Discovering potassium channel blockers from synthetic compound database by using structure-based virtual screening in conjunction with electrophysiological assay

Article abstract of DOI:10.1021/jm060414o

Potassium ion (K⁺) channels are attractive targets for drug discovery because of the essential roles played in biological systems. However, high-throughput screening (HTS) cannot be used to screen K⁺ channel blockers. To overcome this disadvantage of HTS, we have developed a virtual screening approach for discovering novel blockers of K⁺ channels. On the basis of a three-dimensional model of the eukaryotic K⁺ channels, molecular docking-based virtual screening was employed to search the chemical database MDL Available Chemicals Directory (ACD). Compounds were ranked according to their relative binding energy, favorable shape complementarity, and potential to form hydrogen bonds with the outer mouth of the K⁺ channel model. Twenty candidate compounds selected from the virtual screening were examined using the whole-cell voltage-clamp recording in rat dissociated hippocampal neurons. Among them, six compounds (5, 6, 8, 18-20) potently blocked both the delayed rectifier (I_K) and fast transient K⁺ currents (I_A). When applied externally, these six compounds preferentially blocked I_K with potencies 2- to 500-fold higher than that of tetraethylammonium chloride. Intracellular application of the six compounds had no effect on both K⁺ currents. In addition, the interaction models and binding free energy calculations demonstrated that hydrophobic interaction and solvent effects play important roles in the inhibitory activities of these compounds. The results demonstrated that structure-based computer screening strategy could be used to identify novel, structurally diverse compounds targeting the pore binding pocket of the outer mouth of voltage-gated K⁺ channels. This study provides an alternative way of finding new blockers of voltage-gated K⁺ channels, while the techniques for high-throughput screening of K⁺ channel drugs remain in development.

Full text of DOI:10.1021/jm060414o

J. Med. Chem. 2007, 50, 83-93
83
Discovering Potassium Channel Blockers from Synthetic Compound Database by Using
Structure-Based Virtual Screening in Conjunction with Electrophysiological Assay
†
†
†
Hong Liu, Zhao-Bing Gao, Zhiyi Yao, Suxin Zheng, Yang Li, Weiliang Zhu, Xiaojian Tan, Xiaomin Luo, Jianhua Shen,
Kaixian Chen, Guo-Yuan Hu,* and Hualiang Jiang*
Center for Drug DiscoVery and Design, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, P. R. China
ReceiVed April 10, 2006
+
Potassium ion (K ) channels are attractive targets for drug discovery because of the essential roles played
+
in biological systems. However, high-throughput screening (HTS) cannot be used to screen K channel
blockers. To overcome this disadvantage of HTS, we have developed a virtual screening approach for
+
+
discovering novel blockers of K channels. On the basis of a three-dimensional model of the eukaryotic K
channels, molecular docking-based virtual screening was employed to search the chemical database MDL
Available Chemicals Directory (ACD). Compounds were ranked according to their relative binding energy,
favorable shape complementarity, and potential to form hydrogen bonds with the outer mouth of the K+
channel model. Twenty candidate compounds selected from the virtual screening were examined using the
whole-cell voltage-clamp recording in rat dissociated hippocampal neurons. Among them, six compounds
+
(
5, 6, 8, 18-20) potently blocked both the delayed rectifier (I
K
) and fast transient K currents (I
K
A
). When
applied externally, these six compounds preferentially blocked I
with potencies 2- to 500-fold higher than
that of tetraethylammonium chloride. Intracellular application of the six compounds had no effect on both
+
K currents. In addition, the interaction models and binding free energy calculations demonstrated that
hydrophobic interaction and solvent effects play important roles in the inhibitory activities of these compounds.
The results demonstrated that structure-based computer screening strategy could be used to identify novel,
+
structurally diverse compounds targeting the pore binding pocket of the outer mouth of voltage-gated K
+
channels. This study provides an alternative way of finding new blockers of voltage-gated K channels,
+
while the techniques for high-throughput screening of K channel drugs remain in development.
2+
+
1
. Introduction
liVidans,7,8 (2) MthK, a Ca -gated K channel from Metha-
9
+
nobacterium thermoautotrophicum, (3) Kv, a voltage-gated K
channel from Aeropyrum pernix and Pichia pastoris,
4) KirBac, an inward rectifier K channel from Burkholderia
pseudomallei. In general, the functional K channels consist
of four subunits (tetramer), and each subunit contributes two
transmembrane R-helices (denoted M1 and M2 in KcsA, MthK,
and KirBac and denoted S5 and S6 in Kv channels) plus a re-
entrant loop, which forms the central K -selective pore.
The pore-forming regions of both prokaryotic and eukaryotic
K channels are highly conserved, including a pore-helix and
an extended filter region that contains the TVGYG sequence
motif. The structural biology studies clearly elucidate the
molecular mechanisms underlying the K conduction and
giving indisputable evidence that the ion
permeation pathway across the membrane is indeed at the center
of four identical subunits that cluster around the narrowest part
of the pore formed by the P loop. Moreover, the existing
crystal structures of K channels also provide a solid framework
+
Potassium ion (K ) channels consist of a ubiquitous family
10,11
and
of membrane proteins that play critical roles in a wide variety
of physiological processes, such as the regulation of neuronal
excitability, muscle contraction, cell proliferation, and insulin
secretion. The abundant diversity for structures and functions
of K channel family members is supported by sequencing
+
(
1
2
+
1
+
studies of the Caenorhabditis elegans genome. About 100
+
13-15
+
unique K channels genes have been predicted to exist in this
organism.2 Mammalian K channels are classified into three
,3
+
+
groups in terms of their membrane topology: (1) voltage-gated
K
contain six transmembrane domains (TMDs); (2) “leak” K
channels, which have four TMDs; (3) inward-rectifier K
channels (Kir), which contain two TMDs. Each K channel
group consists of multiple subfamily members. Impaired func-
+
2+
+
channels (Kv) and Ca -activated K channels, which
1
6
+
+
+
7
-8,11,14
selectivity,
4
+
+
+
tions of K channel or mutations in the genes encoding K
7
,8
channels have been demonstrated to cause a wide spectrum of
+
human diseases.5,6
for constructing three-dimensional (3D) model of the eukaryotic
K channels for drug discovery.
Structure-based virtual screening (SBVS) approaches based
In addition to the substantial advances in molecular cloning
+
+
and functional studies of K channels, the structural elucidation
of K channels has rapidly progressed. Thus far, the crystal
structures of four bacterial homologues of different mammalian
K channels have been elucidated using X-ray analysis. They
+
on molecular docking have become promising tools for dis-
17-22
covering active compounds.
We successfully constructed
+
+
a three-dimensional model for the eukaryotic voltage-gated K
channels based on the crystal structure of KcsA channel from
Streptomyces liVidans and developed a computational virtual
screening approach to search for new blockers of K channels
from large databases. Four candidate compounds discovered
from the China Natural Products Database using SBVS were
demonstrated to possess a potent K channel blocking effect.
+
are (1) KcsA, a pH-gated K channel from Streptomyces
7
+
*
To whom correspondence should be addressed.For G.-Y.H.: phone,
+
86-21-50806778; fax, +86-21-50807088; e-mail, gyhu@mail.shcnc.ac.cn.
For H.J.: phone, +86-21-50806600, extension 1303; fax, +86-21-50807088;
e-mail, hljiang@mail.shcnc.ac.cn.
†
+
Equivalent contributors.
1
0.1021/jm060414o CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/10/2006

8
4
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Liu et al.
out by using the GCG software.29 The side chains of residues
Arg27, Ile60, Arg64, Glu71, and Arg117 missing in the current
KcsA crystal structure were added using the Biopolymer module
3
0
30
of Insight II. The Biopolymer module of Insight II was also
used to construct the 3D structural model of eukaryotic Shaker
+
K
channels by mutations of Pro55Glu (P55E), Ala57Ser
(A57S), Ile60Lys (I60K), Thr61Ser (T61S), Arg64Asp (R64D),
Leu81Met (L81M), and Tyr82Thr (Y82T) on the X-ray crystal
+
7
structure of the KcsA K channel. After a preliminary
optimization of the side chains, the entire 3D model was
minimized by 200 steps of steepest descent, followed by Powell
minimization to a root-mean-square (rms) energy gradient of
0
.05 kcal/(mol‚Å). The AMBER force field with the Kollman
3
1,32
all-atom charges
was employed during the structural opti-
mization. The quality of the model was checked by the Prostat
3
0
Figure 1. Computational and experimental flow chart for this study.
In rat hippocampal neurons they are 20-1000 times more potent
program in Insight II.
.1.2. Virtual Screening. The ACD database containing the
structural information of 200 000 chemicals (http://www.mdl-
2
+
than the classical delayed rectifier K channel blocker tetra-
24
.
com) was used for the virtual screening. A chemistry space
filter for scoring druglikeness developed in our laboratory was
used to remove the nondruglike structures from the ACD.
DOCK4.0 was employed for the primary screening. Residues
around the motif TXXTVGYG of the K channel at a radius
ethylammonium chloride.23 Continuing this study, we have
26
+
recently used this approach to search for K channel blockers
from the synthetic compounds. In this study, we show that novel
25
+
structurally diverse blockers of K channels can be found in
+
the large chemical database MDL Available Chemicals Direc-
tory (ACD)24 using SBVS in conjunction with chemical
synthesis and electrophysiological assay. Twenty candidate
compounds were selected from the ACD database with relatively
low binding energy, favorable shape complementarity, and/or
of 5 Å were isolated for constructing the grids of docking
screening. During the docking calculation, Kollman all-atom
31
charges were assigned to the protein, and Gasteiger-Marsili
33
charges were assigned to the small molecules in the tidy ACD.
The flexibility of each small molecule was considered during
the virtual screening. The top 300 molecules were selected for
further analysis.
+
potential to form hydrogen bonds with the K channel. The
electrophysiological assay demonstrated that six compounds
+
showed potent blocking effects on voltage-gated K channels
+
The structures of the complexes of the K channel with the
in rat hippocampal neurons.
DOCK selected molecules were optimized automatically by
3
2
running a SPL program encoded into the Sybyl environment
written by us to adjust the orientation and interaction. The
2
. Materials and Methods
The computational and experimental flow chart is shown in
+
binding energy (∆Ebind) between each molecule and K channel
Figure 1. Briefly, 300 candidate molecules were first selected
from the ACD database by using the docking program
was calculated by using
2
5
DOCK4.0 targeting the binding site of a 3D model of the
∆Ebind ) Ecomplex - Echannel - Emol
(1)
+
eukaryotic Shaker K channel constructed by homology model-
ing. Before virtual screening, nondruglike molecules were
filtered out from the ACD database using a druglike analysis
where Ecomplex, Echannel, and Emol are respectively the total
+
energies of the complexes, the K channel, and the small
26
32
filter developed by us. Afterward, the binding models of the
molecules. The Tripos force field was used for structure
+
candidates with the K channel were optimized using molecular
optimization and energy calculation. At the same time, the log P
mechanics, and log P values of the candidates were predicted
using the XlogP program.27 According to the DOCK scoring
results, interaction models (shape complementarities between
values of these DOCK selected molecules were predicted using
the XLOGP program. The top 300 molecules produced from
27
DOCK screening were analyzed for relatively lower binding
energy, favorable shape complementarity, and/or potential in
+
candidates and K channel), and the XlogP values, 20 com-
+
pounds were selected for bioassay. Six active compounds were
discovered through electrophysiological screening. The binding
models and binding free energies of the active compounds to
forming hydrogen bonds with K channel and XlogP values.
According to the DOCK scoring results, interaction models
(shape complementarity and potential in hydrogen bond forma-
tion), and log P values, 20 compounds (1-20) were finally
selected for biological assay, and their primary binding free
+
the K channel were re-estimated by using the MD simulation
and MM/GBSA approach,28 based on which structure-activity
relationship of these active compounds was discussed. The
details of the computations and experiments are described as
follows.
+
energies with the K channel were calculated using AutoDock
3
4
3.0.
2.1.3. Free Energy Calculation. The complex structures of
2
.1. Modeling and Computation. 2.1.1. 3D Model of
the channel with the active compounds (inhibitors) were solvated
by a TIP3P water sphere with a radius of 25 Å. Then the
+
35
Eukaryotic Shaker K Channel. The 3D model of eukaryotic
Shaker K channels was generated on the basis of the crystal
structure of the KcsA K channel retrieved from the Brookhaven
Protein Data Bank (PDB) (http://www.rcsb.org/pdb/) (entry
+
solvent models were equilibrated for 600 ps at 300 K by using
molecular dynamics (MD) simulation, and a 200 ps MD
simulation was run on each model system to sample the
conformations every 4 ps for binding free energy calculations.
Only the solvent and those residues within 15 Å of the inhibitor
were allowed to move during the MD simulation. All MD
simulations were carried out by using the AMBER 7.0
+
7
23
1
BL8) using our previously reported procedure. The se-
+
quences of eukaryotic Shaker K and KcsA channels were
obtained from the SwissProt database (entries P08510 and
Q54397, respectively). A sequence alignment of KcsA and
+
36
37
eukaryotic channels with the Shaker K channels was carried
program, and the AMBER force field (parm98) and general

Potassium Channel Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 85
Scheme 1. Chemical Structures of Compounds 5, 6, 8, and 18-20a
a
Reagents: (a) PhONa, (b) NH2OH·HCl; (c) LAH; (d) 10% hydrochloric acid.
Scheme 2a
a
Reagents: (a) SOCl2, pyridine; (b) K2CO3, KI.
AMBER force field (GAFF)38 were applied on the protein and
the inhibitors, respectively. The partial charges for all the
inhibitors were obtained by fitting the Hartree-Fock calculations
two compounds have been described in Table S1 in
the Supporting Information. Compounds 6, 8, 18, and 19
were synthesized by us. Scheme 1 depicts the sequence of
reactions that led to the preparation of compound 6 by using
2-bromo-1-(2,3-dihydro-1,4-benzodioxin-6-yl)ethanone and so-
dium phenoxide as the starting raw materials. Intermediate
3
9
with Gaussian 98 at the 6-31G* basis set level using RESP.
28
The MM/GBSA method was used to evaluate the binding free
+
energies (∆Gbind) of the inhibitors to the K channel,
1
-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-phenoxyethanone (21)
was synthesized by reacting the commercially available 2-bromo-
-(2,3-dihydro-1,4-benzodioxin-6-yl)ethanone with sodium phen-
∆
Gbind ) Gcomplex - Gprotein - Gligand
1
)
∆EMM + ∆GGB + ∆GNP
(2)
oxide in THF. Then compound 21 was condensed with
hydroxylamine hydrochloride by refluxing in ethanol, which
afforded 1-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-phenoxyetha-
none oxime (22). Compound 22 was reduced by LAH in THF,
providing 1-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-phenoxyethy-
lamine (23). Compound 23 was changed into its hydrochloride,
compound 6, by the addition of 10% hydrochloric acid.
where ∆GGB is the polar solvation energy in the continuum
solvent, computed using a modified generalized Born model
4
0
and where the nonpolar contribution (∆GNP) was estimated using
a simple empirical relation ∆GNP ) σA + b,41 where A is the
solvent-accessible surface area estimated using the MSMS
program, σ and b are empirical parameters, and in this work,
.005 42 and 0.92 kcal/mol were adopted for these two
4
2
Scheme 2 depicts the sequence of reactions that led to the
preparation of compound 8 by using tetraethylene glycol as the
starting raw materials. Tetraethylene glycol was chlorinated
using SOCl2 in the presence of pyridine in 1,4-dioxane,
providing bis[2-(2-chloroethoxy)ethyl] ether (24). Then com-
pound 24 reacted with toluene-4-sulfonamide in DMF, using
KI as a catalyst, which afforded the target compound 8.
0
parameters, respectively. ∆EMM denotes the sum of the molec-
ular mechanical (MM) energies of the molecules.
The starting raw materials for preparing compound 18 were
commercially available 2-[(4-chlorophenyl)sulfonyl]ethanol and
SOCl2. Scheme 3 shows the procedures for preparation of the
target compound. Intermediate 2-[(4-chlorophenyl)sulfonyl]-
ethanol was chlorinated by SOCl2 in the presence of pyridine
in toluene to yield 2-chloro-[(4-chlorophenyl)sulfonyl]ethane
(25). Compound 25 reacted with 1-benzylpiperazine in THF,
catalyzed by KI, providing the target molecule 18.
Compound 19 was synthesized through the route outlined in
Scheme 4, using 5-chloronaphthalene-1-sulfonic acid as the
starting raw material. Intermediate 5-chloronaphthalene-1-
sulfonic acid was chlorinated by PCl5, providing 5-chloronaph-
thalene-1-sulfonyl chloride (26). Compound 26 reacted with 1,2-
diaminoethane in the presence of K2CO3 in THF, giving the
target compound 19.
2
.2. Compounds and Chemical Synthesis. On the basis of
+
virtual screening, six compounds were selected as K channel
inhibitor candidates for further electrophysiological assay.
Compounds 5 and 20 were purchased from the Sigma and
Maybridge companies, respectively. The spectral data of these

8
6
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Liu et al.
Scheme 3a
a
Reagents: (a) SOCl2, pyridine; (b) K2CO3, I2.
Scheme 4a
the six compounds were calculated using the computer software
“
GraphPad Prism 3.0”. Data are presented as mean ( SEM.
3
. Results and Discussion
.1. Identification of Compounds by Virtual Screening.
3
Sequence alignment indicates that the identity between the pore
+
+
regions of KcsA K channel and the Shaker K channels is
36.875% (Figure S1 in the Supporting Information). The
a
Reagents: (a) PCl5; (b) H2NCH2CH2NH2.
+
optimized 3D model of the eukaryotic K channels is very
similar to the X-ray crystal structure of KcsA K channel
+
2
.3. Electrophysiological Assay. 2.3.1. Preparation of
Dissociated Hippocampal Neurons. Dissociated CA1 pyra-
midal neurons were prepared from 5- to 9-day-old Sprague-
Dawley rats as described previously.43 Briefly, transverse
minislices (500 µm) of the hippocampal CA1 region were cut
in an oxygenated ice-cold dissociation solution. The dissociation
solution consisted of the following (in mM): Na2SO4, 82; K2-
SO4, 30; MgCl2, 5; N-(2-hydroxyethyl)piperazine-N′-(2-ethane-
sulfonic acid) (HEPES), 10; glucose, 10; at pH 7.3 with NaOH.
The slices were incubated in the dissociation solution containing
protease XXIII (3 mg/mL) at 32 °C for 8 min and then placed
in the dissociation solution containing trypsin inhibitor type II-S
(Figure S2 in the Supporting Information). We can therefore
use this 3D model for virtual screening. Virtual screening was
24
25
performed on the ACD database by using DOCK 4.0
+
targeting the 3D model of K channel. Before virtual screening,
nondruglike structures were removed by a filter for druglike
26
analysis developed in our laboratory. Because compounds that
+
45
plug the “ion-selective filter” of the K channel may function
as blockers of the channel, the extracellular pore binding site
composed by the residues around the TXXTVGYG motif was
used as the target site for virtual screening. The binding pose
(orientation and conformation) of each molecule was evaluated
by the DOCK scoring function, which consists of the van der
Waals and Coulombic electrostatic interaction energies. The top-
300 candidate molecules were selected from ACD for further
analysis (Figure 2A, Figure S3). The DOCK scoring indicates
that electrostatic interaction dominates the binding between the
(1 mg/mL) and bovine serum albumin (1 mg/mL) at room
temperature under an oxygen atmosphere. Before recording, the
slices were triturated using a series of fire-polished Pasteur
pipettes with decreasing tip diameters. The neurons were placed
in a recording dish and perfused with an external solution
containing the following (in mM): NaCl, 135; KCl, 5; CaCl2,
+
candidates and K channel. This is in agreement with the free
+
1
; MgCl2, 2; HEPES, 10; glucose, 10; tetrodotoxin, 0.001; at
pH 7.3 with NaOH.
.3.2. Whole-Cell Recording. Whole-cell voltage-clamp
energy calculation for the active compounds with the K channel
(see discussion below).
2
However, docking programs and scoring functions have a
tendency to generate a significant number of false positives.17
Accordingly, the structures of the complexes of the top-300
recording was made from pyramidal-shaped neurons using an
Axopatch 200A amplifier (Axon Instruments) at 21-23 °C. The
electrodes (tip resistance of 3-4 MΩ) were filled with a pipet
solution containing the following (in mM): KCl, 140; MgCl2,
+
32
molecules with the K channel were optimized in the Sybyl6.7
32
environment by running a SPL program written by us to adjust
the binding pose, and the binding energies (∆Ebind) were
recalculated by using a more sophisticated method, molecular
mechanics force field (see eq 1 in Materials and Methods). The
1
; CaCl2, 1; HEPES, 10; EGTA, 10; at pH 7.3 with KOH. The
+
neuron was held at -50 mV. Voltage-activated K currents were
elicited with command protocols provided by pClamp 9.0
software via a DigiData-1322A interface (Axon Instruments).
Signals were filtered at 2-10 kHz and sampled at frequencies
of 10-40 kHz. Series resistance was compensated by 75-85%.
Linear leak and residual capacitance currents were subtracted
+
interaction energies of these 300 molecules with the K channel
calculated by DOCK range from -38.33 to -22.76 kcal/mol
and that calculated by Sybyl are from -178.85 to -72.15 kcal/
mol (Figure 2B). More than 50% candidate molecules are
located in the high interaction energy area on the DOCK-Sybyl
plot (top-right part of Figure 2B). These molecules would not
be considered for further analysis. Most of the rest of the
candidate molecules are dispersed at the top-left part of the plot
(red rectangle), and several molecules are displayed in the lower-
middle region of the plot (green rectangle). There are almost
no points at the bottom-left. It is therefore difficult to select
candidates directly from the red and green rectangles, and other
factors have to be considered in compound selection.
+
online. The peak amplitude of the fast transient K current (IA)
was measured. The amplitude of the delayed rectifier K current
+
(IK) was measured with 300 ms latency. For external application,
the candidate compound was dissolved in the external solution,
and delivered to the recorded neuron using RSC-100 rapid
solution changer with a nine-tube head (BioLogic Co. France).
For intracellular application, the compound was dissolved in
the pipet solution and diffused from the pipet into the neuron
after the patch membrane was ruptured.44 The IC50 values of

Potassium Channel Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 87
Table 1. Binding Energies (in kcal/mol), Actual Ranks, and XlogP
a
Values of the 20 Compounds Selected from the 300 Candidates
compd
BE1 b
R1 b
BE2 c
R2 c
BE3 d
R3 d XlogP
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-33.85
-29.10
-32.82
-35.40
-35.16
-34.32
-28.67
-29.51
-29.06
-30.49
-32.96
-33.12
-28.67
-32.21
-33.90
-34.46
-28.51
-35.50
-38.33
-35.16
9
57
13
3
4
7
64
51
57
33
11
10
64
15
9
-82.245 167 -16.57
-84.331 154 -45.68
14
3
7
0.35
-2.09
4.42
0.51
1.29
2.59
-0.89
1.87
-175.019
2
-26.52
-92.112 104 -45.95
-75.437 225 -16.33
2
16
17
22
20
21
1
-96.668
80 -15.23
-91.259 110
6.64
-9.81
-6.14
-132.34
-75.32
12
227
5.05
-1.13
0.15
1
1
1
1
1
1
1
1
1
1
2
-81.235 176 -46.50
59 -27.32
-103.654
6
-83.070 161 -13.74
-83.269 160 -22.71
-87.947 131 -42.21
-77.120 205 -16.34
-87.807 132 -11.31
18
11
4
15
19
10
9
0.96
-0.12
-0.19
-0.49
-1.82
0.19
3.31
2.00
1.04
6
68
2
1
4
-100.82
-75.069 231 -25.57
-100.505 67 -17.00
-78.822 191 -17.29
64 -25.57
13
12
a
Boldfaced and italic values are for the six active compounds discussed
in the text. b The binding energy (BE1) and the actual rank (R1) calculated
c
using DOCK. The binding energy (BE2) and the actual rank (R2) calculated
using Sybyl. d The binding energy (BE3) and the actual rank (R3) calculated
using AutoDock.
Figure 2. Binding energies of the top-300 compounds: (A) DOCK
binding energies (black 9, total binding energy; red b, van de Waals
energy; green 2, electrostatic energy); (B) DOCK predicted binding
.3. K+ Channel-Blocking Effects of Candidate Com-
3
1 2
energies (BE ) versus Sybyl binding energies (BE ) (blue 2, compounds
pounds. 3.3.1. Primary Electrophysiological Assay. In the
primary electrophysiological assay, 20 candidate compounds
selected from the virtual screening were determined using the
whole-cell voltage-clamp recording from dissociated hippoc-
ampal neurons. When applied externally, six candidate com-
pounds (5, 6, 8, 18, 19, and 20) at 100 µM suppressed at least
5
-6, 8, 18-20; red 9, compounds 1-4, 7, 9-17; black [, compounds
2
1-300).
Hydrophobicity is an important druglike property of mol-
23
ecules. As described in our previous study, the MDL Drug
+
4
6
+
one type of the K current (IK or IA) by more than 10%, whereas
Data Report (MDDR) database contains 206 potent K channel
inhibitors, and 10 of them have entered into clinical trail. We
the other 14 compounds at 100 µM and even at 1 mM had no
significant effect. Each of the six compounds more markedly
suppressed IK than IA. The results are shown in Figure 3A and
Table S3 in the Supporting Information.
+
have calculated the log P values for these K channel inhibitors
2
7
by use of the XLOG program. The result indicated that 70%
+
of the K channel inhibitors in MDDR show log P values from
3.3.2. Secondary Electrophysiological Assay. The secondary
0
to 6, and nine of the clinical inhibitors showed log P values
2
3
electrophysiological assay was conducted to determine the
concentration-response relationship for the six candidate
compounds. The result is shown in Figure 3B. The IC50 values
of these compounds obtained via curve-fitting procedures are
presented in Table 2. All six compounds potently blocked both
from 2 to 6. Accordingly, candidate molecules should have
similar log P values compared with existing K channel
+
inhibitors. Synthetically considering the two kinds of interaction
energies and the log P values (is better between 0 and 6) and a
visual check of the interaction models, 20 molecules were
selected for bioassay, 18 from the red rectangle and 2 from the
green one (Figure 2B). The predicted log P values of most of
the 20 selected candidates are within or close to 0-6. Only 4
compounds (2, 7, 10, and 16) are outside the region of the log P
+
types of K currents with IC50 values ranging from a few µM
to nearly 1 mM. The rank order of potency in inhibition of IK
(
8 > 19 > 18 > 6 > 20 > 5) was basically parallel to that of
inhibition of IA (8 > 19 > 20 > 6 > 5 > 18). The ratio of IC50
values in inhibition of I to those of I represented the selectivity
+
profile of the existing K channel inhibitors (Table 1). As will
A
K
+
of a compound for two types of K channels. For the six
compounds, the ratio of IC50 values ranged from 1.9 to 16.3.
Compound 18 shows the highest selectivity for IK (IC50(IK)/
IC50(IA) ) 16.3), whereas compound 5 is the least selective
be seen below, these four compounds are inactive, suggesting
+
the importance of hydrophobicity in the selection of K channel
inhibitors.
3
.2. Compounds Synthesis. The 20 candidate compounds
(
IC50(IK)/IC50(IA) ) 1.9).
3.3.3. Intracellular Application of the Candidate Com-
were purchased from the Sigma and Maybridge companies for
the primary screening. Among the six active compounds, only
enough chemical samples of compounds 5 and 20 could be
supported respectively by the Sigma and Maybridge companies
for further bioassay. The spectral data of these two compounds
have been described in Table S1 in the Supporting Information.
Because the companies could not provide enough samples for
compounds 6, 8, 18, and 19, they were synthesized through the
routes outlined in Schemes 1-4, and the details of the synthetic
procedures and structural characterizations are described in the
Experimental Section. The purities of these six compounds are
listed in Table S2 in the Supporting Information.
pounds. Externally applied chemicals, like verapamil, could
+
diffuse through the membrane and block the K channels at an
4
9
internal binding site. In order to exclude the possibility, the
effects of intracellular application of the six compounds on the
currents were investigated. Each compound was included
in the recording pipet at a concentration that could inhibit IK
+
K
by more than 75% when applied externally and diffused into
4
4
the neuron after the membrane ruptured. As shown in Figure
4, all six compounds had no detectable effect on IK nor did the
compounds affect IA (data not shown). The results suggest that

8
8
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Liu et al.
+
+
Figure 3. Effects of the externally applied six compounds on voltage-activated K currents in rat hippocampal neurons. (A) The total K current
(
+
+
K A
upper record), delayed rectifying K current (I , middle record), and fast transient K current (I , lower record) were recorded in control and in
the presence of compound 5 (500 µM). The traces at the bottom of the upper and middle records are the command voltage steps. The neuron was
+
held at -50 mV. The total K current was elicited by a depolarizing step to +40 mV following a hyperpolarizing prepulse of 500 ms to -110 mV.
I
K
(
K
+
was elicited by a similar protocol in which a 50 ms interval at -50 mV was inserted after the prepulse. I
A
was the subtraction of I
K
from the total
43,47-48
current.
(B) Concentration-response curves of the six compounds in inhibition of I and I . Each symbol represents the mean ( SEM
K A
n ) 5-12).
+
the six compounds bind to an external site of the K channels,
thus blocking the K currents.
compounds possess good hydrophobic profiles similar to those
of the existing K inhibitors. This may be one of the reasons
+
+
3
.4. Structure-Activity Relationship (SAR). If we reap-
why only these 6 compounds are active among the 20
candidates.
praise the virtual screening result with the active compounds,
it can be seen that five active compounds are in the lowest
DOCK interaction energy region and one is in the low Sybyl
interaction energy region (Figure 2B). As mentioned above, most
of the clinical K channel inhibitors showed log P values from
to 6. The log P values of the six active compounds range
The general interaction models of the six compounds with
+
the K channel are presented in Table S4 in the Supporting
Information and Figure 5. The binding free energies of the six
+
+
compounds to the K channel were calculated by the MM/
2
3
28
36
2
GBSA method encoded in the AMBER program, version 7.0,
and the results are listed in Table 3. The result indicates that
from 1.0 to 3.5 (Table 1), indicating that the discovered active

Potassium Channel Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 89
+
Table 2. IC50 Values of the Six Compounds in Inhibition of the
Voltage-Activated K Currents
for these compounds binding to the K channel, and this may
+
be an important reason that these compounds could potentially
+
IC50 (µM)
inhibit K channel. Compounds 5, 6, 8, and 18-20 bind with
n a
the side chains of residues Ser57(A), Tyr78(B-D), Gly79(A,
compd
IK
IA
IC50(IA)/IC50(IK)
+
B, D), Asp80(B, D), Thr82(C, D), and Val84(A) of the K
5
6
8
8
9
0
524 ( 14
67 ( 9
972 ( 34
737 ( 48
28 ( 4
1.9
11
13.3
16.3
6.9
6
6
6
6
6
6
channel through both hydrophobic interactions and electrostatic
interactions (Figure 5 and Table S4 of the Supporting Informa-
tion). The number of hydrophobic contact pairs is not consistent
with inhibitory potency, indicating that the difference in
inhibitory activities of these compounds might be contribution
of complex effects related to hydrogen bonding, hydrophobic
contact, electrostatic interaction, and solvation. The correlation
between the van der Waals binding energies and log IC50 values
2.1 ( 0.5
60 ( 7
1
1
2
980 ( 150
124 ( 9
18 ( 3
83 ( 8
244 ( 26
2.9
a
n: the number of neurons tested.
(
R ) 0.75, Figure 6B) indicates that the van der Waals
interaction demonstrates structure-activity relationship (SAR)
of the six compounds. Although the electrostatic binding energy
for each compound to the K channel is the determinant
+
component in the molecular mechanical energy (Figure 2A and
Table 3), the polar solvation energy penalty is also high, which
counteracts the electrostatic interaction.
Among the six compounds, compound 8 shows the most
potent inhibitory activity (Table 2). In line with the bioassay
result, MM/GBSA calculations indicated that compound 8 binds
+
to the K channel with the lowest binding free energy (Table
3). The components of the binding free energy of this compound
are also different from those of other compounds (Table 3). The
+
electrostatic interaction of compound 8 with the K channel is
much weaker than that of other compounds. This is also in
agreement with the interaction models (Figure 5), indicating
+
that only compound 8 did not form a hydrogen bond to the K
channel. However, the solvation free energy (Gbsur + Gb) of
compound 8 is remarkably smaller than that of other compounds
(
Table 3), suggesting that this compound can easily overcome
+
the bondage of solvent during binding to the K channel. In
addition, the hydrophobic interaction of compound 8 to the K
+
channel reflected by the van der Waals binding energy is also
the strongest among the six compounds. Consequently, com-
prehensive effects of the electrostatic interaction, hydrophobic
interaction, and solvation make compound 8 the most potent
+
inhibitor of the K channel among the six compounds.
Figure 4. Time course of the delayed rectifier K+ current (I
K
) in rat
4
. Conclusions
hippocampal neurons during intracellular application of the six
compounds. In each panel, the normalized I is plotted against the time
K
As mentioned above, high-throughput screening (HTS) has
of intracellular application of the compounds. Each symbol represents
the mean ( SEM (n ) 5). The concentrations chosen for intracellular
K
application were above the IC75 values (causing 75% inhibition of I )
of the same compounds in external application: compound 5 (1 mM),
compound 6 (100 µM), compound 8 (10 µM), compound 18 (100 µM),
compound 19 (30 µM), and compound 20 (300 µM).
+
not been applied to discover K channel blockers because of
the hindrance of the electrophysiological assay. Previously, we
discovered four natural K channel blockers by using docking
based virtual screening in conjunction with an electrophysi-
ological assay. Here, we further demonstrated the effectiveness
of the strategy for discovering K channel blockers from the
synthetic chemicals. The molecular docking program DOCK4.0
was used to screen the chemical database ACD targeting the
3D model of the Shaker K channel. The top-300 molecules
+
23
+
25
the calculated MM/GBSA binding free energies correlate well
2
4
with the inhibitory activities (R ) 0.83), as shown in Figure
+
+
6
A. This relationship suggests that those potential K channel
inhibitors exhibiting stronger binding free energies using this
paradigm would therefore expect to have greater efficacy toward
inhibitory action. The results demonstrated the efficiency of our
strategy for discovering potassium channel blockers by using
virtual screening in conjunction with electrophysiological assay.
Structurally, compounds 5, 6, and 18-20 have similar
chemical scaffolds with amino groups that are easily protonated
to be quaternary ammoniums in vivo. The amino group of
compounds 5, 6, 19, and 20 acts as a donor to form hydrogen
bonds with the O atoms of Gly77(A-D) and Tyr78(A-D) of
were obtained by the scoring function of DOCK. Then the
complexes of these 300 candidates with the K channel model
+
were optimized by molecular mechanics encoded in Sybyl and
the interaction energies were calculated. In addition, the log P
values of these molecules were predicted by the XlogP
2
7
program. According to the interaction energies, the DOCK
scoring result, and the XlogP values, 20 compounds were
3
4
selected and re-estimated using AutoDock. The bioassay
results demonstrated a relatively high hit rate of our virtual
screening approach. Of the 20 candidates, 6 compounds (5, 6,
8, 18-20) were found to suppress at least one type of voltage-
+
the K channel, respectively (Figure 5 and Table S4 in the
+
Supporting Information). These hydrogen bonds play vital roles
activated K current (IK or IA) by more than 10%. Furthermore,

9
0
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Liu et al.
Figure 5. 3D representatives for the general interaction models of compounds 5 (A), 6 (B), 8 (C), 18 (D), 19 (E), and 20 (F) with the potassium
ion channel. The broken lines in red represent hydrogen bonds. This picture was produced by using the PyMOL program.50
Table 3. Binding Free Energies of the Six Compounds to the K+ Channel (kcal/mol)
compd
log IC50
Eele a
EvdW b
EMM c
Gbsur d
Gb e
Gbsol f
Gbind g
5
6
8
8
9
0
-3.28
-4.17
-5.68
-4.22
-4.74
-4.08
-220.06
-219.27
-42.22
-330.40
-234.63
-256.31
-27.10
-23.14
-42.16
-30.14
-33.10
-18.01
-247.15
-242.42
-84.38
-360.53
-267.73
-274.32
-2.37
-2.26
-3.02
-2.79
-2.77
-2.05
245.73
227.70
67.43
352.21
257.64
269.12
243.36
225.44
64.41
349.42
254.86
267.07
-3.80 ( 2.60
-16.98 ( 2.53
-19.97 ( 2.54
-11.11 ( 3.77
-12.86 ( 2.89
-7.25 ( 2.83
1
1
2
a
Nonbonded electrostatic energy. b Nonbonded van der Waals energy. c The molecular mechanical energy ()Eele + EvdW). d Hydrophobic contribution to
e
the solvation free energy, which is determined with solvent accessible surface area terms. Electrostatic contribution to the solvation free energy, which is
calculated with the generalized Born method. f The solvation free energy ()Gbsur + Gb). g The binding free energy()Gbsol + EMM).
the 6 compounds preferentially blocked IK rather than IA with
a ratio of IC50(IA)/IC50(IK) ranging from 1.9 to 16.3 (Figure 3
and Table 2). In addition, the interaction features of these
compounds with the K channel were mapped (Figure 5 and
Table S4 in the Supporting Information) and provide clear clues
for further structural modification for the six compounds. The
Yields were not optimized. Melting points were measured in a
capillary tube and were uncorrected. Infrared (IR) spectra were
determined using a Bruker IFS-48 FT-IR spectrometer. Solids were
prepared with potassium bromide (KBr) disks, and the results were
+
-
1
reported in wavenumbers (cm ). Nuclear magnetic resonance
NMR) spectra were determined using a Bruker AMX-400 spec-
trometer referenced to Me Si. Chemical shifts are reported in parts
(
4
calculated binding free energies of the six compounds to the
per million (ppm, downfield from tetramethylsilane. Splitting
patterns are described as singlet (s), doublet (d), triplet (t), quartet
+
K
channel are in agreement with their inhibitory potency
(Figure 6). The hydrogen bonding and solvation penalty play
(q), multiplet (m), and broad (br). Low- and high-resolution mass
important roles for the binding affinity between the compounds
and the channel. The strategy used in this paper provides a mode
spectra (LRMS and HRMS) were obtained by electric or chemical
ionization (ESI) produced by a MAT-95 spectrometer.
+
for quickly discovering new K channel blockers from large
1-(2,3-Dihydro-1,4-benzodioxin-6-yl)-2-phenoxyethylamine Hy-
+
databases, especially when the HTS of K channel is current
drochloride (6). A solution of phenol sodium salt (1.74 g, 0.015
mol) in THF (20 mL) was added dropwise to a solution of 2-bromo-
not available.
1
-(2,3-dihydro-1,4-benzodioxin-6-yl)ethanone (2.57 g, 0.01 mol)
5
. Experimental Section
in THF (15 mL) and continually refluxed for 3 h with constant
stirring. Thereafter, water (20 mL) was added and stirred at room
temperature for 15 min and then extracted with dichloromethane
The reagents (chemicals) were purchased from Shanghai Chemi-
cal Reagent Company and were used without further purification.

Potassium Channel Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 91
dioxane (25 mL) and then refluxed for 3 h, quenched with ice-
water (120 mL), and extracted with dichloromethane (3 × 20 mL).
2 4
The organic layer was dried over anhydrous Na SO and condensed.
After flash chromatography with ethyl acetate-petroleum (1:10;
v/v), bis[2-(2-chloroethoxy)ethyl] ether (24) was obtained as
yellowish oil (1.27 g), yield 55.0%.
A mixture of 24 (0.231 g, 1.0 mmol), toluene-4-sulfonamide
2 3
(0.428 g, 2.5 mmol), K CO (0.345 g, 2.5 mmol), and KI (0.023
g) in DMF (3 mL) was stirred for 24 h at 100 °C, then quenched
with ice-water, extracted with dichloromethane (3 × 10 mL), dried
2 4
under anhydrous Na SO , and condensed. After flash chromatog-
raphy with ethyl acetate-petroleum (1:3, v/v), compound 8 was
obtained as a white solid (0.388 g), yield 77.6%. IR (KBr): 1596,
-
1 1
1
(
480 cm . H NMR (CDCl
3
): δ 2.30 (s, 2H), 2.38 (s, 6H), 3.48
t, 4H), 3.53 (t, 12H), 7.25 (m, 4H), 7.72 (m, 4H). HRMS (SCI)
+
m/z calcd for M , 500.1651; found, 500.1682. Anal. (C22
Calcd, C 52.78, H 6.44, N 5.60; found, C 52.80, H 6.43, N 5.58.
-Benzyl-4-{2-[(4-chlorophenyl)sulfonyl]ethyl}piperazine (18).
The solution of SOCl (0.354 g, 3.0 mmol) in toluene (10 mL)
32 2 2 7
H N S O ).
1
2
was added dropwise to a solution of 2-[(4-chlorophenyl)sulfonyl]-
ethanol (0.220 g, 1.0 mmol) and 10 drops of pyridine in toluene
(
3
15 mL) in an ice-water bath. Then the mixture was refluxed for
h, then ice-water (10 mL) was added, and it was extracted with
SO , and
dichloromethane (3 × 10 mL), dried under anhydrous Na
2
4
condensed. After flash chromatography with ethyl acetate-
petroleum (1:20, v/v), 2-chloro[(4-chlorophenyl)sulfonyl]ethane (25)
was obtained as a pale oil, 0.21 g, yield 87.5%.
A mixture of 25 (0.238 g, 1 mmol), 1-benzylpiperazine (0.176
g, 1 mmol), K
20 mL) was refluxed for 3 h with constant stirring and condensed.
Then water (20 mL) was added and extracted with dichloromethane
3 × 15 mL). The final organic layer was dried under anhydrous
Na SO
2 3 2
CO (0.207 g, 1.5 mmol), and I (0.02 g) in THF
(
(
Figure 6. (A) Correlation between the calculated binding free energies
2
4
. After flash chromatography with ethyl acetate-petroleum
(GBtotal, kcal/mol, T ) 300 K) of the six compounds and the
(
(
(
2
2
3
1:6, v/v), 1-benzyl-4-{2-[(4-chlorophenyl)sulfonyl]ethyl}piperazine
experimental activities (log IC50). (B) Correlation between van de Waals
energies (EvdW, kcal/mol, T ) 300 K) of the six compounds and the
experimental activities (log IC50).
18) was obtained as a pale semisolid, 0.312 g, yield 82.3%. IR
-
1 1
KBr): 3448, 1579, 1479 cm . H NMR (CDCl
3
): δ 2.62 (t, 8H),
.55 (t, 4H), 3.75 (t, 4H), 3.84 (s, 2H), 7.25 (m, 5H), 7.50 (m,
+
H), 7.83 (m, 2H). HRMS (SCI) m/z calcd for M , 378.1169; found,
78.1182. Anal. (C19 S). Calcd, C 60.23, H 6.12, N 7.39;
H
2 2
23ClN O
(
3 × 20 mL). The final organic layer was dried over anhydrous
found, C 60.23, H 6.15, N 7.39.
N-(2-Aminoethyl)-5-chloronaphthalene-1-sulfonamide (19).
The powder of 5-chloronaphthalene-1-sulfonic acid (2.42 g, 10
Na SO , and the solvent was removed by evaporation under
2
4
vacuum. After flash chromatography with dichloromethane-
petroleum (2:1; v/v), 1-(2,3-dihydro-1,4-benzodioxin-6-yl)-2-phe-
noxyethanone (21) was obtained as a white semisolid (1.86 g), yield
mmol) and PCl (12.1 g) was mixed and heated to 80-90 °C and
5
stirred for 1 h. Then the mixture was stirred at room temperature
68.6%.
overnight. After flash chromatography with ethyl acetate-petroleum
A mixture of 21 (1.35 g, 5 mmol) and hydroxylamine hydro-
(1:8, v/v), 5-chloronaphthalene-1-sulfonyl chloride (26) was gained
chloride (0.70 g, 10 mmol) in ethanol (20 mL) was refluxed for 2
h with constant stirring and then condensed. The crude 1-(2,3-
dihydro-1,4-benzodioxin-6-yl)-2-phenoxyethanone oxime (22) was
obtained as a pale semisolid (1.81 g), yield 88.5%.
as a yellowish oil, 1.6 g, yield 61.5%.
A solution of 1,2-diaminoethane (0.072 g, 1.2 mmol) in THF
(30 mL) was added dropwise to a mixture of 26 (0.26 g, 1 mmol),
2 3
30 mL of THF, K CO (1.3 g) in an ice-water bath, then stirred
To the solution of the crude 22 (1.0 g, 3 mmol) in THF (10
mL), LiAlH
4
(0.56 g 15 mmol) was added in portions. Thereafter,
at room temperature for 1 h and condensed. After flash chroma-
tography with dichloromethane-ethanol (10:1, v/v), 19 was
obtained as a yellow oil (178.4 mg), yield 82.2%. IR (KBr): 1605
the mixture was stirred for 3 h, then ice-water (10 mL) was added
and extracted with dichloromethane (3 × 20 mL). The final organic
-
1 1
layer was dried under anhydrous Na
2
SO
4
and condensed. After flash
cm . H NMR (CDCl
3
): δ 1.60 (s, 2H), 2.35 (t, 2H), 6.85 (s,
chromatography with ethyl acetate-petroleum (1:4; v/v), 1-(2,3-
dihydro-1,4-benzodioxin-6-yl)-2-phenoxyethylamine (23) was ob-
tained as a pale oil (0.52 g), yield 40.6%. A solution of 10%
hydrochloric acid (2 mL) was added dropwise to a cold solution
1H), 7.50 (d, 2H), 8.35 (d, 2H), 8.45 (m, 2H). HRMS (SCI) m/z
+
2 2
calcd for M , 284.0386; found, 284.0373. Anal. (C12H13ClN SO ).
Calcd, C 44.87, H 4.39, N 8.72; found, C 44.76, H 4.71, N 8.44.
(
0-5 °C) of 23 (0.256 g, 1 mmol) in dichloromethane (5 mL).
Acknowledgment. The authors thank Professor I. D. Kuntz
and Professor Arthur J. Olson for their kindness in offering the
DOCK 4.0 and AutoDock 3.0.3 programs. We gratefully
acknowledge financial support from the State Key Program of
Basic Research of China (Grant 2002CB512802), the National
Natural Science Foundation of China (Grants 29725203,
After stirring for 15 min, the salt was filtered and recrystallized in
1
0
cm . H NMR (CDCl ): δ 3.98 (t, 1H), 4.05 (q, 2H), 6.92 (m,
3
6
2
0% hydrochloric acid. Compound 6 was obtained as a pale solid,
.24 g (82.1%), mp 236-238 °C. IR (KBr): 3425, 1589, 1498
-
1 1
+
H), 7.27 (m, 2H). HRMS (SCI) m/z calcd for M , 271.1208; found,
71.1198. Anal. (C16 ‚HCl). Calcd, C 62.44, H 5.89, N 4.55;
H17NO
3
20472094, and 30472086), the Basic Research Project for Talent
found, C 62.40, H 5.61, N 4.52.
,11-Di{[(4-methylphenyl)sulfonyl]amino}-3,6,9-trioxaunde-
cane (8). A solution of SOCl (0.708 g, 6.0 mmol) in 1,4-dioxane
5 mL) was added slowly to a cold solution (0-5 °C) of
tetraethylene glycol (1.94 g, 10 mmol) and pyridine (1 mL) in 1,4-
Research Group from the Science and Technology Commission
of Shanghai, the Key Project from the Science and Technology
Commission of Shanghai (Grants 02DJ14006 and 03ZR14110),
and the Key Project for New Drug Research from CAS.
1
2
(

9
2
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1
Liu et al.
Supporting Information Available: Figures S1-S3 showing
(22) Wang, J. L.; Liu, D. X.; Zhang, Z. J.; Shan, S.; Han, X. B.;
Srinivasula, S. M.; Croce, C. M.; Alnemri, E. S.; Huang, Z. W.
Structure-based discovery of an organic compound that binds Bcl-2
protein and induces apoptosis of tumor cells. Proc. Natl. Acad. Sci.
U.S.A. 2000, 97, 7124-7129.
+
the sequence alignment of the KcsA and Shaker K channel pore
regions, the structure superposition of the 3D model of eukaryotic
+
+
Shaker K channel with the X-ray crystal structure of KcsA K
+
channel, and the log P value distributions of the K channel
(
23) Liu, H.; Li, Y.; Song, M.; Tan, X.; Cheng, F.; Zheng, S.; Shen, J.;
Luo, X.; Ji, R.; Yue, J.; Hu, G.; Jiang, H.; Chen, K. Structure-based
discovery of potassium channel blockers from natural products:
virtual screening and electrophysiological assay testing. Chem. Biol.
2003, 10, 1103-1113.
inhibitors in the top-300 compounds picked from the ACD database;
1
Tables S1-S4 listing the IR, H NMR, and MS spectral data,
element analysis results, HPLC analysis results, the effects of the
selected 20 compounds on the voltage-dependent K+ currents in
rat hippocampal neurons, and the hydrogen-bonding pairs and the
(24) AVailable Chemicals Directory Compound Database; MDL Informa-
+
hydrophobic contacts of compounds 5, 6, 8, 18-20 with the K
tion Systems, Inc., 2003 (http://www.mdl.com).
(
25) (a) Ewing, T.; Kuntz, I. D. Critical evaluation of search algorithms
for automated molecular docking and database screening. J. Comput.
Chem. 1997, 18, 1175-1189. (b) Kuntz, I. D. Structure-based
strategies for drug design discovery. Science 1992, 257, 1078-1082.
26) Zheng, S.; Luo, X.; Chen, G.; Zhu, W.; Shen, J.; Chen, K.; Jiang, H.
A new rapid and effective chemistry space filter in recognizing a
druglike database J. Chem. Inf. Model. 2005, 45, 856-862.
channel derived from AutoDock simulations. This material is
available free of charge via the Internet at http://pubs.acs.org.
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