Supporting Information
Crystal structure-based virtual screening for fragment-like ligands of the human histamine H1 receptor Chris de Graaf1*, Albert J. Kooistra1*, Henry F. Vischer1, Vsevolod Katritch2, Martien Kuijer1, Mitsunori Shiroishi3,4, So Iwata3,5,6,7, Tatsuro Shimamura3,8, Raymond C. Stevens2, Iwan J.P. de Esch1, Rob Leurs1#
1
Leiden/Amsterdam Center for Drug Research (LACDR), Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands 2
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, GAC-1200, La Jolla, CA 92037 USA 3
Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan 4
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan 5
Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK 6
Diamond: Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK. 7
Kyoto: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan.
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8
Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501
*
These authors contributed equally to this work
#
Corresponding author; phone: +31 20 5987600; fax: +31 20 5987610; e-mail:
[email protected] S2
Supporting information: Table of contents Supplementary figure S1. PLANTS/IFP score distribution Supplementary figure S2. Kernel density of PLANTS/IFP scores for the ChEMBLdb actives Supplementary figure S3. Venn diagram illustrating the compound selection Supplementary figure S4. SCA-plot of the ECFP-4 and ROCS Comboscore Supplementary figure S5. H1R radioligand displacement curves Supplementary figure S6. InsP accumulation barplot Supplementary figure S7. H3R radioligand displacement curves Supplementary figure S8. H4R radioligand displacement curves Supplementary Table 1. The distribution of physicochemical properties Supplementary Table 2. Known H1R active compounds within the top selection Supplementary Table 3. Histamine H3 (H3R) and H4 (H4R) receptor binding affinities Supplementary Table 4. Supplier information for each of the validated compounds Supplementary Table 5. Purity data for each of the validated compounds as measured by LC-MS.
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scores (B) for known active compounds, the decoys as well as the selected compounds.
PLANTS−score distribution
0.04
0.05
0.06
CNS actives ChEMBLdb Selected Fragment Library Decoys
0.01
4
0.02
6
Density
8
10
12
CNS actives ChEMBLdb Selected Fragment Library Decoys
0.00
2
Density
B
IFP−score distribution
0.03
A
0
.%S1%
Supplementary figure S1. The distribution of the PLANTS-scores (A) and IFP-
0.3
0.4
0.5
0.6
0.7
IFP−score
0.8
0.9
1.0
−140 −120 −100
−80
−60
−40
PLANTS−score
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−20
0
Supplementary Figure S2. The kernel density in a contour plot corresponding to the scatterplot of the PLANTS scores versus the IFP scores for known actives from the ChEMBLdb.
Kernel density −40 −60
−60
−80
−80
−100
−100
−120
PLANTS
0.10 −40
−120
0.08
0.06
0.04
0.02
0.00 0.3
0.4 0.40.5 0.50.60.6 0.7 0.3 0.7 0.8 0.8
0.9 1.0 1.0 0.9
IFP
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Supplementary Figure S3. The number of compounds present at each step in the virtual screening workflow (Figure 1C) and overlap between these compound sets is reported in a Venn diagram.
Fragment)library) 108790& D3.32)Filter) 95.147&
PLANTS)≤)D90) Model)cutoff)) 6416&
611&
IFP)≥)0.75) Model)cutoff)) 2274&
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Supplementary Figure S4. Scatter plot of ECFP-4 Tanimoto similarity vs. ROCS Comboscore for the active hits (blue) and all other fragments (gray). The horizontal dotted line indicates the cutoff of 1.4 for the ROCS ComboScore as proposed by Blum et al.1 The vertical dotted lines indicate the cutoff of 0.26 and 0.40 for ECFP-4 as proposed by Steffen et al.2 and Wawer et al.3 respectively.
0.5
Doxepin similarity Fragments Selected
0.3 0.2
●
●
0.1
●
●
●
● ●● ●● ● ● ● ●
●
● ●
●
●
0.0
ECFP−4
0.4
●
0.4
0.6
0.8
1.0
1.2
1.4
1.6
ROCS
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log [ligand] M
log [ligand] M
80 60 40 20 0
2 10 11 12 13 14
3
100
[ H]-mepyramine specific binding (%)
3
[ H]-mepyramine specific binding (%)
Supplementary Figure S5. H1R radioligand displacement binding curves 100 80 60 40 20 0
2 15 16 17 18 19
-12-11-10 -9 -8 -7 -6 -5 -4
-12-11-10 -9 -8 -7 -6 -5 -4
log [ligand] M
log [ligand] M
suppl
1) doxepine 2) mepyramine 3) VUF13816 4) VUF13810 5) VUF13806 6) VUF13803 7) VUF13798 8) VUF13795 9) VUF13794
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binding curves H3R & H4R 3 [3H]-InsP accumulation (dpm) N [methylH]-histamine 3 N [methylH]-histamine % specific binding % specific binding
FIGURE 2x: 10 uM ligand for 1 hr on 293T H1R => InsP (pooled n=2) 100
[3H]-histamine [ H]-histamine % specific binding % specific binding
100
Supplementary Figure S6. Barplot of the inositol phosphates accumulation (agonist80 80 100 100 mode) 6080 4000 4060 3000 10002040
750
6080
His Imitit His 13 Imetit 15 13 14
020
0 -12-11-10 -9 -8 -7 -6 -5 -4
500
0
log [ligand] M
250
0
2040
3
020
4060
His 4 His 18 4 JNJ7777120 18 JNJ7777120
-12-11-10 -9 -8 -7 -6 -5 -4
log [ligand] M
-12-11-10 -9 -8 -7 -6 -5 -4
-12-11-10 -9 -8 -7 -6 -5 -4
log [ligand] M
log [ligand] M
BF HA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
N [methyl- H]-histamine 3 3 [ H]-histamine [3H]-InsP accumulationN(dpm) H]-histamine [methyl% specific binding 3 [% H]-histamine specific binding % specific binding % specific binding
Supplementary Figure S7. H3R radioligand displacement curves 100 100 80
3
suppl60FIGURE: 80 20 uM ligandHis for 15 min preincub 4060 then 10 uM ligand Imitit + 0.1 uM histamine for 1 hr on 293T H1R => InsP His 13Imetit 1513
2040
3500 020
3000 0 14 -12-11-10 -9 -8 -7 -6 -5 -4 2500 log [ligand] -12-11-10 -9 -8 -7M-6 -5 -4 2000 log [ligand] M 1500 1000
Supplementary Figure S8. H4R radioligand displacement curves 500
0 100 BF
BF
1
2
100 80
3
4
5
6
7
8
9
+ 0.1 µM histamine
6080 4060 2040
original files: 020
His 4 His 184 HV110817 293T H1 InsP SBVFS-1.xlsx JNJ7777120 18
HV110817 293T H1 InsP SBVFS-2.pzf 0 JNJ7777120 -12-11-10 -9 -8 -7 -6 -5 -4
log [ligand] -12-11-10 -9 -8 -7M-6 -5 -4 log [ligand] M
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Supplementary Table 1. The distribution of the physicochemical properties of the ChEMBLdb actives, the CNS actives, the decoy set and the fragment-screening library.
H-acceptors H-donors Heavy atoms Rings MW
H-acceptors H-donors Heavy atoms Rings MW
ChEMBLdb actives SD MIN MAX 1.33 1 6 0.97 0 4 6.54 13 48 1.15 1 7 92.82 195.31 682.85 Decoys AVG SD MIN MAX 1.85 0.53 0 3 1.04 0.29 0 3 22.78 2.30 14 30 3.00 0.69 2 5 328.16 20.96 294.35 399.58 AVG 3.49 0.81 27.76 3.60 387.19
AVG 1.22 0.36 24.90 3.69 351.31 AVG 1.66 0.59 17.97 2.38 258.64
CNS actives SD MIN 1.05 0 0.52 0 4.56 13 0.81 2 64.67 197.69 Fragments MIN MAX 0.00 6 0.00 6 6.00 22 1.00 6 83.11 506.06
MAX 4 2 35 5 478.59 SD 1 1 3 1 51.17
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Supplementary Table 2. Known active compounds found in the sub selection after the model and consistency cutoffs. Fragment
Known active
pKi
pIC50
ZINC00006157
Epinastine
8.85
-
ZINC00000504
Mianserin
8.38
-
ZINC00002227
Triprolidine
8.70
-
ZINC01249433
CHEMBL10602
5.29
-
ZINC01723265
CHEMBL609579
5.56
-
ZINC00010402
Promazine
-
7.9
ZINC00000931
Amoxapine
7.4
-
ZINC00020245
Imipramine
-
7.5
ZINC01530611
Desipramine
-
6.7
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Supplementary Table 3. Histamine H3 (H3R) and H4 (H4R) receptor binding affinities of validated fragment-like H1R hits. Compound 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
pKi H3Ra,b
pKi H4Ra,c
