Structural Characterization and Computer-Aided Optimization of a Small-Molecule Inhibitor of the Arp2/3 Complex, a Key Regulator of the Actin Cytoskeleton

Article abstract of DOI:10.1002/cmdc.201200104

CK-666 (1) is a recently discovered small-molecule inhibitor of the actin-related protein 2/3 (Arp2/3) complex, a key actin cytoskeleton regulator with roles in bacterial pathogenesis and cancer cell motility. Although 1 is commercially available, the crystal structure of Arp2/3 complex with 1 bound has not been reported, making its mechanism of action uncertain. Furthermore, its relatively low potency increases its potential for off-target effects invivo, complicating interpretation of its influence in cell biological studies and precluding its clinical use. Herein we report the crystal structure of 1 bound to Arp2/3 complex, which reveals that 1 binds between the Arp2 and Arp3 subunits to stabilize the inactive conformation of the complex. Based on the crystal structure, we used computational docking and free-energy perturbation calculations of monosubstituted derivatives of 1 to guide optimization efforts. Biochemical assays of ten newly synthesized compounds led to the identification of compound 2, which exhibits a threefold increase in inhibitory activity invitro relative to 1. In addition, our computational analyses unveiled a surface groove at the interface of the Arp2 and Arp3 subunits that can be exploited for additional structure-based optimization.

Full text of DOI:10.1002/cmdc.201200104

MED
DOI: 10.1002/cmdc.201200104
Structural Characterization and Computer-Aided
Optimization of a Small-Molecule Inhibitor of the Arp2/3
Complex, a Key Regulator of the Actin Cytoskeleton
Andrew W. Baggett,[a] Zoe Cournia,[b] Min Suk Han,[a] George Patargias,[b] Adam C. Glass,[a]
Shih-Yuan Liu,[a] and Brad J. Nolen*[a]
CK-666 (1) is a recently discovered small-molecule inhibitor of
the actin-related protein 2/3 (Arp2/3) complex, a key actin cy-
toskeleton regulator with roles in bacterial pathogenesis and
cancer cell motility. Although 1 is commercially available, the
crystal structure of Arp2/3 complex with 1 bound has not
been reported, making its mechanism of action uncertain. Fur-
thermore, its relatively low potency increases its potential for
off-target effects in vivo, complicating interpretation of its in-
fluence in cell biological studies and precluding its clinical use.
Herein we report the crystal structure of 1 bound to Arp2/3
complex, which reveals that 1 binds between the Arp2 and
Arp3 subunits to stabilize the inactive conformation of the
complex. Based on the crystal structure, we used computation-
al docking and free-energy perturbation calculations of mono-
substituted derivatives of 1 to guide optimization efforts. Bio-
chemical assays of ten newly synthesized compounds led to
the identification of compound 2, which exhibits a threefold
increase in inhibitory activity in vitro relative to 1. In addition,
our computational analyses unveiled a surface groove at the
interface of the Arp2 and Arp3 subunits that can be exploited
for additional structure-based optimization.
Introduction
Many cellular processes such as motility, endocytosis, and divi-
sion require the assembly of actin filaments from a pool of
globular, monomeric actin.[1] Formation of the actin polymer
depends on a slow nucleation step,[2] and cells use a handful
of actin nucleating factors that are responsive to cellular sig-
naling pathways to provide precise spatiotemporal regulation
of the assembly of actin filaments.[3] Actin-related protein 2/3
(Arp2/3) complex is a seven-subunit ~225 kDa ATPase that is
a key actin nucleator.[4] It functions by attaching itself to the
side of a preexisting actin filament and nucleating a new
(daughter) filament to create a Y-shaped branch. While it is al-
ready known that Arp2/3 complex is required for many cellular
processes, knockouts of Arp2/3 complex subunits in multicellu-
lar organisms are lethal, so roles of the complex late in devel-
opment or in adult stages remain largely undiscovered.[5] RNA
interference experiments could circumvent this problem, but
are relatively slow acting, irreversible, and cannot be used in
some biological contexts. Therefore, fast-acting, simple, and re-
versible small-molecule inhibitors have the potential to
become powerful tools to study Arp2/3 complex in vivo. Such
inhibitors may also have clinical value, as many bacterial and
viral pathogens usurp host cell Arp2/3 complex for invasion or
intracellular motility.[6] In addition, tumor cell migration is
thought to require Arp2/3 complex, and Arp2/3 overexpression
contributes to pathogenesis, growth, and invasion of carcino-
mas.[7] Therefore, Arp2/3 complex inhibitors also hold promise
as antitumor agents.[8]
indole attached at the 3-position by a short amide linker to an
aromatic ring, and both inhibit Arp2/3 complex in actin poly-
merization assays in vitro. Importantly, these compounds are
cell-permeable and inhibit Listeria actin comet tail formation in
infected SKOV3 cells and podosome formation in THP-1-de-
rived monocyte cells, processes previously shown to require
Arp2/3 complex.[6,9,10] An X-ray crystal structure of 12 bound to
inactive Arp2/3 complex showed that it binds at the interface
between the Arp2 and Arp3 subunits to block a conformational
rearrangement of these subunits required for activation.[9]
Therefore, 12 represents a novel example of an allosteric inter-
facial inhibitor, a molecule which binds at subunit interfaces of
a macromolecular assembly to block conformational dynamics
critical for function.[11] Compound 1, the more potent of the
two compounds, is commercially available and has already
been used in a number of in vivo studies of Arp2/3 complex
function.[12] However, the half-maximal inhibitory concentration
(IC50 value) for 1 is in the low micromolar range, and undesira-
bly high concentrations are necessary for complete inhibition
of the complex, increasing the probability of off-target effects.
[a] A. W. Baggett,+ M. S. Han, Dr. A. C. Glass, Prof. S.-Y. Liu, Prof. B. J. Nolen
Department of Chemistry, University of Oregon
Eugene, OR 97403-1253 (USA)
E-mail: bnolen@uoregon.edu
[b] Dr. Z. Cournia,+ Dr. G. Patargias
Biomedical Research Foundation, Academy of Athens
4 Soranou Ephessiou, 11527 Athens (Greece)
[+] These authors contributed equally to this work.
The discovery of two related small-molecule inhibitors of
Arp2/3 complex, CK-636 (12) and 1, was recently reported
(Figure 1).[9] Both compounds include a 2-methyl-substituted
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200104.
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An Arp2/3 Complex Inhibitor
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Figure 1. a) Previously synthesized Arp2/3 complex inhibitors 1 and 12. Compound 1 was used as a parent scaffold for substitutions indicated in Scheme 1.
b) Crystal structure of 1 bound to Bos taurus Arp2/3 complex; 1 is shown as spheres and binds at the interface of the Arp3 (orange) and Arp2 (red) subunits.
The five other subunits in the complex are labeled ARPC1–5. c) Comparison of 1 (grey carbon atoms) with 12 (turquoise carbon atoms) showing a close-up of
the binding pocket at the interface of Arp2 and Arp3. Coordinates for 12 for this image were generated by overlaying Arp2 and Arp3 from 3DXK.pdb onto
the structure of 1 and applying the transformation to 12.
The potential of Arp2/3 complex inhibitors to become power-
ful basic research tools and the possibility of their clinical value
led us to optimize parent compound 1. We first solved the
crystal structure of 1 bound to Arp2/3 complex and computa-
tionally docked analogues of 1 to predict the effect of various
substitutions on binding. We then synthesized and tested the
analogues in an actin polymerization assay to determine their
effect on Arp2/3 complex-mediated nucleation. As docking re-
sults did not correlate with experimental findings, we subse-
quently turned to the more sophisticated free-energy pertur-
bation calculations for binding affinity predictions, which corre-
lated well with experimental findings. In addition to yielding
an inhibitor with threefold improved inhibition, this work pro-
vides significant insight into further structure-based optimiza-
tion of Arp2/3 complex inhibitors. Specifically, we identify
atoms within the scaffold of 1 that cannot tolerate substitu-
tions and reveal a position (R4) on 1, which is a promising site
for future optimization efforts, because it provides opportuni-
ties to exploit a groove at the interface of Arp2 and Arp3. Fi-
nally, the work presented herein provides new insight into the
use of computational docking and free-energy perturbation
calculations to aid drug design.
rine atom pointing in the opposite direction, toward the b7/
aC loop of Arp3. Refinement of this conformation followed by
Fo–Fc difference electron density map generation showed the
fluorine in 4.8 s negative density, indicating that our original
model is correct. The amide oxygen atom of 1 hydrogen
bonds with Ala203 of Arp2, and the indole nitrogen forms a hy-
drogen bond with Asp248, a conserved residue in Arp2. Both
of these polar contacts are also present in the crystal structure
of Arp2/3 complex with 12 bound. Compound 1 buries 16 ꢁ2
more solvent-exposed surface area at the interface than 12,
which may explain the decreased IC50 value of 1 relative to 12
in inhibiting Arp2/3 complex in in vitro actin polymerization
assays.[9] Binding of 1 causes minor conformational changes
similar to those caused by 12, including rotation of Arg250Arp2
out of the binding pocket and a subtle movement of the loop
connecting aE and aF in Arp2 toward the binding pocket. Be-
cause both 12 and 1 bind at an interface between Arp2 and
Arp3 present only in the inactive conformation, both inhibitors
probably function by blocking the 25 ꢁ movement of the Arp2
subunit thought to be required for activation of the com-
plex.[13]
Calculation of Glide docking scores and physicochemical
properties
Results
Glide docking and scoring has been previously validated in vir-
tual screening exercises;[14] however, we considered it necessa-
ry to test its performance with the Arp2/3 complex. We calcu-
lated Glide binding poses for inhibitors 12 and 1 and com-
pared them with the respective inhibitor-bound Arp2/3 crystal
structures. The RMSD values between non-hydrogen atoms of
the Glide-calculated pose and the crystal structures of 12 or
1 bound to Arp2/3 are 0.31 and 0.42 ꢁ, respectively, indicating
close agreement between docking and the crystal structure
(Supporting Information figure 2). The docked poses of 12 or
1 engage in intermolecular interactions identical to those ob-
served in the crystal structures, including hydrogen bonds be-
X-ray crystal structure of 1 bound to Arp2/3 complex
Crystals of the apoenzyme form of bovine Arp2/3 complex
soaked in 500 mm compound 1 diffracted to a resolution of
2.48 ꢁ (Supporting Information table 1). Compound 1 binds to
the Arp2–Arp3 interface in a manner identical to that of 12,
except for the fluorobenzene moiety in 1, which replaces the
thiophene ring in 12 (Figure 1). The asymmetric density distri-
bution of the fluorobezene ring indicates that the fluorine
atom is directed toward b12 and b13 in subdomain 4 of Arp2
(Supporting Information figure 1). To verify the correct assign-
ment of the ring flip, we also modeled the ring with the fluo-
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B. J. Nolen et al.
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tween the indole amine hydro-
gen and the side chain of
Asp248Arp2
,
and between the
amide oxygen in the linker and
the backbone NH of Ala203Arp2
.
One difference is that the fluo-
rine atom of 1 is flipped in the
docked pose with respect to its
position in the crystal structure.
However, the calculations sug-
gest the two poses are nearly
isoenergetic, and we observed
the fluorobenzene ring flip in
a number of the Glide-docked
analogues.
Half of the analogues were
predicted to bind better than
1 based on their docking scores
(Table 1). Hydroxy and methyl
substitutions at position R1 and
Scheme 1. Reagents and conditions: a) POCl3, DMF; b) CH3NO2, NH4OAc; c) 1. NaBH(OCH3)3, THF; 2. Pd/C, NH4CO2H,
CH3OH; d) NEt3, THF; e) BBr3, CH2Cl2.
Chemistry
Table 1. Biochemical and computational data of compounds tested.
Inhibitor candidates predicted to have similar, better, or
worse activity than 1 were synthesized to provide a body
of data to guide analysis of structure–activity relationships
and refinement of the computational studies (Scheme 1).
Table 1 shows the structures of all synthesized inhibitor
candidates along with their experimentally determined
IC50 values.
[b]
[c]
Compd R1 R2 R3 R4 IC50 [mm][a] GScore [kcalmolꢀ1
]
DDGb [kcalmolꢀ1
]
1
2
H
H
F
F
H
H
H
H
H
H
H
Cl
H
H
Cl
H
H
OH
H
12ꢂ1
4ꢂ1
ꢀ7.10
ꢀ8.40
ꢀ6.85
ꢀ7.00
ꢀ7.97
ꢀ7.04
ꢀ7.92
ꢀ6.95
ꢀ8.46
ꢀ7.66
ꢀ7.66
n.a.
ꢀ0.55ꢂ0.15
3.6ꢂ0.16
3
4
5
6
7
8
9
10
11
15
Cl
H
OH
H
H
H
OCH3
H
H
H
F
F
F
F
H
F
F
Cl
H
H
18ꢂ5
OCH3 19ꢂ6
ꢀ1.25ꢂ0.4
ꢀ1.3ꢂ0.27
0.12ꢂ0.12
ꢀ0.65ꢂ0.13
1.9ꢂ0.14
H
Cl
Cl
H
H
H
H
H
32ꢂ4
32ꢂ18
181ꢂ85
238ꢂ91
n.i.
n.i.
n.i.
n.a.
3.3ꢂ0.5
Biochemistry
3.9ꢂ0.15
2.5ꢂ0.17
To measure the potency of the analogues, we used a stan-
dard assay to measure the rate of actin polymerization. In
this assay, the increase in fluorescence of pyrene-labeled
actin subunits upon polymerization is used to monitor
the time course of filament formation.[17] Arp2/3 complex,
together with its activator protein, N-WASp-VCA, increases
the rate of polymer formation by nucleating branched fil-
aments and increasing the number of elongating filament
ends (Figure 2). Therefore, this assay can be used to deter-
mine the nucleation activity of Arp2/3 complex. We used Bos
taurus Arp2/3 complex in our assays because it can be isolated
in large quantities. Residues in the binding cleft of 1 are con-
served from yeast to humans; thus, optimization of the inhibi-
tors against the bovine complex will very likely improve IC50
values for inhibition of the complex from any species, includ-
ing Homo sapiens.[9] Figure 2a shows that 1 decreased Arp2/3
complex nucleation activity in a concentration-dependent
manner, with the addition of 200 mm inhibitor decreasing the
maximum polymerization to a rate similar to that observed in
the absence of Arp2/3 complex. The IC50 value of 1 calculated
under these conditions is 12 mm, in agreement with previously
reported values.[9] Of the ten derivative compounds, three
showed no inhibition toward Arp2/3 complex. The IC50 values
of the other compounds are compared with that of 1 in
ꢀ0.53ꢂ0.09
[a] IC50 values were determined from the actin polymerization assay. [b] Docking
scores were calculated with the Glide 5.7 XP scoring function; n.i.=no inhibi-
tion, n.a.=not applicable. [c] Differences in free energies of binding (DDGb)
were calculated from the free-energy perturbation calculations.
chloro or hydroxy groups at R4 yielded the compounds with
the lowest scores, suggesting the R1 and R4 positions are
promising optimization sites. Compound 2 had the best dock-
ing score, with a GScore 1.3 kcalmolꢀ1 lower than that of 1.
Calculation of the physicochemical properties for 2–11 showed
that like 1, each of the analogues have predicted octanol/
water partition coefficients, aqueous solubility, and Caco-2 cell
permeability within the optimal range for marketed drug com-
pounds (Supporting Information table 2).[15] In addition, all
compounds comply with the Lipinski rule of five and the Jor-
gensen rule of three, with the exception of 3, which has
a slightly lower predicted solubility (logS=ꢀ6.0) than the solu-
bility limit of the Jorgensen rule of three (logSꢁꢀ5.7).[16]
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An Arp2/3 Complex Inhibitor
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Figure 2. Biochemical characterization of 1 and analogues. a) Time course of the polymerization of 30% pyrene-labeled actin in the presence of 5 nm Arp2/3
complex, 200 nm N-WASp-VCA, and either DMSO for a range of concentrations of 1. A control reaction (actin alone) lacking only Arp2/3 complex shows the
intrinsic nucleation rate of actin. b) Comparison of actin polymerization time courses in the presence of equal concentrations of 1 versus 2; conditions are
identical to those described for panel a). c) Plot of maximal polymerization rate versus inhibitor concentration for 1, 2, and 5. Data were fit as described in
the Experimental Section.
Table 1: 7 and 8 showed a 15–20-fold increase in IC50, 5 and 6
showed approximately threefold increased IC50 values, and
compounds 3 and 4 had IC50 values nearly double that of 1.
Compound 2, derivatized from 1 by replacement of a hydrogen
atom at R4 with an OH group, showed a threefold improved
IC50 value (Figure 2b,c).
table 2). Compound 2 has a hydroxy substitution at R4, a posi-
tion which allows functional groups to project into an extend-
ed groove formed at the interface of Arp2 and Arp3. Figure 3a
illustrates that in the Glide-docked structure of 2, the fluoro-
benzene ring flips and moves toward the Arp2 subunit, allow-
ing the hydroxy group at R4 to form a hydrogen bond with the
carboxyl oxygen of Ile251 of Arp2. The movement of the ring
requires a 338 deviation from planarity for the amide bond in
the linker region of the inhibitor. In the FEP-calculated struc-
ture the amide is within 5.48 of planarity, and the R4 hydroxy
group is close to the Thr119Arp3 side chain, but a hydrogen
bond with the backbone amide of Asn122Arp3 locks the threo-
nine hydroxy in a rotamer incapable of bonding to the R4 hy-
droxy group. Therefore, in the FEP calculations, R4 hydroxy hy-
drogen bonds only to solvent, explaining the modest decrease
in calculated FEP binding energy relative to 1, which agrees
more closely with the biochemical data than the Glide docking
calculation. We note that examination of the crystal structure
and all computational models reveal that binding requires de-
solvation of the nitrogen in the amide linker without a compen-
sating hydrogen bond to the protein, suggesting that increas-
ing the hydrophobicity of the linker may increase the net bind-
ing energy.
Free-energy perturbation calculations
The Glide XP scores showed poor correlation with the assay
data for analogues 1–14 (Table 1), indicating an inability to
predict binding affinities for a series of close analogues in this
system. Therefore, we initiated free-energy perturbation (FEP)
calculations, a robust methodology for evaluating differences
in free energy of binding of different ligands. The differences
in free energy of binding affinity (DDGb) presented in Table 1
correlate well with experimental results. In particular, all inac-
tive analogues of 1 were correctly identified by FEP calcula-
tions, which yielded large positive differences in free energy of
binding with respect to 1. Moreover, analogue 2 was identified
as a better binder in accordance with a threefold improvement
in the IC50 value. For three compounds (4, 5, and 7), the FEP
calculations did not correctly predict the experimental results;
a detailed discussion follows in the next section. Results in
Table 1 are shown as DDG(CK-666!analogue), for which negative
values indicate more favorable predicted binding.
In compound 4, a methoxy group replaces hydrogen at po-
sition R4. The Glide-docked and FEP-calculated poses show the
methyl group making favorable van der Waals contacts with
either the methyl group of Thr119Arp3 or the aliphatic portion
of Arg250Arp2, respectively (Supporting Information figure 3).
Accordingly, compound 4 showed a lower DDG value than 2
in the FEP calculations, despite the fact that it has a fivefold
higher IC50 value than 2. The FEP-generated pose shows the
methoxy oxygen wedged in an aliphatic cleft lined by the
methyl group of Thr119Arp3 and the hydrophobic part of
Arg250Arp2, where it is partially shielded from solvent (Support-
ing Information figure 3). It hydrogen bonds to a single solvent
molecule and leaves a lone pair of electrons unsatisfied. This
indicates a desolvation penalty that may not be fully account-
Discussion
Herein we present the crystal structure of 1 bound to Arp2/3
complex and show that it binds to the same pocket at the in-
terface of Arp3 and Arp2 as 12, a previously described inhibi-
tor, indicating that 1 and 12 use the same mechanism of inhib-
ition.[9] Ten analogues of 1 were designed and synthesized,
leading to the discovery of 2, a compound with threefold
better inhibition of Arp2/3 complex than 1 and a promising
physicochemical profile (Table 1 and Supporting Information
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chains Asp209 and Thr212 of
Arp2 by Glide docking and FEP
calculations, but these interac-
tions require the indole ring to
distort slightly, which may out-
weigh energetic benefits from
the predicted hydrogen bonding
(Figure 3b). Replacing the R1 hy-
droxy group of 1 with a methoxy
group gave compound 9, which
also binds with
a distorted
indole ring and does not hydro-
gen bond to the side chain of
Asp209Arp2 in the FEP pose (Fig-
ure 3b). While the oxygen atom
of the methoxy substituent
maintains one hydrogen bond
with Thr212Arp2
,
the methyl
group excludes water around
the R1 site, leaving the oxygen
with one unsatisfied lone-pair
hydrogen bond acceptor. The
partial desolvation penalty of
the methoxy and strain of the
indole ring likely contribute to
the severe decrease in binding
affinity for 9, which is also pre-
dicted by the FEP calculations.
Placing a chlorine at position
R1 to give 3 increased the IC50
value only slightly relative to 1,
despite the fact that the FEP cal-
Figure 3. Binding pocket of 1 with overlapping docked analogues 2, 5, and 9. a) Stereo image showing a compari-
son of 1 and the docked and FEP poses of 2. Arp3 (blue carbon atoms) and Arp2 (green carbon atoms) from the
crystal structure of Arp2/3 with bound 1 (3UKR.pdb, light-blue carbon atoms) are shown in line representation
overlaid with the Glide-docked pose (teal carbon atoms) and the FEP-docked pose (pink) of 2. Selected hydrogen
bonds are shown as black dotted lines. b) Stereo image showing a comparison of 1 and the FEP-generated poses
of 5 and 9. Colors are identical to those of panel a), except 5 is shown with orange carbon atoms, and 9 is shown
with purple carbon atoms.
ed for by the FEP calculations. Replacing the methoxy oxygen
with a carbon atom would decrease the desolvation penalty,
and is a promising future optimization strategy.
culation predicted this substitution would be highly unfavora-
ble (DDG= +3.6 kcalmolꢀ1). This discrepancy may be due to
the inability of the force field to account for the halogen bond
that appears to form between the chlorine and the neighbor-
ing carbonyl group of Asp209Arp2 and the side chain of
Thr212Arp2. Oxygen atoms from both of these side chains are
equidistant from the chlorine atom at 3.1 ꢁ, which is close to
the experimentally observed value for a halogen bond.[18]
Derivatives 7, 10, and 11, which feature monochloro-substi-
tuted benzene rings at the R2, R3, and R4 positions, respectively,
all showed greatly decreased or no inhibition. Substitution of
chlorine at the para position (7) causes the benzene ring to tilt
by 30.58 and move away from the side chain of Thr119Arp3 to
relieve steric clash (Supporting Information figure 5). The FEP
calculation predicted this substitution to have little or no influ-
ence on binding. The importance of the fluorine at the ortho
position is evident in 10, as its replacement with chlorine com-
pletely abolishes inhibition. The FEP calculation correctly pre-
dicted the poor binding of this compound, and the FEP-calcu-
lated structures show that the bulky chlorine substituent
causes a slight repositioning of the benzene ring and the
amide linker (Supporting Information figure 5), that may de-
crease shape complementarity. Further evidence for the impor-
tance of the fluorine atom at the R2 position is provided by
compounds 6 and 8, which have unfavorable substitutions at
Compound 6 has a chlorine atom in position R4 and exhibit-
ed an approximate threefold increase in IC50 value relative to
1. Steric clash between the chlorine and the Thr119Arp3 methyl
group repositions the fluorobenzene ring in both the Glide-
docked and FEP-generated poses, although the direction of
movement is the opposite in the two computations (Support-
ing Information figure 4). In the FEP pose, the side chain of
Arg250 moves to accommodate the chlorine, whereas the side
chains are fixed in the Glide docking, explaining the difference
in the conformations favored for each method. These data sug-
gest that the fluorobenzene ring can be repositioned within
the pocket without a major decrease in affinity, indicating that
the protein functional groups that interact with the R4 sub-
stituent may vary depending on the bulkiness of the R4 sub-
stituent. Taken together, the results for compounds 2, 4, and 6
strongly suggest that position R4 in the fluorobenzene ring is
a promising site for future optimization.
Derivatives 9 and 5 feature substitutions at the R1 position
and were predicted by docking to bind better than 1. Com-
pound 5, with a hydroxy group at R1, exhibited an IC50 value
almost threefold higher than that of 1 (Table 1). The hydroxy
group at R1 was predicted to hydrogen bond to the side
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1
3H); 13C NMR (150 MHz, (CD3)2CO): d=164.02, 161.18 (d, JCF
=
the para and meta positions, such as 7 or 11, but due to the
ortho-fluorine atom, bind better than the monochloro com-
pounds.
246.0), 135.33, 135.21, 133.68 (d, JCF =9.2 Hz), 132.14, 131.09,
125.48 (d, JCF =3.5 Hz), 124.84, 123.98 (d, 2JCF =13.2 Hz), 121.17,
118.01, 116.90 (d, 2JCF =23.6 Hz), 112.55, 109.18, 41.37, 24.92,
11.58 ppm; FTIR (thin film): n˜ =3278, 2929, 1648, 1614, 1531,
1026 cmꢀ1; HRMS (EI) calcd for C18H16N2OFCl [M]+ 330.09352,
330.09289 found.
Our analysis suggests that the weak correlation between the
binding affinities predicted from docking calculations and the
measured inhibition constants is at least partly due to the fact
that protein and water molecules are kept rigid in the docking
process. FEP calculations allow for a dynamic evolution of the
system, which, in several instances, resulted in changes in the
positions of side chains or water molecules to accommodate
the structures of the bound analogues. This induced-fit effect
appears to be a critical contributor to the DDG of binding. The
correlation between the FEP calculations and the measured in-
hibition constants demonstrate the predictive value of the FEP
calculations in this system and will allow us to streamline the
development of improved inhibitors of Arp2/3 complex for
basic research and potential clinical applications.
2-Fluoro-4-methoxy-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benza-
mide (4): 4 (0.790 g, 84.4%) was synthesized as a pink oil from 2-
(2-methyl-1H-indol-3-yl)ethanamine (0.500 g, 2.87 mmol) using the
general procedure described above. 1H NMR (300 MHz, (CD3)2CO):
d=9.85 (brs, 1H), 7.91 (t, 3JHH =9 Hz, 1H), 7.56 (dd, 3JHH =8.7,
1.8 Hz, 1H), 7.35 (brs, 1H), 7.26 (dd, 3JHH =8.1, 1.8 Hz, 1H), 6.98
(quin.d, JHH =7.5, 1.5 Hz, 2H), 6.85 (dd, JHH =8.7, 2.4 Hz, 1H), 6.75
(dd, JHH =13.8, 2.4 Hz, 1H), 3.87 (s, 3H), 3.62 (qd, JHH =7.2, 0.9 Hz,
2H), 3.02 (t, JHH =7.2 Hz, 2H), 2.40 ppm (s, 3H); 13C NMR (150 MHz,
3
3
3
3
3
(CD3)2CO): d=163.27 (d,
JCF =12.2 Hz), 162.59 (d, JCF =3.5 Hz),
161.39 (d, 1JCF =246.0 Hz), 135.86, 132.46 (d, JCF =4.5 Hz), 132.11,
128.83, 120.28, 118.42, 117.59, 114.70 (d, 2JCF =13.2 Hz), 110.57,
110.26, 108.09, 101.24 (d, 2JCF =27.6 Hz), 55.40, 40.38, 24.16,
10.54 ppm; FTIR (thin film): n˜ =3402, 3294, 2920, 1620, 1499, 1270,
952, 743 cmꢀ1; HRMS (EI) calcd for C19H19N2O2F [M]+ 326.14306,
326.14214 found.
Experimental Section
Chemistry: The synthesis in Scheme 1 proceeds through standard
protocols to produce inhibitor candidates.[19] Commercially avail-
able indoles are formylated under Vilsmeier conditions to produce
aldehydes, which undergo the Henry reaction followed by conden-
sation to yield nitroolefins. Reduction with NaBH(OCH3)3 followed
by basic aqueous workup and subsequent reduction with 10 wt%
Pd/C in the presence of NH4CO2H in CH3OH yields amines. Acid
chloride coupling completes the synthesis of 1, 3–4, and 6–11,
with subsequent methoxy deprotection using BBr3 required for 2
and 5.[20] Compounds 1, 3–4, and 7–11 were all >98% pure as de-
termined by HPLC analysis. Compound 2 was determined to be
>97% pure, compound 5 was determined to be >95% pure, and
compound 6 was determined to be >96% pure by HPLC analysis.
4-Chloro-2-fluoro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide
(6): 6 (0.103 g, 7.2%) was synthesized as a white solid from 2-(2-
methyl-1H-indol-3-yl)ethanamine (0.758 g, 4.35 mmol) using the
general procedure described above. 1H NMR (300 MHz, (CD3)2CO):
3
d=9.85 (brs, 1H), 7.88 (t, JHH =7.8 Hz, 1H), 7.60 (brs, 1H), 7.55 (d,
3JHH =7.8 Hz, 1H), 7.33 (m, 2H), 7.26 (d, JHH =7.8 Hz), 6.98 (quin.d,
3
3
3JHH =7.2, 1.5 Hz, 2H), 3.63 (q, 3JHH =7.2 Hz, 2H), 3.03 (t, JHH
=
7.2 Hz, 2H), 2.40 ppm (s, 3H); 13C NMR (150 MHz, (CD3)2CO): d=
162.11, 159.97 (d, 1JCF =246.0 Hz), 136.90 (d, JCF =11 Hz), 135.85,
132.38 (d, JCF =4.0 Hz), 122.10 (d, 2JCF =13.8 Hz) 132.16, 128.82,
2
124.90 (d, JCF =3.5 Hz), 120.30, 118.44, 117.54, 116.52 (d, JCF
=
27.6 Hz), 110.28, 107.93, 40.53, 24.03, 10.54 ppm; FTIR (thin film):
n˜ =3397, 3312, 2928, 1649, 1530, 1076, 901, 742 cmꢀ1; HRMS (EI)
calcd for C18H16N2OFCl [M]+ 330.09352, 330.09261 found.
General procedure for 1, 3, 4, 6, 7, 8, 9, 10, and 11: Indole
amines prepared according to published methods were stirred in
THF (1 mL per 25 mg compound) with NEt3 (1.1 equiv) and acid
chloride (1.1 equiv) at room temperature for 30 min. At the conclu-
sion of the reaction, H2O was added, and the resulting solution
was partitioned with Et2O. The organic phase was washed with sa-
turated aqueous NaCl, dried with MgSO4, and the solvent was re-
moved under reduced pressure to yield crude solid product. Purifi-
cation on a silica gel column using 2:1 Et2O/hexanes as the eluent
followed by removal of solvent under reduced pressure yielded
compounds in decent yields.
4-Chloro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide (7):
7
(0.049 g, 54.4%) was synthesized as a white solid from 2-(2-methyl-
1H-indol-3-yl)ethanamine (0.050 g, 0.29 mmol) using the general
1
procedure described above. H NMR (300 MHz, (CD3)2CO): d=9.88
3
(brs, 1H), 7.99 (brs, 1H), 7.90 (m, 2H), 7.50 (m, 3H), 7.27 (d, JHH
=
=
3
7.2 Hz, 1H), 6.98 (quin.d, 3JHH =7.2, 1.2 Hz, 2H), 3.60 (q, JHH
3
6.6 Hz, 2H), 3.01 (t, JHH =6.6 Hz, 2H), 2.35 ppm (s, 3H).
3-Chloro-2-fluoro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide
(8): 8 (0.131 g, 39.6%) was synthesized as a white solid from 2-(2-
methyl-1H-indol-3-yl)ethanamine (0.175 g, 1.00 mmol) using the
general procedure described above. 1H NMR (300 MHz, (CD3)2CO):
2-Fluoro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide
(1):
1 (0.050 g, 42.7%) was synthesized as a white solid from 2-(2-
methyl-1H-indol-3-yl)ethanamine (0.069 g, 0.40 mmol) using the
general procedure described above. 1H NMR (300 MHz, (CD3)2CO):
3
d=9.85 (brs, 1H), 7.65 (m, 3H), 7.55 (d, JHH =7.8 Hz, 1H), 7.28 (m,
3
3
3
2H), 6.98 (quin.d, JHH =7.8 Hz, 2H), 3.63 (q, JHH =7.2 Hz, 2H), 3.04
(t, 3JHH =7.2 Hz, 2H), 2.40 ppm (s, 3H); 13C NMR (150 MHz,
(CD3)2CO): d=162.33, 155.26 (d, 1JCF =246.0 Hz), 135.85, 132.57,
132,16, 129.42 (d, JCF =2.3 Hz), 128.83, 125.46 (d, 2JCF =14.3 Hz),
125.13 (d, JCF =4.5 Hz), 120.45 (d, 2JCF =15.8 Hz), 120.29, 118.43,
117.53, 110.27, 107.92, 40.56, 24.05, 10.55 ppm; FTIR (thin film): n˜ =
3398, 3292, 1649, 1529, 1451, 1300, 1240, 745 cmꢀ1; HRMS (EI)
calcd for C18H16N2OFCl [M]+ 330.09352, 330.09398 found.
d=9.91 (brs, 1H), 7.93 (td, JHH =7.8, 1.8 Hz, 1H), 7.64 (s, 1H), 7.59
3
3
(d, JHH =6.6 Hz, 1H), 7.54 (m, 1H), 7.30 (m, 2H), 7.21 (ddd, JHH
=
11.4, 8.1, 1.2 Hz, 1H), 7.02 (quin.d, 3JHH =6.9, 1.5 Hz, 2H), 3.68 (q,
3JHH =7.2 Hz, 2H), 3.07 (t, JHH =7.2 Hz, 2H), 2.42 ppm (s, 3H).
3
N-(2-(5-Chloro-2-methyl-1H-indol-3-yl)ethyl)-2-fluorobenzamide
(3): 3 (0.040 g, 31.7%) was synthesized as a white solid from 2-(5-
chloro-2-methyl-1H-indol-3-yl)ethanamine (0.080 g, 0.38 mmol)
using the general procedure described above. 1H NMR (300 MHz,
3
(CD3)2CO): d=10.09 (brs, 1H), 7.92 (td, JHH =7.8, 1.8 Hz, 1H), 7.61
2-Fluoro-N-(2-(6-methoxy-2-methyl-1H-indol-3-yl)ethyl)benza-
mide (9): 9 (0.103 g, 42.9%) was synthesized as a clear oil from 2-
(brs, 1H), 7.59 (d, 3JHH =2.1 Hz, 1H), 7.55 (m, 1H), 7.30 (m, 2H), 7.22
(ddd, 3JHH =11.4, 8.4, 0.9 Hz, 1H), 7.01 (dd, 3JHH =8.4, 2.4 Hz, 1H),
3.64 (q, 3JHH =7.2 Hz, 2H), 3.04 (t, 3JHH =7.2 Hz, 2H), 2.43 ppm (s,
(5-methoxy-2-methyl-1H-indol-3-yl)ethanamine
0.73 mmol) using the general procedure described above. H NMR
(0.150 g,
1
ChemMedChem 2012, 7, 1286 – 1294
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B. J. Nolen et al.
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3
(300 MHz, (CD3)2CO): d=9.70 (brs, 1H), 7.90 (td, JHH =7.5, 1.8 Hz,
161.42 (d, 2JCF =12.8 Hz), 135.86, 132.70 (d, JCF =4.7 Hz), 132.10,
3
3
1H), 7.53 (m, 3H), 7.28 (td, JHH =7.5, 0.9 Hz, 1H), 7.19 (ddd, JHH
=
=
128.82, 120.28, 118.41, 117.59, 113.72 (d, 2JCF =13.2 Hz), 111.84 (d,
11.7, 8.1, 0.9 Hz, 1H), 7.15 (d, 3JHH =8.4 Hz, 1H), 7.09 (d, JHH
J
CF =2.3 Hz), 110.25, 108.10, 102.56 (d, JCF =26.4 Hz), 40.35, 24.18,
3
2
2.4 Hz, 1H), 6.66 (d, 3JHH =8.4, 2.4 Hz, 1H), 3.76 (s, 3H), 3.63 (q,
3JHH =6.3 Hz, 2H), 3.00 (t, 3JHH =6.3 Hz, 2H), 2.376 ppm (s, 3H);
13C NMR (150 MHz, (CD3)2CO): d=162.95 (d, JCF =2.4 Hz), 160.19 (d,
10.53 ppm; FTIR (thin film): n˜ =3401, 2926, 1620, 1502, 1287,
741 cmꢀ1 HRMS (EI) calcd for C18H17N2O2F [M]+ 312.12741,
;
312.12653 found.
1JCF =246.0 Hz), 153.84, 132.90, 132.67 (d, JCF =9.2 Hz), 131.16,
2-Fluoro-N-(2-(6-hydroxy-2-methyl-1H-indol-3-yl)ethyl)benza-
mide (5): 5 (0.043 g, 30.9%) was synthesized as a white solid from
2-fluoro-N-(2-(6-methoxy-2-methyl-1H-indol-3-yl)ethyl)benzamide
(0.145 g, 0.44 mmol) using the general procedure described above.
130.90, 129.29, 124.49, 122.99 (d, 2JCF =13.4 Hz), 115.90 (d, JCF
=
2
23.6 Hz), 110.81, 109.99, 107.97, 100.07, 54.96, 40.37, 24.13,
10.64 ppm; FTIR (thin film): n˜ =3299, 2932, 1643, 1482, 1215, 1028,
756 cmꢀ1
326.14365 found.
;
HRMS (EI) calcd for C19H19N2O2F [M]+ 326.14306,
3
1H NMR (300 MHz, (CD3)2CO): d=9.57 (brs, 1H), 7.90 (td, JHH =7.5,
3
1.2 Hz, 1H), 7.55 (m, 2H), 7.29 (td, JHH =7.2, 1.2 Hz, 1H), 7.20 (ddd,
3JHH =11.4, 8.4, 1.2 Hz, 1H), 7.08, (d, 3JHH =8.4 Hz, 1H), 6.97 (d,
2-Chloro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide (10): 10
(0.050 g, 55.6%) was synthesized as a clear oil from 2-(2-methyl-
1H-indol-3-yl)ethanamine (0.050 g, 0.29 mmol) using the general
3
3JHH =2.4 Hz, 1H), 6.60 (dd, 3JHH =8.4, 2.4 Hz, 1H), 3.60 (q, JHH
=
6.9 Hz, 2H), 2.95 (t, 3JHH =6.9 Hz, 2H), 2.35 ppm (s, 3H); 13C NMR
1
1
(150 MHz, (CD3)2CO): d=162.95 (d, JCF =2.3 Hz), 160.18 (d, JCF
=
procedure described above. H NMR (300 MHz, (CD3)2CO): d=9.85
3
246.0 Hz), 150.65, 132.78, 132.63 (d, JCF =8.6 Hz), 131.12, 130.52,
129.59, 124.47, 123.07 (d, 2JCF =13.2 Hz), 115.90 (d, 2JCF =23.6 Hz),
110.56, 110.06, 107.33, 102.20, 40.29, 24.25, 10.62 ppm; FTIR (thin
(brs, 1H), 7.55 (m, 2H), 7.37 (m, 4H), 6.99 (quin, JHH =6.6 Hz, 2H),
3.61 (q, 3JHH =7.2 Hz, 2H), 3.04 (t, 3JHH =7.2 Hz, 2H), 2.42 ppm (s,
3H); 13C NMR (150 MHz, (CD3)2CO): d=166.16, 137.26, 135.83,
132.10, 130.52, 129.73, 129.16, 128.90, 126.82, 120.25, 118.42,
117.59, 110.26, 107.99, 40.29, 24.23, 10.66 ppm; FTIR (thin film): n˜ =
3385, 3056, 2932, 1643, 1383, 1026, 743 cmꢀ1; HRMS (EI) calcd for
C18H17N2OCl [M]+ 312.10295, 312.10365 found.
film): n˜ =3383, 3048, 2931, 1642, 1372, 1207, 1028, 756 cmꢀ1
;
HRMS (EI) calcd for C18H17FN2O2 [M]+ 312.12741, found 312.12790.
Protein preparation: Bos taurus Arp2/3 complex was purified from
calf thymus as previously described.[21] Rabbit skeletal muscle actin
was purified from acetone powder purchased from Pel-freeze
using established methods.[22] Purified actin was labeled on Cys375
with pyrene-iodoacetamide following the procedure of Pollard.[23]
Human N-WASp-VCA (residues 428–505) was overexpressed in
E. coli BL21(DE3) cells and purified as described by Nolen et al.[9]
3-Chloro-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide (11): 11
(0.042 g, 46.7%) was synthesized as a white solid from 2-(2-methyl-
1H-indol-3-yl)ethanamine (0.050 g, 0.29 mmol) using the general
1
procedure described above. H NMR (300 MHz, (CD3)2CO): d=9.84
3
3
(brs, 1H), 7.96 (brs, 1H), 7.88 (t, JHH =1.5 Hz, 1H), 7.82 (dt, JHH
=
7.2, 1.5 Hz, 1H), 7.49 (m, 3H), 7.26 (d, 3JHH =7.2 Hz), 6.98 (quin.d,
Pyrene actin polymerization assays: A solution of 2 mL 0.5 mm
MgCl2 and 2 mm EGTA was added to 20 mL 15 mm 30% pyrene-la-
beled actin in 2 mm Tris pH 8.0, 0.1m ATP, 0.5 mm DTT, and 0.1 mm
CaCl2. Polymerization was initiated by adding 78 mL of a solution
containing Arp2/3 complex and N-WASp-VCA to bring the final
buffer concentrations to 10 mm imidazole pH 7.0, 50 mm KCl,
1 mm EGTA, 1 mm MgCl2 and 1 mm DTT. The fluorescence was
measured every 10 s for 45 min using an excitation wavelength of
365 nm and an emission wavelength of 407 nm on a Tecan Safire2
plate reader. We tested each of the inhibitor analogues using
a maximum concentration of 200 mm, as all compounds were solu-
ble at this concentration. The maximum polymerization rate was
determined by measuring the slope of each polymerization curve
at each time point and converted from RFUsꢀ1 to (nm actin)sꢀ1 as-
suming the total polymer concentration at equilibrium is the total
actin concentration minus 0.1 mm, the critical concentration. Plots
of maximum polymerization rate versus inhibitor concentration
were fit using Equation (1):
3JHH =7.5, 0.9 Hz, 2H), 3.60 (q, 3JHH =7.2 Hz, 2H), 3.01 (t, JHH
=
3
7.2 Hz, 2H), 2.36 ppm (s, 3H); 13C NMR (150 MHz, (CD3)2CO): d=
164.98, 137.32, 135.82, 133.83, 132.09, 130.74, 130.01, 128.90,
127.16, 125.55, 120.26, 118.43, 117.52, 110.28, 108.15, 40.60, 24.08,
10.57 ppm; FTIR (thin film): n˜ =3403, 3306, 1641, 1544, 1436, 1300,
742 cmꢀ1
312.10318 found.
;
HRMS (EI) calcd for C18H17N2OCl [M]+ 312.10295,
General procedure for 2 and 5: Indole amides prepared according
to the general procedure described above, were stirred in CH2Cl2
(~1 mL per 15 mg compound) under N2 at ꢀ788C. BBr3 (1m in
CH2Cl2, 5 equiv per methoxy group) was added by cannula to the
stirred solutions. The reaction was allowed to warm to room tem-
perature following addition and stirred overnight. After 18 h,
excess BBr3 was quenched by addition of H2O followed by 2m
NaOH until no visible reaction was evident, and pH strips indicated
strong basicity. The aqueous phase was acidified with 6m HCl and
the phases were separated. The aqueous layer was extracted twice
with 10 mL CH2Cl2, and the organic phases were combined. Sol-
vent was removed by rotary evaporation, yielding an oil as crude
product. Purification on a silica gel column using 6:1 Et2O/hexanes
as eluent followed by removal of solvent under reduced pressure
yielded deprotected compounds in moderate to low yields.
"
#
ðmax ꢀ minÞ
rate ¼ max ꢀ
ð1Þ
IC50
1 þ
=
inhibitor
X-ray crystallography: Crystals of Bos taurus Arp2/3 complex were
grown by hanging-drop vapor diffusion as previously described.[21]
Crystals were transferred to soaking solution containing 18% PEG
8000, 50 mm HEPES pH 7.5, 100 mm potassium thiocyanate, 20%
glycerol, and either 0.5 mm CK-666, 0.5 mm CK-869, or 0.5 mm CK-
869 plus 2 mm ATP and 2 mm CaCl2 and soaked at 48C for 16 h.
Data were collected at beamline 5.0.1 at the Advanced Light
Source in Berkeley, CA (USA). Phases were solved by molecular
placement using the apo-Arp2/3 complex as a starting model
(1K8K.pdb), and the structures were refined using crystallography
and NMR system[24] using inhibitor parameter files generated using
2-Fluoro-4-hydroxy-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benza-
mide (2): 2 (0.036 g, 9.4%) was synthesized as a white solid from
2-fluoro-4-methoxy-N-(2-(2-methyl-1H-indol-3-yl)ethyl)benzamide
(0.400 g, 1.23 mmol) using the general procedure described above.
1H NMR (300 MHz, (CD3)2CO): d=9.85 (brs, 1H), 9.36 (brs, 1H), 7.86
3
3
(t, JHH =8.7 Hz, 1H), 7.56 (d, JHH =7.2 Hz, 1H), 7.31 (brs, 1H), 7.26
3
3
(dd, JHH =6.9, 1.5 Hz, 1H), 6.98 (quin.d, JHH =7.5, 1.5 Hz, 2H), 6.77
3
3
(dd, JHH =8.7, 2.4 Hz, 1H), 6.61 (dd, JHH =13.5, 2.4 Hz, 1H), 3.61 (q,
3JHH =7.2 Hz, 2H), 3.01 (t, 3JHH =7.2 Hz, 2H), 2.39 ppm (s, 3H);
13C NMR (150 MHz, (CD3)2CO): d=162.78, 161.51 (d, JCF =246.0 Hz),
1
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ChemMedChem 2012, 7, 1286 – 1294

An Arp2/3 Complex Inhibitor
MED
the prodrg server.[25] Coordinates were deposited in the RCSB Pro-
tein Data Bank with accession code 3UKR.
both the protein side chains and backbone remained fixed. All MC
simulations were run at 298 K.
The FEP calculations were performed by employing a simple over-
lap (OS) sampling scheme[32] with 11 windows, as described previ-
ously.[33] A window refers to a MC simulation at one point along
the mutation coordinate l, which interconverts two ligands as
l goes from 0 to 1. The spacing between windows was 0.1. Each
window for the unbound ligand in water consisted of 30ꢂ106
(30M) configurations of MC equilibration for the first window fol-
lowed by 40M configurations of averaging. For the bound calcula-
tions, the equilibration period was 20M configurations followed by
30M configurations of averaging. The reported uncertainties for
the free-energy changes were obtained from the fluctuation in
separate averages over batches of 2M configurations. Equation (2)
is used, in which m is the number of batches, qi is the average of
property q for the ith batch, and <q> is the overall average for q.
Docking calculations: The crystal structure of Arp2/3 complexed
with 1 (PDB ID: 3UKR) was employed in the docking calculations
performed with Glide 5.7 (Schrçdinger, LLC). The crystal structure
with substrate 12 (PDB ID: 3DXK) was also used for validation of
the docking algorithm. Both crystal structures were initially pre-
pared within the “Protein Preparation Wizard” module of the Maes-
tro software suite (Schrçdinger, LLC). Initially, bond orders were as-
signed, hydrogen atoms were added, and water molecules were
deleted beyond 5 ꢁ of the ligand. Missing side chains were filled in
with the “Prime” module of Maestro. The hydrogen-bonding net-
work was then optimized by reorienting atoms within hydroxy and
thiol groups, water molecules, amide groups of asparagine and
glutamine, and the imidazole ring in histidine. Protonation states
of histidine, aspartic acid, and glutamic acid and tautomeric states
of histidine were predicted. These optimizations are necessary be-
cause the orientation of atoms within these functional groups
cannot be determined from the X-ray structure. Finally, the com-
plex was submitted to a series of restrained, partial minimizations
using the OPLS-AA force field.[26] The ligand structures 1–11 were
drawn in ChemDraw (Cambridgesoft) and energy minimized in
vacuum with the Fletcher–Powell algorithm as implemented in
BOSS[27] with an energy tolerance of 0.0001 kcalmolꢀ1. All struc-
tures were then docked and scored using the Glide standard-preci-
sion (SP) mode;[28] they were subsequently re-docked and re-
scored using the Glide extra-precision (XP) mode.[29] For the dock-
ing calculations, the protein structure remained fixed, while the
ligand was fully flexible. To accommodate differently sized ligands
and to decrease the penalties for close ligand–protein contacts be-
cause the protein is not flexible, the van der Waals radii for nonpo-
lar ligand atoms were scaled by a factor of 0.8. Receptor atoms
were not scaled. For the grid generation and ligand docking proce-
dures, default Glide settings were used.
m
X
2
s2 ¼
ðqi ꢀ hqiÞ =mðm ꢀ 1Þ
ð2Þ
i
To monitor the convergence of the results, we carried out several
forward and reverse mutations to ensure that hysteresis is kept
minimal (i.e., for compounds 6, 7, 8, 10, 11, see Supporting Infor-
mation table 3).
For compounds 7, 8, 10, 11, which bear ortho and meta substitu-
tions, we carried out FEP calculations for both equivalent positions
on the rings. In simulations within a protein environment, the
finite accessible simulation time does not allow sampling of ring
flips. Therefore, equivalent ortho and meta substitutions were sam-
pled in separate simulations. For example, Supporting Information
figure 6 shows compound 11 in the two possible orientations. We
found no significant differences for mutations involving these
equivalent positions with respect to the difference in free energy
of binding as calculated from the FEP simulations.
Free-energy perturbation calculations: FEP calculations were car-
ried out in the context of Monte Carlo (MC) statistical mechanics
simulations to predict relative free energies of binding. During
a FEP calculation, a substituent on the lead compound A (e.g. fluo-
rine) is transformed into a different substituent (e.g. chlorine) to
form compound B inside the binding pocket of the protein as well
as in the aqueous phase. The difference in free-energy changes for
the ligands A and B in protein and in water gives the relative free
energy of binding: DDGb =DGBꢀDGA. If DDGb <0, the binding of
compound B is favored with respect to A. For the FEP calculations
initial structures of the analogues were generated with the ligand
growing program BOMB[30] in the binding site of 1 in the Arp2/3
complex (PDB ID: 3UKR). To minimize computation time, a reduced
model of the protein was used, consisting of the 215 amino acid
residues closest to the binding site, which corresponds to a 23-ꢁ
radius around the binding site. A few remote side chains were neu-
tralized so that there was no net charge for the protein. The pro-
tein–ligand and ligand-only systems were solvated in 25-ꢁ caps,
and the MC/FEP calculations were executed with MCPRO.[27] The
energetics for the systems were described classically with the
OPLS-AA force field for the protein, OPLS/CM1A for the ligands,
and TIP4P for water molecules.[26,31] After system construction, the
complex was subjected to conjugate gradient optimization with
MCPRO. For the MC simulations, all degrees of freedom were sam-
pled for the ligand, while the TIP4P water molecules only translat-
ed and rotated. Bond angles and dihedral angles for protein side
chains in a radius of 10 ꢁ around the binding site were also sam-
pled, while the backbone was kept fixed. Beyond the 10 ꢁ radius,
Multistage FEP: For moderately large perturbations, the configura-
tional ensembles representative of molecules A and B are relatively
disparate, and an FEP calculation runs the risk of not converging. It
is therefore recommended to break the transformation pathway
into intermediate states, which ensure that the ensembles of mole-
cules A and B overlap. In this study, for mutations that introduce
large changes in the overall molecular conformation, structure and
characteristics of the ligand, intermediate steps were introduced,
using the Multistage FEP (MFEP) method. MFEP is based on the
idea of splitting a large perturbation into a series of smaller ones
that are suitable for the single-stage FEP calculations. To split the
perturbation, one or more intermediate molecules are constructed
in addition to compounds A and B. Because free energy is a state
variable, the overall DG is given by the sum of the changes for
each step. For the mutation of the methoxy group to hydrogen
(analogues 4 and 9), we chose to initially perturb the methoxy to
hydroxy, and then the hydroxy group to hydrogen. For example,
for compound 4, first a transformation of 4 to 2 was performed
and then a transformation from 2 to 1. The values reported in
Table 1 for these analogues are the sums of these transformations.
For analogue 7, the DDG results from a concurrent transformation
of the fluorine substituent at R2 into hydrogen and of the chlorine
substituent at R4 into hydrogen.
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B. J. Nolen et al.
MED
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Acknowledgements
We thank the Oregon Medical Research Foundation (Grant
105DG2610189 to B.J.N.) and the National Institutes of Health
(Grant 5R01GM09291703 to B.J.N.) G.P. acknowledges a postdoc-
toral fellowship awarded by the Greek Ministry of Education, Na-
tional Strategic Reference Framework (NSRF) 2007–2013 and co-
funded by the European Regional Development Fund and nation-
al resources. All calculations were performed at BRFAA using an
Intel Xeon cluster funded from a European Economic Area Grant.
Funding for the University of Oregon Chemistry Research and In-
strumentation Services has been furnished in part by the NSF
(CHE-0923589).
Keywords: actin · amides · Arp2/3 complex · cancer · free-
energy perturbation
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Published online on May 23, 2012
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