 2-[4-(4-
trifluoromethylphenoxy)phenoxy]propionyl chloride or 2-[4-(5-trifluo-
romethyl-2-pyridyloxy)phenoxy]propionyl chloride followed by acetone
cyanohydrin-catalyzed rearrangement gave V and VI, respectively.
Compound VII. In an analogous manner, acylation of 5,5-dimethyl-
1,3-cyclohexanedione with 2-[4-(4-trifluoromethylphenoxy)phenoxy]-
propionyl chloride and subsequent rearrangement gave VII.
All of the preceding compounds were purified and were completely
characterized by proton nuclear magnetic resonance (NMR) and mass
spectrometry (MS).
Plant Material. The sources of the resistant and susceptible seed
used in this study have been described previously (12, 17, 18). Seeds
of all biotypes were germinated and grown in vermiculite in a growth
chamber at 20/15 °C (day/night) in a 16-h photoperiod at 75% relative
humidity. Young leaf tissue was collected from plants at the 2-3 leaf
stage, frozen in liquid nitrogen, and stored at -80 °C.
ACCase Extraction and Assay. ACCase was extracted as previously
described (23). Three grams of frozen tissue was homogenized in 30

5146 J. Agric. Food Chem., Vol. 52, No. 16, 2004
Shukla et al.
Figure 1. Chemical structures of experimental compounds used in the study. Compounds I−III are based on a CHD core structure; compounds IV−VII
are CHD−AOPP hybrids.
mL of extraction buffer [100 mM Tricine (pH 7.5), 20% (v/v) glycerol,
50 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM dithio-
threitol] and centrifuged at 27 000g for 15 min. The supernatant was
brought to 45% ammonium sulfate saturation and stirred for 30 min at
4 °C, followed by a 30 min centrifugation at 27 000g. The supernatant
was discarded, and the pellet was resuspended in 700 µL of elution
buffer [100 mM Tricine (pH 8.3), 10% (v/v) glycerol, 50 mM KCl, 1
mM dithiothreitol] and desalted on a Sephadex G-25 column, pre-
equilibrated with elution buffer.
ACCase was assayed as previously described (11). The enzyme
extract was incubated at 32 °C in assay buffer [20 mM Tricine-KOH
(pH 8.3), 10 mM KCl, 5 mM ATP, 2 mM MgCl2, 0.2 mg (w/v) BSA,
2.5 mM dithiothreitol, 3.7 mM NaHCO3 (including 0.185 MBq of
NaH14CO3)], along with appropriate concentrations of compounds
I-VII. Acetyl CoA (0.25 mM final concentration) was added to initiate
the reaction, and after 10 min, concentrated HCl was added to stop the
reaction. Aliquots (50 µL) of the assay solutions were transferred onto
2.2-cm filter paper disks and dried under an infrared lamp. The acid-
and heat-stable products were then quantified by liquid scintillation
spectroscopy. Three samples per biotype were assayed in triplicate for
each concentration of a compound. I50 values were calculated from the
linear equation using the two concentrations bracketing 50% inhibition.
respectively) (Table 2). Compounds III and V were the most
potent against the S3 goosegrass ACCase (I50 ) 4.3 and 4.6
µM, respectively), and compound IV the least inhibitory (I50
)
76 µM), with the I50 values of other compounds falling between
these extremes. Compound IV inhibited the susceptible maize
(S4) ACCase but was much less potent than two known ACCase
inhibitors, sethoxydim and fluazifop (Table 3).
Inhibition of Resistant ACCase by Experimental Com-
pounds. The activities of the same experimental compounds
were tested against ACCase from resistant biotypes of green
foxtail (two different biotypes), goosegrass, and maize. In almost
all cases, the I50 values obtained with the resistant ACCase were
higher than with the corresponding sensitive ACCase (Tables
2 and 3), indicating reduced binding of these compounds to the
resistant ACCases. In the case of ACCase from green foxtail
biotype R1, the I50 values in most cases were considerably higher
than those for the corresponding sensitive ACCase, S1 (see
Table 2, R1/S1 I50 ratios of 1.3-17). Only with compound IV
were the I50 values similar in both the resistant and susceptible
biotypes (R1/S1 I50 ratio ) 1.3). However, compound IV was
a weak inhibitor of the sensitive ACCase in general. Similar
results were obtained with ACCase from resistant green foxtail
biotype R2, with only compounds I and IV showing similar
inhibitory activity against ACCase from R2 and the correspond-
ing S biotype (R2/S1 I50 ratios ) 1.7 and 1.0, respectively;
Table 2). However, as indicated previously, the I50 values for
compounds I and IV against the sensitive ACCase were
relatively high.
RESULTS
The specific activities of ACCase extracted from the resistant
biotypes were not different from those of their respective
susceptible counterparts (green foxtail, 77-79 nmol CO2 fixed
min-1 mg-1 protein; goosegrass, 12-19 nmol C fixed min-1
g
f wt-1). This is in agreement with previous results (17, 21, 23).
Inhibition of Susceptible ACCase by Experimental Com-
pounds. Compounds I-VII inhibited ACCase from the sensi-
tive biotypes of green foxtail (S1) and goosegrass (S3) (Figures
2 and 3; Table 2). However, the extent of inhibition varied
among the compounds. Compound V was the most potent in-
hibitor of S1 ACCase, with an I50 of 0.9 µM, whereas com-
pounds I and IV were the least potent (I50 ) 54 and 51 µM,
Slightly different results were obtained when the test com-
pounds were assayed against ACCase from the resistant goose-
grass biotype (R3). In this case, the R/S I50 ratios were very
low (1.0-1.7) for compounds V, VI, and VII and slightly higher
(3.0) for compound IV (Table 2). Again, the I50 value for com-
pound IV against the S ACCase was relatively high (76 µM).

Resistant ACCase Mutants
J. Agric. Food Chem., Vol. 52, No. 16, 2004 5147
Figure 2. Inhibition of ACCase from susceptible (S1) and resistant (R1
and R2) biotypes of green foxtail. Vertical bars represent ± standard error
(SE).
Figure 3. Inhibition of ACCase from susceptible (S3) and resistant (R3)
biotypes of goosegrass. Vertical bars represent ±SE.
Table 2. I50 Values and R/S I50 Ratios for ACCase Herbicide-Sensitive
and -Resistant Biotypes of Green Foxtail (S1, R1, R2) and
Goosegrass (S3, R3), Assayed in Vitro with Experimental ACCase
Inhibitors
As expected, ACCase from the susceptible maize line (S4)
was sensitive to sethoxydim and fluazifop, whereas that
from the resistant maize line (R4) was resistant to inhibition
by these herbicides, especially to sethoxydim (Table 3). Al-
though compound IV was not a particularly potent inhibitor
of susceptible maize ACCase, it was a stronger inhibitor of
the resistant maize ACCase than either sethoxydim or fluazi-
fop (Table 3). This resulted in a much lower R/S I50 ratio
for compound IV than for either of these two known herbi-
cides.
green foxtail
I50 (µM)
R2
89
goosegrass
I50 (µM)
R3 R3/S3
I
50 (µM)
compound S1
R1
R1/S1
R2/S1 S3
1.7 38
4.6
13
I
II
54
2.5
3.5
51
>100
38
59
>1.9
15
407
5.7 58
4.3 29
11
10
6.6
12
45
54
III
IV
V
VI
VII
17
67
1.3
9.0
18
1.0 76
7.1 4.6
20
14
227
7.8
3.0
1.7
1.1
1.0
0.9
1.8
3.5
8.1
32
24
6.4
36
50
DISCUSSION
28
17
31
16
6.9
The ideal ACCase inhibitor from a study such as this would
have two essential characteristics: inhibition of a range of
“sensitive” ACCases from different grass species and equal
inhibition of “resistant” ACCases from weeds that have evolved
resistance to AOPP and CHD herbicides. In other words, the
ideal candidate would be a potent inhibitor of a wide range of
ACCase isoforms and not be readily desensitized by frequent
mutations that give rise to resistant forms of ACCase.
mercialized herbicides (compare the S1 and S3 I50 values in
Table 1 with those in Table 2). In only a few cases were the
I50 values of the experimental compounds in the low micromolar
range. Therefore, none of these compounds would satisfy the
primary criterion for ideal activity described above. However,
some did inhibit ACCase significantly at relatively low con-
centrations (e.g., compound V), indicating that they are at least
useful leads in new inhibitor development. This is not surprising,
In general, the experimental compounds tested were not
particularly potent ACCase inhibitors compared to some com-

5148 J. Agric. Food Chem., Vol. 52, No. 16, 2004
Shukla et al.
(32). The compounds thus produced, which included compound
VII, had 100× and 1-10× greater binding affinity with
ACCase than CHD and AOPP herbicides, respectively. How-
ever, the authors suggested that these compounds probably
would not overcome target-site-based herbicide resistance based
on an altered herbicide binding site.
Table 3. I50 Values and R/S I50 Ratios for ACCase from Wild-Type
(S4) and Sethoxydim-Resistant (R4) Biotypes of Maize, Assayed in
Vitro
I50 (µM)
inhibitor
S4
18
0.9
1.5
R4
40
>100
55
R4/S4
compound IV
sethoxydim
fluazifop
2.2
>111
37
Since resistant ACCases often remain relatively susceptible
to one or more AOPP or CHD herbicides (see Table 1 for
examples), it is possible that AOPP-CHD hybrid molecules
may be more potent inhibitors of resistant forms of ACCase.
The AOPP-CHD hybrid structures used in this study (com-
pounds V, VI, and VII) were relatively potent inhibitors of
the S1 and S3 ACCases (Table 2). In particular, compound
V demonstrated consistently high activity against the suscep-
tible ACCases S1 and S3 and was also a relatively strong
inhibitor of the three resistant ACCases against which it
was assayed (R1, R2, R3; Table 2). Compounds VI and VII
were equally potent inhibitors of the R and S ACCases
from goosegrass. In contrast, while they were active against
ACCase from the susceptible green foxtail biotype (S1), they
were less effective against the resistant green foxtail ACCases
(Table 2).
given that all the experimental compounds are closely related
to known classes of ACCase inhibitors.
The more important question in this study addressed the
second criterion, that is, would these compounds be equally
potent against resistant ACCase isoforms? This goal might be
more achievable if there was only one resistant form of ACCase.
However, at least four different patterns of resistance to ACCase
inhibitors have been observed in different weed biotypes in
which resistance is conferred by an altered form of ACCase
(28). These include high-level resistance to sethoxydim and low-
level resistance to other AOPP and CHD herbicides, high-level
resistance to fluazifop and low-level resistance to other AOPP
and CHD herbicides, relatively high-level resistance to all AOPP
and CHD herbicides, and resistance to AOPP but not to CHD
herbicides. Collectively, these results suggest that at least four
different forms of ACCase occur in different resistant weed
populations. Therefore, the new molecules sought must inhibit
a range of ACCase isoforms with different structural, steric, or
electrostatic properties in or around the herbicide binding
domain.
Only one compound, number IV, had somewhat similar
activity against the resistant and susceptible forms of ACCase
used in this study. Unfortunately, this compound had inherently
low activity even against the susceptible ACCase isoforms (I50
values of 18-76 µM; Tables 2 and 3), which would preclude
it from further development as a herbicide. However, it has value
as a new lead to develop a broad-based inhibitor for all isoforms
of ACCase. For this compound, meeting the second require-
ment for comparable activity against resistant and susceptible
ACCases is negated by its low inhibitory activity in general.
The results do suggest, however, that it may be possible to
identify new inhibitors that bind equally to resistant and
susceptible forms of the enzyme. The challenge is to identify
molecules that do so in the low micromolar range. Although it
is based on a CHD core and is homologous to the 3-aryltetramic
class of ACCase inhibitors (29, 30), compound IV lacks the
aliphatic substituents of more typical CHDs and 3-aryltetramic
acids. Further structural modifications to this molecule, bringing
it closer to other CHDs and 3-aryltetramic acids, may lead to a
more potent molecule that would inhibit the S and R ACCases
at the same low concentration.
Although compound V shares some structural similarities
with fluazifop, it was much more potent against the resis-
tant goosegrass ACCase than fluazifop (I50 values of 7.8
and >500 µM for compound V and fluazifop, respectively;
Tables 1 and 2). Presumably, the CHD moiety in the hybrid
structure contributes to the high potency of this compound.
Although this compound did not satisfy the criteria for high
overall activity, it is also a potential lead for further synthesis
and evaluation.
Based on amino acid sequence comparisons of plastidic
ACCase from herbicide-sensitive and -resistant plants, it has
become clear that an Ile to Leu change close to the highly
conserved motif of the carboxyltransferase domain of
ACCase plays an important role in the development of re-
sistance to ACCase inhibitor herbicides. Zhang and Devine
(33) reported such a mutation in the plastidic ACCase from
herbicide-resistant green foxtail, and Zagnitko et al. (25) re-
ported a similar mutation in maize and annual ryegrass. The
latter authors suggested that this mutation changes the interac-
tion of the herbicides with the enzyme without compromising
the enzyme activity. More recently, De´lye et al. (34) re-
ported that an Ile to Asn substitution was responsible for
resistance in blackgrass. We can speculate that this change,
and possibly other amino acid substitutions not yet identified,
may reduce binding of some inhibitors more than others,
depending on how the altered charge and steric parameters
change the affinity of the inhibitors for the binding niche on
the enzyme. However, without knowing the three-dimensional
structure of the binding niche, it is difficult to say more about
the effect of specific mutations on inhibitor interaction with the
enzyme.
Derivatives of CHD herbicides (based on modification of the
alkyl chains) have been used to study their effect on whole
plants, de novo fatty acid biosynthesis, and inhibition of
ACCase in order to better understand structure-activity rela-
tionships of CHD herbicides (31). Several new compounds were
2 orders of magnitude more effective than known CHD
herbicides in vitro; however, these compounds were not very
effective at the whole-plant level. Any new inhibitors developed
through a rational design approach must also meet other critical
requirements for biological activity, including adequate foliar
penetration, transport through the plant, and stability in the plant
tissue.
None of the experimental compounds examined in this
work was completely effective against the range of ACCase
enzymes tested. However, the activity of compound V sug-
gests it may be possible to develop potent ACCase inhibitors
that are effective against both susceptible and resistant forms
of the enzyme. The inclusion of herbicide-resistant target en-
zymes in screening protocols is one approach to identifying
new molecules with broad-based potency against isoforms of
target enzymes for which there are known differences in
susceptibility.
A novel class of cyclic triketones were synthesized by
attaching the cyclic portion of CHD oximes to AOPP herbicides

Resistant ACCase Mutants
J. Agric. Food Chem., Vol. 52, No. 16, 2004 5149
ABBREVIATIONS USED
(15) Preston, C.; Tardif, F. J.; Christopher, J. T.; Powles, S. B.
Multiple resistance to dissimilar herbicide chemistries in a
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ACKNOWLEDGMENT
We acknowledge chemical synthetic assistance from Weiming
Fan, Arthur Steiger, and Stefan Blarer and technical assistance
from Corinne Biesenthal and Susan Marles. We dedicate this
work to F. Dana Hess, whose enthusiasm and intellect inspired
us all.
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Received for review July 7, 2003. Revised manuscript received June 4,
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from the Natural Sciences and Engineering Research Council of Canada
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JF034738M