Interactive Exploration of Chemical Space with Scaffold Hunter€¦ · Interactive Exploration of...

Interactive Exploration of Chemical Space with Scaffold Hunter

Stefan Wetzel, Karsten Klein, Steffen Renner, Daniel Rauh, Tudor I. Oprea, Petra Mutzel, and

Herbert Waldmann*

Dipl.-Chem. S. Wetzel, Dr. S. Renner, Prof. Dr. H. Waldmann

Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Str. 11, D –

44227 Dortmund, Germany and Chemical Biology, Technical University of Dortmund, D-

44227 Dortmund, Germany, Tel: +49 231 133 2400; Fax: +49 231 133 2499, E-mail:

[email protected]

Dipl.-Inform. K. Klein, Prof. Dr. P. Mutzel

Chair of Algorithm Engineering, Faculty of Computer Sciences, Technical University

Dortmund, Otto-Hahn-Str. 14, D-44227 Dortmund, Germany, Tel: +49 231 755 7700, E-mail:

[email protected]

Dr. Daniel Rauh, Chemical Genomics Centre of the Max-Planck Society, Otto-Hahn-Str. 15,

D-44227 Dortmund, Germany, [email protected]

Prof. Dr. Tudor I. Oprea, University of New Mexico School of Medicine, Division of

Biocomputing, MSC11 6145, Research Incubator Building, Suite 170, 2703 Frontier, NE, 1

University of New Mexico

Albuquerque, NM 87131, United States of America

Supplementary Methods

General: Compounds employed in the pyruvate kinase screen were purchased from ChemDiv

and Aurora Fine Chemicals Ltd. as 10mM solutions in DMSO.1, 2

Supplementary Movies: Supplementary Movies were generated using CamStudio 2.03, with

the Lagrith lossless codec 1.3.15.4 Post processing was done with Adobe Premiere Pro CS3.5

Nature Chemical Biology: doi:10.1038/nchembio.187

Supplementary Movie 1 – Scaffold Hunter – A guided Tour

Supplementary Movie 2 – Exploring Virtual Scaffolds Data sources:

WOMBAT: WOMBAT is a database containing small molecules and their annotated biological

activity extracted from contemporary literature. The 2007.1 version used in this analysis

contains more than 186,000 molecules and 370,000 annotated bioactivities. More information

can be obtained from www.sunsetmolecular.com.

PubChem: PubChem is a database providing information on the biological activities of small

molecules. It is part of the NIH’s moelcular libraries part of the roadmap initiative aiming at

providing researchers with publicly available HTS data sets for biomedical research. As of

February 19th 2009 it contained more than 10.5 million unique chemical structures and over

1300 bioassays. For more information see: http://pubchem.ncbi.nlm.nih.gov/.

For our analysis we used the set of the Pyruvate Kinase Screen (assay id 361). AC50 and log

AC50 values are provided for all compounds. Inactive compounds have values in the range of

2.9 x 10-5 M to 6.7 x 10-3 M and -4.55 to -2.24, respectively.

UniProt: UniProt is a comprehensive web resource for protein data, e.g. sequences and

annotation.6 Its identifier is a well recognized standard to unambiguously identify proteins and

to link protein data from different sources. It can be found at: http://www.uniprot.org/.

Biochemical assays: Lactate dehydrogenase was obtained from USB Corporation, Cleveland,

USA. Pyruvate kinase from Bacillus stearothermophilus, ribose-5-phosphate (R5P), potassium

dihydrogen phosphate, potassium chloride, magnesium sulphate, imidazole and

phosphoenolpyruvic acid monopotassium salt (PEP) were bought from Sigma Aldrich.

Adenosine-5’-diphosphate disodium salt (ADP), � -nicotinamide adenine dinucleotide (NADH)

and DMSO were purchased from Serva Electrophoresis GmbH, Heidelberg, Germany. Sodium


pyruvate was purchased from Alfa Aesar GmbH, Germany. All measurements were conducted

in transparent 384 well small volume plates from Greiner with a total volume of 14 � L and

carried out in triplicate using a Tecan infinite M200 plate reader set to absorbance at 340 nm.

Plates were measured for 40 minutes. Pipetting was done using a Caliper Zymark Sciclone

ALH 500 pipetting robot. All measurements were evaluated using Microsoft Excel and IDBS

XLfit. The values reported are the average values and their standard deviation.

Pyruvate kinase assay: The lactate dehydrogenase (LDH) coupled pyruvate kinase assay was

set up according to protocols from PubChem7, 8 and Sigma.9 Firstly, 1 � L of compound solution

in DMSO was dissolved in 49 � L of a solution containing all components except the pyruvate

kinase substrate PEP. Of this mix, 4x7 � L were transferred to the small volume measurement

plate. After incubation for 15 minutes at 30°C, 7 � L of PEP solution were added to each well

and the plate was measured in a continuous kinetics mode. Final assay concentrations were 50

mM imidazole (pH 7.2), 0.6 mM NADH, 0.4 mM ADP, 0.14 nM R5P, 50 mM potassium

chloride, 7mM magnesium sulphate, 0.01% Tween 20, 0.2 U LDH, 0.07 U pyruvate kinase,

0.05% BSA and 2 mM PEP.

The data was treated as described by Inglese et al.8 except for normalization. Activity data were

normalized to a negative control without PEP instead of a negative control with the pyruvate

kinase inhibitor luteolin.8 Compounds with less than 50% or more than 130% activity in the

pyruvate kinase assay were confirmed in a concentration dependent measurement using 11

concentrations with a 2-fold dilution starting at 100 � M. The IC50 values derived from these

experiments are reported together with their standard deviation for three independent

measurements.

Lactate dehydrogenase assay: To rule out false positives by inhibition of the LDH reporter

system, all compounds were screened for possible inhibition of LDH with a protocol adapted

from Sigma-Aldrich.10 For this purpose, 1 � L of compound solution in DMSO was dissolved in


49 � L of a solution containing all components except pyruvate. Of this mix, 4x7 � L were

transferred to the small volume measurement plate. After incubation for 15 minutes at 37°C,

7 � L of pyruvate solution were added to each well. Final concentrations in this assay were

38 mM potassium dihydrogen phosphate pH 7.6, 0.9 mM NADH, 0.01% Tween 20, 0.002 U

LDH, 0.05% BSA and 1.2 mM sodium pyruvate.

Database generation and tree construction

“Scaffold Tree Generator“, a separate program, generates the scaffold hierarchy data structure

and database in one step from a standard SD file format. The graphical user interface makes

database generation an easy task, such that Scaffold Hunter can be efficiently and reliably run

also by users with non-expert cheminformatic knowledge. The SDF format is a standard open

file format supported by most chemical drawing programs including ChemDraw (ChemDraw

2006, CambridgeSoft Corporation,www.cambridgesoft.com) and ISISDraw (IsisDraw 2.5,

MDL Information Systems Inc.,www.mdl.com) and used by a wide variety of databases, e.g.,

PubChem and compound vendor catalogues. The process of the database generation is depicted

in Supplementary Figure 1.


Supplementary Figure 1: Visualization of the scaffold tree database generation. After scaffold

isolation, all possible parent scaffolds are generated and one parent-child pair is selected. If this

parent scaffold can be deconstructed further, successive pairs are formed using the generated

parent scaffold as a new child, and the resulting branch is generated. Combination of all

branches yields the scaffold tree, which is written to a database. For exploration of gaps in

chemical space, Scaffold Hunter reads the scaffolds from the database that pass the initial user-

defined filter and displays the tree resulting from them. Annotation of bioactivity, for example,

by color shading, identifies promising virtual scaffolds shown in gray.

Scaffold Hunter first reads the scaffold data from an SQL database and automatically constructs

and displays the tree. Chemical structures are mapped on the nodes which are stored in the

database as scalable vector graphics (SVG) images. The branches, which reflect the

substructure relationships between scaffolds, are represented by radial lines whereas concentric

circles in the background mark the hierarchy levels of the tree. Virtual scaffolds are colored in

grey.

Analysis of PubChem with annotation from WOMBAT to identify virtual scaffolds


The analysis of PubChem and WOMBAT (see the main text) yielded 60 different targets

present in both PubChem and in the WOMBAT database, corresponding to 199 individual

assays in PubChem (see Supplementary Table 1). Of these 199 assays, in 102 cases

concentration-dependent measurements were carried out and IC50, EC50 or GI50 values were

calculated for 46 different targets. These values represent the concentration at half-maximal

inhibition (IC50), at half-maximal response (EC50), and at half-maximal growth inhibition (GI50),

e.g. in cells. We further analyzed the scaffold tree comprising of all compounds screened on a

particular target (screened at one fixed concentration as well as concentration-dependent). We

then started from the scaffolds representing the active compounds as defined by PubChem and

selected their virtual parents, i.e. scaffolds that are not represent by compounds for which

screening data are available against this particular target in PubChem (see Supplementary Table

2).


Supplementary Figure 2: Representative example for a virtual scaffold from a Serotonin 5-

HT1a receptor screen in PubChem, the Murcko families and their corresponding compounds

found in the WOMBAT search. For a complete list of examples, please see Supplementary

Table 3.

Control screen of compounds based on virtual scaffolds from branches of the pyruvate kinase

tree mainly representing inactive compounds

For the control screen we selected and tested 88 compounds from branches where parent and

child scaffolds represent mainly inactive compounds, i.e. they exhibit a mean log AC50 > -2.5.

The 88 compounds selected represent six different scaffolds and are shown in the

Supplementary Table 7. A screen conducted at 100 � M compound concentration indeed

confirmed that even at this very high concentration in all cases more than 70% of the enzymatic

activity remained. Thus, these compounds are weak pyruvate kinase inhibitors at best.


Retrospective analysis of virtual scaffolds from PubChem screens Supplementary Table 1: PubChem Assays whose targets are also present in the WOMBAT11 database.

UniProt Accession Code

Uni Prot Name UniProt Description PubChem Assay ID

PubChem Assay Name

O14920 IKKB_HUMAN Inhibitor of nuclear factor kappa-B kinase subunit

beta 1282 In Vitro Kinase Assay Using Purified Enzyme IKK-beta

O75116 ROC2_HUMAN Rho-associated protein

kinase 2 604

Primary biochemical high-throughput screening assay for inhibitors of Rho kinase 2 (Rhok2)


kinase 2 644 Dose-response biochemical assay of inhibitors of Rho kinase 2 (Rock2)


kinase 2 791 Dose-response biochemical assay of Rho kinase 2 (Rock2) inhibitors

O95136 EDG5_HUMAN Sphingosine 1-phosphate

receptor 2 729

Primary Cell-Based High-Throughput Screening to Identify Agonists of the Sphingosine 1-phosphate receptor 2 (S1P2)


receptor 2 736

Primary Cell-Based High-Throughput Screening to Identify Antagonists of the Sphingosine 1-phosphate receptor 2 (S1P2)


receptor 2 851 Dose Response Cell Based Assay for Antagonists of the S1P2 Receptor


receptor 2 854 Dose Response Cell Based Assay for Agonists of the S1P2 Receptor


receptor 2 872

Dose Response Cell Based Assay for Agonists of the S1P2 Receptor of Purchased Analogues


receptor 2 1232

Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 2 (S1P2): Purchased Analogues

O95477 ABCA1_HUMAN ATP-binding cassette sub-

family B member 1 377 MDR-1

O96013 PAK4_HUMAN Serine/threonine-protein

kinase PAK 4 389 Compound Screen Assay, Human PAK4

P00352 DHA1_HUMAN Retinal dehydrogenase 1 1030 Concentration-Response Assay for Inhibitors of aldehyde dehydrogenase 1 (ALDH1A1)

P00533 EGFR_HUMAN Epidermal growth factor

receptor 578

Literature data for small-molecule inhibitors of EGF-R_Tyrosine _Kinase_Homo_sapiens

P00734 THRB_HUMAN Prothrombin 1046 Thrombin 1536 HTS

P00734 THRB_HUMAN Prothrombin 1215 Thrombin 1536 HTS Dose Response Confirmation

P00748 FA12_HUMAN Coagulation factor XII 684 Factor XIIa Mixture HTS

P00748 FA12_HUMAN Coagulation factor XII 701 Factor XIIa Single Well HTS

P00748 FA12_HUMAN Coagulation factor XII 716 Factor XIIa Dose Response Confirmation




PubChem Assay Name

P00748 FA12_HUMAN Coagulation factor XII 728 Factor XIIa Dose Response Confirmation from Single Well HTS

P00748 FA12_HUMAN Coagulation factor XII 800 Factor XIIa 1536 HTS

P00748 FA12_HUMAN Coagulation factor XII 852 Factor XIIa 1536 HTS Dose Response Confirmation

P01112 RASH_HUMAN GTPase HRas 759 HTS to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtype

P01112 RASH_HUMAN GTPase HRas 1335 Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras wildtype

P01112 RASH_HUMAN GTPase HRas 1341 Multiplexed dose response to identify specific small molecule inhibitors of Ras and Ras-related GTPases specifically Ras activated mutant

P03372 ESR1_HUMAN Estrogen receptor 629 HTS of Estrogen Receptor- alpha Coactivator Binding inhibitors

P03372 ESR1_HUMAN Estrogen receptor 639 HTS of Estrogen Receptor- alpha Coactivator Binding Potentiators

P03372 ESR1_HUMAN Estrogen receptor 694 HTS of LOPAC library for Estrogen Receptor-alpha Coactivator Binding inhibitors

P03372 ESR1_HUMAN Estrogen receptor 713 Estrogen Receptor-alpha Coactivator Binding Inhibitors Dose Response Confirmation

P03372 ESR1_HUMAN Estrogen receptor 737 Estrogen Receptor-alpha Coactivator Binding Potentiators Dose Response Confirmation

P03372 ESR1_HUMAN Estrogen receptor 1078 Estrogen Receptor-alpha Coactivator Binding Inhibitors Reporter Gene Dose Response

P03372 ESR1_HUMAN Estrogen receptor 1079 Estrogen Receptor-alpha Coactivator Binding Inhibitors ELISA Secondary Assay

P03372 ESR1_HUMAN Estrogen receptor 1211 Estrogen Receptor (alpha) binding: Primary Screen

P03372 ESR1_HUMAN Estrogen receptor 1223 Estrogen Receptor (alpha) binding: Dose Response of Primary Screen Assay

P03468 NRAM_IAPUE Neuraminidase 493 Literature data for small-molecule inhibitors of Influenza_A_virus_(A_Puerto_Rico_8_34(H1N1))

P03474 NRAM_INBLE Neuraminidase 489 Literature data for small-molecule inhibitors of Influenza_B_Virus_(B_Lee_40)

P03951 FA11_HUMAN Coagulation factor XI 679 Factor XIa Dose Response Confirmation

P03951 FA11_HUMAN Coagulation factor XI 680 Factor XIa Mixture HTS

P03951 FA11_HUMAN Coagulation factor XI 687 Factor XIa Single Well HTS

P03951 FA11_HUMAN Coagulation factor XI 721 Factor XIa Dose Response Confirmation from Single Well Screen

P03951 FA11_HUMAN Coagulation factor XI 798 Factor XIa 1536 HTS

P03951 FA11_HUMAN Coagulation factor XI 846 Factor XIa 1536 HTS Dose Response Confirmation

P05067 A4_HUMAN Amyloid beta A4 protein 956 Beta amyloid ThS binding assay

P05771 KPCB_HUMAN Protein kinase C beta type 1280 In Vitro Kinase Assay Using Purified Enzyme PKC-beta

P06280 AGAL_HUMAN Alpha-galactosidase A 992 Counterscreen for Glucocerebrosidase Inhibitors: qHTS Assay for Human alpha-Galactosidase at pH 4.5




PubChem Assay Name

P06820 NRAM_IATOK Neuraminidase 490 Literature data for small-molecule inhibitors of Influenza_A_virus_(A_Tokyo_3_67(H2N2))

P07711 CATL_HUMAN cathepsin L1 preproprotein 460 Cathepsin L

P07711 CATL_HUMAN cathepsin L1 preproprotein 825 Cathepsin L dose-response confirmation

P07858 CATB_HUMAN Cathepsin B 453 Cathepsin B

P07858 CATB_HUMAN Cathepsin B 488 Cathepsin B compound mixture screening

P07858 CATB_HUMAN Cathepsin B 523 Cathepsin B Inhibitor Series SAR Study

P07858 CATB_HUMAN Cathepsin B 820 Cathepsin B dose-response confirmation

P07858 CATB_HUMAN Cathepsin B 830 Cathepsin B mixture HTS dose-response confirmation

P08311 CATG_HUMAN Cathepsin G 581 Cathepsin G

P08311 CATG_HUMAN Cathepsin G 832 Cathepsin G dose-response confirmation

P08908 5HT1A_HUMAN 5-hydroxytryptamine

receptor 1A 567

Primary HTS assay for 5-Hydroxytryptamine (Serotonin) Receptor Subtype 1a (5HT1a) agonists


receptor 1A 612

Primary HTS assay for 5-Hydroxytryptamine (Serotonin) Receptor Subtype 1a (5HT1a) antagonists


receptor 1A 613

Dose Response assay for 5-Hydroxytryptamine (Serotonin) Receptor Subtype 1a (5HT1a) agonists


receptor 1A 718

Dose Response assay for agonists of 5-Hydroxytryptamine (Serotonin) Receptor Subtype 1A (5HT1A)


receptor 1A 755

Dose Response Cell Based Assay for Antagonists of the 5-Hydroxytryptamine Receptor Subtype 1A (5HT1A)

P09871 C1S_HUMAN Complement C1s subcomponent

538 Complement factor C1s


787 Complement factor C1s IC50 from mixture screen


829 Complement C1s ELISA

P10415 BCL2_HUMAN Apoptosis regulator Bcl-2 950 Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2.

P10415 BCL2_HUMAN Apoptosis regulator Bcl-2 1328 Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Bcl-2

P10635 CPD6_HUMAN Cytochrome P450 2D6 891 qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2D6

P11229 ACM1_HUMAN Muscarinic acetylcholine

receptor M1 626

Discovery of Novel Allosteric Modulators of the M1 Muscarinic Receptor: Agonist Primary Screen


receptor M1 628

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Primary Screen


receptor M1 677

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Confirmation Screen




PubChem Assay Name


receptor M1 678

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Secondary Assay 1


receptor M1 859

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Assay


receptor M1 860

Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Counterscreen


receptor M1 943

qHTS Assay for Antagonists of Acetylcholine Muscarinic M1 Receptor: Kinetic Measurement of Intracellular Calcium Response


receptor M1 944

qHTS Assay for Antagonists of Acetylcholine Muscarinic M1 Receptor: Measurement of IP-One Response

P11309 PIM1_HUMAN Proto-oncogene

serine/threonine-protein kinase Pim-1

393 Compound Screen Assay, Human PIM1

P11712 CPC9_HUMAN Cytochrome P450 2C9 883 qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2C9

P12931 SRC_HUMAN Proto-oncogene tyrosine-

protein kinase Src 363 Literature data for small-molecule inhibitors of Human Src.

P13612 ITA4_HUMAN Integrin alpha-4 528 Allosteric Agonists for the VLA-4 Integrin

P13612 ITA4_HUMAN Integrin alpha-4 529 Allosteric Antagonists for the VLA-4 Integrin

P13612 ITA4_HUMAN Integrin alpha-4 576 Auto-fluorescence of compounds effecting screening of VLA-4 Integrin

P13612 ITA4_HUMAN Integrin alpha-4 702 Dose-Response of Allosteric Antagonists for the VLA-4 Integrin

P13612 ITA4_HUMAN Integrin alpha-4 703 Dose-Response of Allosteric Agonists for the VLA-4 Integrin

P15917 LEF_BACAN Lethal factor 912 qHTS Assay for Anthrax Lethal Toxin Internalization

P15917 LEF_BACAN Lethal factor 942 Confirmation Concentration-Response Assay for Anthrax Lethal Toxin Internalization

P16473 TSHR_HUMAN Thyrotropin receptor 926 qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor

P16473 TSHR_HUMAN Thyrotropin receptor 933 Secondary Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: HTRF Activity Detection

P16473 TSHR_HUMAN Thyrotropin receptor 938 qHTS Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293

P16473 TSHR_HUMAN Thyrotropin receptor 939 Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor

P16473 TSHR_HUMAN Thyrotropin receptor 953 Confirmation Concentration-Response Assay for Agonists of the Thyroid Stimulating Hormone Receptor: Activators of Intracellular cAMP Concentrations in Parental HEK 293

P17612 KAPA_HUMAN cAMP-dependent protein kinase catalytic subunit

alpha isoform 1 524

Primary biochemical high-throughput screening assay for inhibitors of protein kinase A (PKA) activity

P17612 KAPA_HUMAN cAMP-dependent protein 548 Dose-response biochemical assay for inhibitors of protein kinase A (PKA)




PubChem Assay Name

kinase catalytic subunit alpha isoform 1

activity

P19838 KBF1_HUMAN Nuclear factor NF-kappa-B

p105 subunit 895

qHTS Assay for Identification of Small Molecule Antagonists for NFkB Signaling Pathway

P19838 KBF1_HUMAN Nuclear factor NF-kappa-B

p105 subunit 928

qHTS Assay for Identification of Small Molecule Agonists for NFkB Signaling Pathway

P21453 EDG1_HUMAN sphingosine-1-phosphate

receptor 1 449

Primary HTS and Confirmation Assays for S1P1 Agonists and Agonism Potentiators

P21453 EDG1_HUMAN Sphingosine 1-phosphate

receptor 1 1044

Primary cell-based high-throughput screening assay to identify agonists of Sphingosine 1-Phosphate receptor 1 (S1P1)


receptor 1 1248



receptor 1 1363

Cell-membrane dose response assay to identify antagonists of the Sphingosine 1-Phosphate receptor 1 (S1P1)

P21462 FPR1_HUMAN formylpeptide receptor

(FPR) family 362 Formylpeptide Receptor Ligand Binding Assay


(FPR) family 441

Primary HTS Assay for Formylpeptide Receptor-Like-1 (FPRL1) Ligands and Primary HTS Counter-Screen Assay for Formylpeptide Receptor (FPR) Ligands

P21462 FPR1_HUMAN fMet-Leu-Phe receptor 519 Dose Response Assay for Formylpeptide Receptor (FPR) Ligands and Dose Response Counter-Screen Assay for Formylpeptide-Like-1 (FPRL1) Ligands


(FPR) family 699

Secondary Calcium Response Assay for Formylpeptide Receptor (FPR) Antagonists


(FPR) family 722

Formylpeptide Receptor (FPR) Ligand Structure Activity Relationship (SAR) Analysis : Primary HTS Assay


(FPR) family 724

Formylpeptide Receptor (FPR) Ligand Structure Activity Relationship (SAR) Analysis : Dose Response Assay


(FPR) family 762

Formylpeptide Receptor (FPRL1) Ligand Structure Activity Relationship (SAR) Analysis : FPR Dose Response Counterscreen Assay


(FPR) family 763

Formylpeptide Receptor-Like-1 (FPRL1) Ligand Structure Activity Relationship (SAR) Analysis : FPR Primary HTS Assay Counterscreen

P21462 FPR1_HUMAN fMet-Leu-Phe receptor 805 Assay for Formylpeptide Receptor Family Ligands: Target Formylpeptide Receptor


(FPR) family 863

Formylpeptide Receptor (FPR) Structure Activity Relationship (SAR) Analysis: Secondary Calcium Response Assay for Antagonists

P21728 DRD1_HUMAN D(1) dopamine receptor 641 Allosteric Modulators of D1 Receptors: Primary Screen

P21728 DRD1_HUMAN D(1) dopamine receptor 642 Allosteric Modulators of D1 Receptors: Confirmation Screen

P21728 DRD1_HUMAN D(1) dopamine receptor 646 Allosteric Modulators of D1 Receptors: Secondary Assay 1


P21918 DRD5_HUMAN D(1) dopamine receptor 641 Allosteric Modulators of D1 Receptors: Primary Screen




PubChem Assay Name

P21918 DRD5_HUMAN D(1) dopamine receptor 642 Allosteric Modulators of D1 Receptors: Confirmation Screen



P21918 DRD5_HUMAN D(1B) dopamine receptor 857 Allosteric Modulators of D1 Receptors: Dose-dependent Counterscreen

P21918 DRD5_HUMAN D(1B) dopamine receptor 858 Allosteric Modulators of D1 Receptors: Dose-dependent Assay

P25774 CATS_HUMAN cathepsin S 501 Cathepsin S

P25774 CATS_HUMAN cathepsin S 831 Cathepsin S dose-response confirmation

P28566 5HT1E_HUMAN 5-hydroxytryptamine

receptor 1E 571

Primary Cell Based High Throughput Screening Assay for Antagonists of the 5-Hydroxytryptamine Receptor Subtype 1E (5HT1E)


receptor 1E 574

Primary Cell Based High Throughput Screening Assay for Agonists of the 5-Hydroxytryptamine Receptor Subtype 1E (5HT1E)


receptor 1E 726

Dose Response Cell Based High Throughput Screening Assay for Agonists of the 5-Hydroxytryptamine Receptor Subtype 1E (5HT1E)


receptor 1E 749

Dose Response Cell Based Assay for Antagonists of the 5-Hydroxytryptamine Receptor Subtype 1E (5HT1E

P28845 DHI1_HUMAN 11-beta-hydroxysteroid

dehydrogenase 1 398 Compound Screen Assay, Human HSD11B1

P29466 I1BC_HUMAN Interleukin-1 beta

convertase 888

Concentration-Response Counterscreen for Redox Active Inhibitors of Caspase-1: Catalase


convertase 896

Confirmation Concentration-Response Assay for Allosteric/Competitive Inhibitors of Caspase-1


convertase 900 qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-1

P30305 MPI2_HUMAN cell division cycle 25B

isoform 2 368 Cdc25B Catalytic Domain protein tyrosine phosphatase HTS

P30305 MPI2_HUMAN M-phase inducer phosphatase 2

443 Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Dose Response Confirmation and Secondary Selectivity/Specificity Assay


isoform 2 569

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase HTS Dose Response Confirmation Assay


isoform 2 649

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in 25 mM DTT


isoform 2 650

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response Reproducibility Assay


isoform 2 668

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in the presence of Catalase.


isoform 2 669

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in 25 mM DTT and Catalase.


isoform 2 670

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in the presence of Beta-Mercaptoethanol.




PubChem Assay Name


isoform 2 671

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment Dose Response assay in the presence of Glutathione.


isoform 2 682

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment: Compound Dose Dependent Redox Cycling H2O2 Generation in 0.5 mM DTT


isoform 2 683

Cdc25B Catalytic Domain Protein Tyrosine Phosphatase Probe Assessment: Compound Dose Dependent Redox Cycling H2O2 Generation in 1.0 mM DTT

P33261 CPCJ_HUMAN Cytochrome P450 2C19 899 qHTS Assay for Inhibitors and Substrates of Cytochrome P450 2C19

P43116 PE2R2_HUMAN Prostaglandin E2 receptor

EP2 subtype 940 Modulators of the EP2 prostaglandin E2 receptor - Primary Screening

P43116 PE2R2_HUMAN Prostaglandin E2 receptor

EP2 subtype 1080 Modulators of the EP2 prostaglandin EP2 receptor - Primary Counterscreens

P43367 CAN2_PIG Calpain-2 catalytic subunit 478 Calpain activity in endothelial cells

P43367 CAN2_PIG Calpain-2 catalytic subunit 1236 uHTS for Calpain Inhibitors

P49146 NPY2R_HUMAN Neuropeptide Y receptor

type 2 1255

Counterscreen assay for antagonists of neuropeptide Y receptor Y1 (NPY-Y1): Cell-based high throughput assay to measure NPY-Y2 antagonism


type 2 1257

Cell-based high throughput confirmation assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)


type 2 1272

Dose response cell-based screening assay for antagonists of neuropeptide Y receptor Y2 (NPY-Y2)


type 2 1278

Dose response counterscreen assay for neuropeptide Y receptor Y1 (NPY-Y1): Cell-based high throughput assay to measure NPY-Y2 antagonism


type 2 1359

Primary cell-based high-throughput screening assay for potentiators or agonists of NPY-Y2

P49888 SUOE_HUMAN Estrogen sulfotransferase 747 Basic StarGazer Assay on Human SULT1E1

P53350 PLK1_HUMAN Serine/threonine-protein

kinase PLK1 619 Fluorescence polarization assay for PLK1 inhibitors


kinase PLK1 693

High throughput fluorescence polarization-based assay to screen for small molecule inhibitors of the Polo box domain (PBD) of Plk1.


kinase PLK1 744 Fluorescence polarization assay for PLK1 confirmation assay


kinase PLK1 756 Fluorescence Polarization assay for Plk1: compound interference assay


kinase PLK1 785 Fluorescence Polarization assay for Plk1: IC50 Dose Response Assay


kinase PLK1 876

Concentration Response Redox Cycling H2O2 Generation assay to characterize small molecule inhibitors identified in the Polo box domain (PBD) of Plk1 Primary HTS.


kinase PLK1 877

Concentration Response fluorescence polarization-based assay to confirm small molecule inhibitors identified in the Polo box domain (PBD) of Plk1




PubChem Assay Name

Primary HTS.


kinase PLK1 936

Concentration Response Redox Cycling H2O2 Generation assay, 1 mM DTT, to characterize small molecule inhibitors identified in the Polo box domain (PBD) of Plk1 Primary HTS.


kinase PLK1 1053

High throughput fluorescence polarization-based assay to screen for small molecule inhibitors of the Polo box domain (PBD) of Plk1 to follow up on purchased Analogs of Selected Hits from the screen.


kinase PLK1 1057

Concentration Response fluorescence polarization-based assay to test PMLSC Chemistry Core synthesized analogs of confirmed hits from the Polo box domain (PBD) of Plk1 Primary HTS.


kinase PLK1 1082

Concentration Response fluorescence polarization-based assay to test PMLSC Chemistry Core synthesized analogs of 861574, a confirmed hit from the Polo box domain (PBD) of Plk1 Primary HTS.


kinase PLK1 1083

Concentration Response fluorescence polarization-based assay to test purchased Analogs of Selected Hits from the Polo box domain (PBD) of Plk1 Primary HTS.


kinase PLK1 1264 Fluorescence polarization assay for PLK1 analogues - primary screen repeat


kinase PLK1 1383 PLK1 counterscreening assay for identified PKD inhibitors

P53779 MK10_HUMAN Mitogen-activated protein

kinase 10 530 JNK3 AlphaScreen Assay


kinase 10 746

Primary biochemical high-throughput screening assay for inhibitors of the c-Jun N-Terminal Kinase 3 (JNK3)


kinase 10 1284

Dose response biochemical screening assay for inhibitors of c-Jun N-Terminal Kinase 3 (JNK3)

P54760 EPHB4_HUMAN Ephrin type-B receptor 4 720 Primary biochemical high-throughput screening assay for antagonists of the interaction between the Eph receptor B4 (EphB4) and its ligand ephrin-B2 via TNYL-RAW peptide probe

P54760 EPHB4_HUMAN Ephrin type-B receptor 4 835 Dose-response biochemical assay for antagonists of the interaction between the Eph receptor B4 (EphB4) and its ligand ephrin-B2 via TNYL-RAW peptide

P55210 ICE7_HUMAN Caspase-7 889 qHTS Assay for Allosteric/Competitive Inhibitors of Caspase-7

P55210 ICE7_HUMAN Caspase-7 897 Confirmation Concentration-Response Assay for Allosteric/Competitive Inhibitors of Caspase-7

Q04759 KPCT_HUMAN Protein kinase C theta type 1283 In Vitro Kinase Assay Using Purified Enzyme PKC-theta

Q05397 FAK1_HUMAN Focal adhesion kinase 1 727 Primary biochemical high-throughput screening assay for inhibitors of Focal Adhesion Kinase (FAK)

Q05397 FAK1_HUMAN Focal adhesion kinase 1 794 Confirmation biochemical assay for inhibitors of Focal Adhesion Kinase (FAK)

Q05397 FAK1_HUMAN Focal adhesion kinase 1 810 Dose-response biochemical assay for inhibitors of Focal Adhesion Kinase




PubChem Assay Name

(FAK)

Q07820 MCL1_HUMAN Induced myeloid leukemia cell differentiation protein

Mcl-1 1009

Multiplexed high-throughput screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1


Mcl-1 1021 uHTS of Mcl-1/Bid interaction inhibitors


Mcl-1 1022 uHTS of Mcl-1/Noxa interaction inhibitors


Mcl-1 1329

Multiplexed dose response screen for small molecule regulators of Bcl-2 family protein interactions, specifically Bim-Mcl-1.

Q15139 KPCM_HUMAN Serine/threonine-protein

kinase D1 797 Fluorescence polarization assay for PKD inhibitors


kinase D1 1286

Fluorescence polarization assay for PKD inhibitors - repeat of primary screen on hits identified from 140K library primary screen


kinase D1 1315

Fluorescence polarization for PKD inhibitors - interference assay (140K library campaign)


kinase D1 1342

Fluorescence polarization assay for PKD inhibitiors-interference assay-57K HTS campaign


kinase D1 1378 AKT counterscreen of confirmed PKD inhibitors-57K library

Q92731 ESR2_HUMAN Estrogen receptor beta 633 HTS for Estrogen Receptor-beta Coactivator Binding inhibitors

Q92731 ESR2_HUMAN Estrogen receptor beta 733 Estrogen Receptor-beta Coactivator Binding Inhibitors Dose Response Confirmation

Q92731 ESR2_HUMAN Estrogen receptor beta 1060 Estrogen Receptor-beta Coactivator Binding Inhibitors ELISA Secondary Assay

Q92731 ESR2_HUMAN Estrogen receptor beta 1210 Estrogen Receptor (beta) binding: Primary Screen

Q92731 ESR2_HUMAN Estrogen receptor beta 1221 Estrogen Receptor (beta) binding: Dose Response of Primary Screen Assay

Q99500 EDG3_HUMAN sphingosine-1-phosphate

receptor 3 373 S1P3 Agonist Primary HTS and Confirmation Assays


receptor 3 439 S1P3 Agonist Dose-Response Potency Assay


receptor 3 484 Dose Response Assay for S1P3 Antagonists


receptor 3 485 Primary HTS Assay for S1P3 Antagonists

Q99500 EDG3_HUMAN sphingosine-1-phosphate 730 S1P3 Dose Response Assay Counterscreen for 5-




PubChem Assay Name

receptor 3 Hydroxytryptamine(Serotonin) Receptor Subtype 1E Agonists


receptor 3 776

S1P3 Dose Response Assay Counterscreen for 5-Hydroxytryptamine(Serotonin) Receptor Subtype 1E Antagonists


receptor 3 1192


Q99527 CML2_HUMAN G-protein coupled estrogen

receptor 1 1212 GPR 30 Binding: Primary Screen


receptor 1 1213 CALCIUM_SIGNALING_VIA_GPR30: Primary Screen


receptor 1 1224 GPR 30 Binding: Dose Response

Q9P1W9 PIM2_HUMAN Serine/threonine-protein

kinase Pim-2 505 Compound Screen Assay, Human PIM2


Supplementary Table 2: Statistics of the analyzed PubChem targets giving the number of compound screened in PubChem, the number of actives found

(exclusively in concentration dependant-manner) and the number of parent scaffolds that are virtual and non-virtual scaffolds in PubChem.

SwissProt ID

SwissProt Name SwissProt Short Description

Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

O75116 ROCK2_HUMAN Rho-associated protein kinase 2 (EC 2.7.11.1) (Rho-associated,

coiled-coil-containing protein kinase 2) (p164 ROCK-2) (Rho kinase 2) [Gene: ROCK2 or KIAA0619] - Homo sapiens (Human)

229 88 11 76

O95136 EDG5_HUMAN

Sphingosine 1-phosphate receptor Edg-5 (S1P receptor Edg-5) (Endothelial differentiation G-protein coupled receptor 5) (Sphingosine

1-phosphate receptor 2) (S1P2) [Gene: EDG5] - Homo sapiens (Human)

202 80 38 41

P00352 AL1A1_HUMAN

Retinal dehydrogenase 1 (EC 1.2.1.36) (RalDH1) (RALDH 1) (Aldehyde dehydrogenase family 1 member A1) (Aldehyde

dehydrogenase, cytosolic) (ALHDII) (ALDH-E1) [Gene: ALDH1A1 or ALDC or ALDH1 or PUMB1] - Homo sapiens (Human)

1238 488 263 131

P00734 THRB_HUMAN

Prothrombin precursor (EC 3.4.21.5) (Coagulation factor II) [Contains: Activation peptide fragment 1; Activation peptide fragment 2;

Thrombin light chain; Thrombin heavy chain] [Gene: F2] - Homo sapiens (Human)

529 235 31 200

P00748 FA12_HUMAN

Coagulation factor XII precursor (EC 3.4.21.38) (Hageman factor) (HAF) [Contains: Coagulation factor XIIa heavy chain; Beta-factor XIIa

part 1; Beta-factor XIIa part 2; Coagulation factor XIIa light chain] [Gene: F12] - Homo sapiens (Human)

774 224 25 196

P01112 RASH_HUMAN GTPase HRas precursor (Transforming protein p21) (p21ras) (H-Ras-1) (c-H-ras) (Ha-Ras) [Gene: HRAS or HRAS1] - Homo sapiens (Human)

634 58 17 39


SwissProt ID


Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

P03372 ESR1_HUMAN Estrogen receptor (ER) (Estradiol receptor) (ER-alpha) (Nuclear

receptor subfamily 3 group A member 1) [Gene: ESR1 or ESR or NR3A1] - Homo sapiens (Human)

577 296 123 172

P03468 NRAM_I34A1 Neuraminidase (EC 3.2.1.18) [Gene: NA] - Influenza A virus (strain

A/Puerto Rico/8/1934 H1N1) 53 52 14 5

P03474 NRAM_INBLE Neuraminidase (EC 3.2.1.18) [Gene: NA] - Influenza B virus (strain

B/Lee/1940) 96 80 24 10

P03951 FA11_HUMAN

Coagulation factor XI precursor (EC 3.4.21.27) (Plasma thromboplastin antecedent) (PTA) (FXI) [Contains: Coagulation factor XIa heavy

chain; Coagulation factor XIa light chain] [Gene: F11] - Homo sapiens (Human)

379 146 19 125

P06820 NRAM_I67A0 Neuraminidase (EC 3.2.1.18) [Gene: NA] - Influenza A virus (strain

A/Tokyo/3/1967 H2N2) 14 14 6 0

P07858 CATB_HUMAN Cathepsin B precursor (EC 3.4.22.1) (Cathepsin B1) (APP secretase) (APPS) [Contains: Cathepsin B light chain; Cathepsin B heavy chain]

[Gene: CTSB or CPSB] - Homo sapiens (Human) 107 96 27 64

P08311 CATG_HUMAN Cathepsin G precursor (EC 3.4.21.20) (CG) [Gene: CTSG] - Homo

sapiens (Human) 93 71 20 51

P08908 5HT1A_HUMAN 5-hydroxytryptamine receptor 1A (5-HT-1A) (Serotonin receptor 1A) (5-

HT1A) (G-21) [Gene: HTR1A or ADRB2RL1 or ADRBRL1] - Homo sapiens (Human)

431 49 16 32

P10415 BCL2_HUMAN Apoptosis regulator Bcl-2 [Gene: BCL2] - Homo sapiens (Human) 834 9 4 5

P10635 CP2D6_HUMAN Cytochrome P450 2D6 (EC 1.14.14.1) (CYPIID6) (P450-DB1)

(Debrisoquine 4-hydroxylase) [Gene: CYP2D6 or CYP2DL1] - Homo sapiens (Human)

9391 3444 1259 1996

P11229 ACM1_HUMAN Muscarinic acetylcholine receptor M1 [Gene: CHRM1] - Homo sapiens

(Human) 1238 1091 428 496


SwissProt ID


Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

P11712 CP2C9_HUMAN

Cytochrome P450 2C9 (EC 1.14.13.80) ((R)-limonene 6-monooxygenase) (EC 1.14.13.48) ((S)-limonene 6-monooxygenase) (EC 1.14.13.49) ((S)-limonene 7-monooxygenase) (CYPIIC9) (P450 PB-1) (P450 MP-4/MP-8) (S-mephenytoin 4-hydroxylase) (P-450MP)

[Gene: CYP2C9 or CYP2C10] - Homo sapiens (Human)

9391 2656 964 1554

P12931 SRC_HUMAN Proto-oncogene tyrosine-protein kinase Src (EC 2.7.10.2) (p60-Src) (c-

Src) (pp60c-src) [Gene: SRC or SRC1] - Homo sapiens (Human) 749 592 329 263

P13612 ITA4_HUMAN Integrin alpha-4 precursor (Integrin alpha-IV) (VLA-4) (CD49d antigen)

[Gene: ITGA4 or CD49D] - Homo sapiens (Human) 62 34 16 17

P15917 LEF_BACAN Lethal factor precursor (EC 3.4.24.83) (LF) (Anthrax lethal toxin

endopeptidase component) [Gene: lef or pXO1-107 or BXA0172 or GBAA_pXO1_0172] - Bacillus anthracis

68995 6274 1990 4028

P16473 TSHR_HUMAN Thyrotropin receptor precursor (TSH-R) (Thyroid-stimulating hormone

receptor) [Gene: TSHR or LGR3] - Homo sapiens (Human) 72086 27221 8214 17885

P17612 KAPCA_HUMAN cAMP-dependent protein kinase catalytic subunit alpha (EC 2.7.11.11) (PKA C-alpha) [Gene: PRKACA or PKACA] - Homo sapiens (Human)

94 62 21 40

P19838 NFKB1_HUMAN Nuclear factor NF-kappa-B p105 subunit (DNA-binding factor KBF1) (EBP-1) [Contains: Nuclear factor NF-kappa-B p50 subunit] [Gene:

NFKB1] - Homo sapiens (Human) 1238 986 524 278

P21453 EDG1_HUMAN Sphingosine 1-phosphate receptor Edg-1 (Sphingosine 1-phosphate

receptor 1) (S1P1) [Gene: EDG1] - Homo sapiens (Human) 11 6 0 4


SwissProt ID


Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

P21462 FPR1_HUMAN fMet-Leu-Phe receptor (fMLP receptor) (N-formyl peptide receptor) (FPR) (N-formylpeptide chemoattractant receptor) [Gene: FPR1] -

Homo sapiens (Human) 330 39 15 23

P21918 DRD5_HUMAN D(1B) dopamine receptor (D(5) dopamine receptor) (D1beta dopamine

receptor) [Gene: DRD5 or DRD1B or DRD1L2] - Homo sapiens (Human)

517 324 223 85

P25774 CATS_HUMAN Cathepsin S precursor (EC 3.4.22.27) [Gene: CTSS] - Homo sapiens

(Human) 129 34 12 21

P28566 5HT1E_HUMAN 5-hydroxytryptamine receptor 1E (5-HT-1E) (Serotonin receptor 1E) (5-

HT1E) (S31) [Gene: HTR1E] - Homo sapiens (Human) 95 51 15 36

P29466 CASP1_HUMAN

Caspase-1 precursor (EC 3.4.22.36) (CASP-1) (Interleukin-1 beta convertase) (IL-1BC) (IL-1 beta-converting enzyme) (ICE) (Interleukin-1

beta-converting enzyme) (p45) [Contains: Caspase-1 subunit p20; Caspase-1 subunit p10] [Gene: CASP1 or IL1BC or IL1BCE] - Homo

sapiens (Human)

73922 2892 896 1953

P30305 MPIP2_HUMAN M-phase inducer phosphatase 2 (EC 3.1.3.48) (Dual specificity phosphatase Cdc25B) [Gene: CDC25B or CDC25HU2] - Homo

sapiens (Human) 87 151 58 92

P33261 CP2CJ_HUMAN

Cytochrome P450 2C19 (EC 1.14.13.80) ((R)-limonene 6-monooxygenase) (EC 1.14.13.48) ((S)-limonene 6-monooxygenase)

(EC 1.14.13.49) ((S)-limonene 7-monooxygenase) (CYPIIC19) (P450-11A) (Mephenytoin 4-hydroxylase) (CYPIIC17) (P450-254C) [Gene:

CYP2C19] - Homo sapiens (Human)

9391 3190 1132 1888

P49146 NPY2R_HUMAN Neuropeptide Y receptor type 2 (NPY2-R) (NPY-Y2 receptor) [Gene:

NPY2R] - Homo sapiens (Human) 182 80 18 61


SwissProt ID


Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

P53350 PLK1_HUMAN Serine/threonine-protein kinase PLK1 (EC 2.7.11.21) (Polo-like kinase

1) (PLK-1) (Serine/threonine-protein kinase 13) (STPK13) [Gene: PLK1 or PLK] - Homo sapiens (Human)

366 141 38 100

P53779 MK10_HUMAN

Mitogen-activated protein kinase 10 (EC 2.7.11.24) (Stress-activated protein kinase JNK3) (c-Jun N-terminal kinase 3) (MAP kinase p49

3F12) [Gene: MAPK10 or JNK3 or JNK3A or PRKM10] - Homo sapiens (Human)

10378 1256 364 717

P54760 EPHB4_HUMAN Ephrin type-B receptor 4 precursor (EC 2.7.10.1) (Tyrosine-protein

kinase receptor HTK) (Tyrosine-protein kinase TYRO11) [Gene: EPHB4 or HTK or TYRO11] - Homo sapiens (Human)

128 5 2 3

P55210 CASP7_HUMAN

Caspase-7 precursor (EC 3.4.22.60) (CASP-7) (ICE-like apoptotic protease 3) (ICE-LAP3) (Apoptotic protease Mch-3) (CMH-1) [Contains:

Caspase-7 subunit p20; Caspase-7 subunit p11] [Gene: CASP7 or MCH3] - Homo sapiens (Human)

73834 2302 688 1593

Q05397 FAK1_HUMAN Focal adhesion kinase 1 (EC 2.7.10.2) (FADK 1) (pp125FAK) (Protein-

tyrosine kinase 2) [Gene: PTK2 or FAK or FAK1] - Homo sapiens (Human)

210 109 57 51

Q07820 MCL1_HUMAN Induced myeloid leukemia cell differentiation protein Mcl-1 (Bcl-2-

related protein EAT/mcl1) (mcl1/EAT) [Gene: MCL1] - Homo sapiens (Human)

834 3 2 1

Q15139 KPCD1_HUMAN Serine/threonine-protein kinase D1 (EC 2.7.11.13) (nPKC-D1) (Protein

kinase D) (Protein kinase C mu type) (nPKC-mu) [Gene: PRKD1 or PKD or PKD1 or PRKCM] - Homo sapiens (Human)

18 17 3 13

Q99500 EDG3_HUMAN

Sphingosine 1-phosphate receptor Edg-3 (S1P receptor Edg-3) (Endothelial differentiation G-protein coupled receptor 3) (Sphingosine

1-phosphate receptor 3) (S1P3) [Gene: EDG3] - Homo sapiens (Human)

367 51 17 34


SwissProt ID


Cpds screened

in PubChem

Active Compounds

PubChem Virtual

Scaffolds

PubChem Non

Virtual Scaffolds

Q99527 GPER_HUMAN

G-protein coupled estrogen receptor 1 (Membrane estrogen receptor) (mER) (G-protein coupled receptor 30) (Chemokine receptor-like 2) (IL8-related receptor DRY12) (Flow-induced endothelial G-protein

coupled receptor 1) (FEG-1) (Lymphocyte-derived G-protein coupled receptor) (LYGPR) (GPCR-BR) [Gene: GPER or CEPR or CMKRL2 or

DRY12 or GPR30] - Homo sapiens (Human)

10 3 1 2


Supplementary Table 3: Data for virtual scaffolds from PubChem present in compounds

described as active against the same molecular target in the WOMBAT database.

Entry Target (Swiss Prot ID)

Virtual scaffold from screen in PubChem

Number of scaffold

families found to be active in WOMBAT

Number of compounds

represented by these scaffolds

1 Estrogen

Receptor-alpha (P03372)

9 169

2 Estrogen

Receptor-alpha (P03372)

6 74

3 5-HT Receptor

Subtype 1A (P08908)

4 9

4 5-HT Receptor

Subtype 1A (P08908)

1 2

5 Acetylcholine

Muscarinic M1 Receptor (P11229) 1 1

6 Acetylcholine

Muscarinic M1 Receptor (P11229)

1 1

7 Acetylcholine


54 131

8 Acetylcholine


12 25

9 Acetylcholine

Muscarinic M1 Receptor (P11229) 1 1

10 Acetylcholine


9 98

11 Acetylcholine


1 1

12

Thyroid Stimulating

Hormone Receptor (P16473)

1 1

13

Allosteric Modulators of D1

Receptors (P21918)

1 1


14

Allosteric Modulators of D1

Receptors (P21918)

2 2


Supplementary Table 4: Virtual scaffolds from PubChem Screens, corresponding Murcko-

families of actives found in WOMBAT as well as the number of compounds represented by

each Murcko-Family

Entry PubChem virtual

scaffold Murcko scaffold of WOMBAT actives

Target Swiss Prot ID

Number of actives

1

P03372 1

2

P03372 4

3

P03372 4

4

P03372 2

5

P03372 2





Number of actives

6

P03372 2

7

P03372 2

8

P03372 2

9

P03372 2

10

P03372 1

11

P03372 1

12

P03372 118

13

P03372 34

14

P03372 1

15

P03372 67

16

P08908 2

17

P08908 3

18

P08908 3

19

P08908 2





Number of actives

20

P08908 1

21

P11229 1

22

P11229 2

23

P11229 2





Number of actives

24

P11229 2

25

P11229 2

26

P11229 2

27

P11229 2





Number of actives

28

P11229 1

29

P11229 3

30

P11229 2





Number of actives

31

P11229 2

32

P11229 4

33

P11229 1

34

P11229 1

35 P11229 1

36

P11229 1

37

P11229 1

38

P11229 48

39

P11229 2

40

P11229 4

41

P11229

42

P11229 1





Number of actives

43

P11229 1

44

P11229 1

45

P11229 2

46

P11229 18

47 P11229 9

48

P11229 1

49

P11229 19

50

P11229 1

51

P11229 25

52

P11229 1

53

P11229 1

54

P11229 1

55

P11229 1

56

P11229 1

57

P11229 1

58

P11229 1





Number of actives

59

P11229 1

60

P11229 3

61

P11229 1

62

P11229 1

63

P11229 1

64

P11229 1

65

P11229 1

66

P11229 3

67

P11229 1

68

P11229 1

69

P11229 1

70

P11229 1

71

P11229 1

72

P11229 1

73

P11229 1

74

P11229 1





Number of actives

75

P11229 1

76 P11229 3

77 P11229 1

78 P11229 1

79

P11229 2

80

P11229 1

81

P11229 1

82

P11229 2





Number of actives

83

P11229 1

84

P11229 2

85

P11229 2

86

P11229 2

87

P11229 2

88

P11229 2





Number of actives

89

P11229 2

90

P11229 2

91

P11229 2

92

P11229 2

93

P11229 2

94

P11229 2





Number of actives

95

P11229 2

296

P11229 2

97

P11229 2

98

P11229 4

99

P11229 2





Number of actives

100

P11229 2

101

P16473 1

102

P21918 1

103

P21918 1

104

P21918 1


Prospective analysis of the PubChem pyruvate kinase screen and experimental

validation

Supplementary Table 5: Structures and hierarchy level of virtual scaffolds with potent

neighbour scaffolds. The average log AC50 for all the compounds represented by either the

parent or the child scaffold(s) is given as well. The table is sorted by ascending log AC50 of the

child scaffolds and the parent scaffolds. Scaffold selected for compound acquisition and

subsequent experimental screening are marked in green.

Number Structures Scaffold hierarchy level

Avg. log AC50 of parent

scaffold(s)

Avg. log AC50 of child

scaffold(s)

1

3 -2.47 -7.26

2

3 -3.06 -6.79

3

3 -2.47 -6.38

4

2 -2.29 -6.27

5

3 -3.20 -6.13

6

4 -3.64 -6.12

7

3 -2.34 -6.07

8

4 -3.84 -6.07

9

3 -2.37 -6.05

10

4 -5.02 -6.03

11

3 -2.99 -5.94




scaffold(s)


scaffold(s)

12

3 -3.02 -5.90

13

3 -2.99 -5.84

14

2 -2.36 -5.83

15

4 -5.02 -5.82

16

3 -2.32 -5.73

17

4 -2.79 -5.72

18

4 -5.02 -5.68

19

3 -2.24 -5.66

20

3 -2.32 -5.64

21

2 -2.45 -5.62

22

3 -2.99 -5.61

23

3 -2.37 -5.60




scaffold(s)


scaffold(s)

24

4 -2.79 -5.56

25

3 -2.34 -5.55

26

3 -2.37 -5.55

27

2 -2.31 -5.50

28

3 -2.32 -5.50

29

2 -2.27 -5.47

30

3 -2.51 -5.46

31

4 -2.24 -5.42

32

2 -2.24 -5.40

33

3 -2.24 -5.38

34

2 -2.26 -5.37

35

3 -2.24 -5.35

36

3 -2.59 -5.35




scaffold(s)


scaffold(s)

37

4 -2.24 -5.33

38

3 -2.75 -5.33

39

3 -2.24 -5.25

40

3 -2.32 -5.25

41

3 -2.24 -5.24

42

4 -3.16 -5.24

43

3 -2.99 -5.23

44

3 -2.24 -5.22

45

4 -2.59 -5.22

46

3 -2.52 -5.21

47

3 -3.06 -5.21

48

2 -2.40 -5.20




scaffold(s)


scaffold(s)

49

4 -3.79 -5.19

50

2 -2.24 -5.16

51

3 -2.32 -5.16

52

3 -2.94 -5.16

53

4 -2.24 -5.15

54

2 -2.24 -5.11

55

3 -2.29 -5.09

56

4 -3.39 -5.07

57

3 -2.34 -5.05

58

3 -2.42 -5.05

59

2 -2.24 -5.04

60

3 -2.24 -5.04




scaffold(s)


scaffold(s)

61

4 -4.30 -5.03

62

3 -2.37 -5.02

63

3 -3.21 -5.02

64

3 -2.75 -5.01

65

4 -4.76 -5.01


Supplementary Table 6: Compound structure, vendor compound ID, source and PubChem SID.

Number Structure Virtual scaffold Vendor cpd. ID Source PubChem

SID

1

kbsenon-0012203

Aurora Fine Chemicals 57571219

2

kcd-124573 Aurora Fine Chemicals 57571220

3

kcheb-067808


4

kcheb-070443


5

kcheb-072091


6

kcheb-079810


7

kcheb-100864


8

kuk-303437 Aurora Fine Chemicals 57571226

9


10


11




SID

12


13


14


15

kchi-145021


16

ken-252537 Aurora Fine Chemicals 57571234

17


18


19


20




SID

21


22


23


24


25


26


27


28


29




SID

30

C505-0827 ChemDiv 57571141

31

E676-1525 ChemDiv 57571148

32

E676-1559 ChemDiv 57571149

33

E676-1574 ChemDiv 57571150

34

E676-1627 ChemDiv 57571151

35

E676-1637 ChemDiv 57571152

36

E676-1644 ChemDiv 57571153

37

E676-1645 ChemDiv 57571154

38

E676-4324 ChemDiv 57571155

39

E676-4359 ChemDiv 57571156

40

E676-4364 ChemDiv 57571157

41

E676-4404 ChemDiv 57571158



SID

42

E676-4435 ChemDiv 57571159

43

E677-1525 ChemDiv 57571160

44

E677-1565 ChemDiv 57571161

45

G786-0905 ChemDiv 57571164

46

G786-0908 ChemDiv 57571165

47

G786-0909 ChemDiv 57571166

48

G786-0911 ChemDiv 57571167

49

G786-0916 ChemDiv 57571168

50

G786-0919 ChemDiv 57571169

51

7756-0336 ChemDiv 57571205

52

7756-0337 ChemDiv 57571206

53

7756-0340 ChemDiv 57571207



SID

54

7756-0761 ChemDiv 57571208

55

7756-0771 ChemDiv 57571209

56

C177-0121 ChemDiv 57571210

57

C184-0754 ChemDiv 57571211

58

C184-0757 ChemDiv 57571212

59

C184-0761 ChemDiv 57571213

60

C184-0762 ChemDiv 57571214

61

C184-0892 ChemDiv 57571215



SID

62

C184-0898 ChemDiv 57571216

63

C184-0918 ChemDiv 57571217

64

C239-0716 ChemDiv 57571218

65

D094-0010 ChemDiv 57571142

66

D094-0011 ChemDiv 57571143

67

D094-0013 ChemDiv 57571144

68

D094-0014 ChemDiv 57571145

69

D094-0015 ChemDiv 57571146

70

D094-0016 ChemDiv 57571147



SID

71

F019-1236 ChemDiv 57571162

72

F019-1314 ChemDiv 57571163

73

K242-1013 ChemDiv 57571170

74

K242-1015 ChemDiv 57571171

75

K242-1019 ChemDiv 57571172

76

K242-1020 ChemDiv 57571173

77

K242-1021 ChemDiv 57571174



SID

78

K242-1037 ChemDiv 57571175

79

K242-1041 ChemDiv 57571176

80

K242-1095 ChemDiv 57571177

81

K242-1096 ChemDiv 57571178

82

K242-1097 ChemDiv 57571179

83

K242-1098 ChemDiv 57571180

84

K242-1099 ChemDiv 57571181



SID

85

K242-1100 ChemDiv 57571182

86

K242-1101 ChemDiv 57571183

87

K242-1102 ChemDiv 57571184

88

K242-1103 ChemDiv 57571185

89

K242-1104 ChemDiv 57571186

90

K242-1108 ChemDiv 57571187

91

K242-1109 ChemDiv 57571188



SID

92

K242-1110 ChemDiv 57571189

93

K242-1111 ChemDiv 57571190

94

K242-1112 ChemDiv 57571191

95

K242-1119 ChemDiv 57571192

96

K242-1126 ChemDiv 57571193

97

K242-1127 ChemDiv 57571194

98

K242-1128 ChemDiv 57571195



SID

99

K242-1129 ChemDiv 57571196

100

K242-1130 ChemDiv 57571197

101

K242-1138 ChemDiv 57571198

102

K242-1139 ChemDiv 57571199

103

K242-1140 ChemDiv 57571200

104

K242-1141 ChemDiv 57571201

105

K242-1142 ChemDiv 57571202



SID

106

K242-1143 ChemDiv 57571203

107

K242-1148 ChemDiv 57571204

Supplementary Table 7: Virtual scaffolds identified and selected from the scaffold tree

(generated with Scaffold Hunter) for the pyruvate kinase screen. Number of compounds

purchased for each scaffold.

Num. Virtual

scaffold

Number of compounds

purchased

1 21

2

57

3

14

4

15


Supplementary Table 8: Hits identified in the pyruvate kinase assay with an IC50 of 10 � M or

less (PubChem assay id 1687).

Num. Structure PubChem SID Activity type IC50 [µM]

5

57571161 Inhibitor 8.9 +/- 1.1

6

57571143 Inhibitor 2.0 +/- 0.3

7

57571145 Inhibitor 10.5 +/- 1.6

8

57571142 Inhibitor 1.0 +/- 0.1

9

57571146 Inhibitor 1.7 +/- 0.3

10

57571147 Inhibitor 2.2 +/- 0.3

11

57571219 Activator 4.9 +/- 0.6

12

57571225 Activator 5.4 +/- 0.6

13

57571232 Activator 7.6 +/- 0.6


Supplementary Table 9: Selected and tested compounds incorporating virtual scaffolds from

mainly inactive branches of the scaffold tree.

No. Structure Virtual Scaffold Vendor Compound Id Producer

1

C240-0044 ChemDiv

2

C358-0046 ChemDiv

3

C240-0169 ChemDiv

4

C358-0169 ChemDiv

5

C358-0091 ChemDiv

6

C289-0037 ChemDiv



7

C289-0046 ChemDiv

8

C240-0047 ChemDiv

9

C240-0170 ChemDiv

10

C066-2311 ChemDiv

11

C066-2239 ChemDiv

12

C066-2160 ChemDiv

13

C647-0466 ChemDiv



14

C647-0110 ChemDiv

15

C647-0467 ChemDiv

16

C647-0390 ChemDiv

17

C647-0227 ChemDiv

18

C647-0387 ChemDiv

19

C647-0192 ChemDiv

20

C647-0472 ChemDiv

21

C647-0386 ChemDiv



22

C647-0347 ChemDiv

23

C647-0345 ChemDiv

24

C647-0268 ChemDiv

25

C647-0468 ChemDiv

26

C647-0351 ChemDiv

27

C647-0426 ChemDiv

28

C647-0108 ChemDiv

29

C647-0187 ChemDiv



30

C647-0392 ChemDiv

31

C647-0225 ChemDiv

32

C647-0105 ChemDiv

33

C614-0595 ChemDiv

34

C614-0593 ChemDiv

35

C614-0921 ChemDiv

36

C614-0571 ChemDiv

37

C614-0570 ChemDiv

38

C614-0985 ChemDiv



39

C614-0591 ChemDiv

40

C614-0592 ChemDiv

41

C614-0979 ChemDiv

42

C614-0569 ChemDiv

43

C614-0918 ChemDiv

44

C614-0923 ChemDiv

45

C614-0577 ChemDiv

46

C614-0573 ChemDiv



47

C614-0920 ChemDiv

48

C614-0917 ChemDiv

49

C614-0913 ChemDiv

50

C614-0910 ChemDiv

51

C614-0578 ChemDiv

52

C614-6013 ChemDiv

53

C614-0572 ChemDiv

54

C614-0589 ChemDiv

55

C614-0933 ChemDiv



56

C614-0574 ChemDiv

57

C614-0598 ChemDiv

58

C614-0907 ChemDiv

59

C614-0901 ChemDiv

60

C614-0586 ChemDiv

61

C614-0935 ChemDiv

62

C567-0017 ChemDiv



63

C567-0407 ChemDiv

64

C567-0857 ChemDiv

65

C567-0967 ChemDiv

66

C567-0257 ChemDiv

67

C567-0057 ChemDiv



68

C567-0467 ChemDiv

69

C567-0767 ChemDiv

70

C567-0107 ChemDiv

71

C567-0447 ChemDiv

72

C594-0015 ChemDiv



73

C594-0040 ChemDiv

74

C619-0133 ChemDiv

75

C594-0018 ChemDiv

76

C619-0164 ChemDiv

77

C594-0036 ChemDiv



78

C619-0208 ChemDiv

79

C619-0193 ChemDiv

80

C594-0010 ChemDiv

81

C066-3140 ChemDiv

82

C619-0082 ChemDiv

83

C619-0166 ChemDiv

84

C660-0117 ChemDiv

85

C660-0125 ChemDiv



86

C619-0202 ChemDiv

87

C594-0021 ChemDiv

88

C594-0076 ChemDiv


1. Aurora Fine Chemicals Ltd., Reininghausstr. 49, A-8020 Graz, Austria, E-mail:

[email protected],http://www.aurorafinechemicals.com/ 2. ChemDiv Inc., 6605 Nancy Ridge Drive, San Diego, CA 92121 USA,

[email protected],www.chemdiv.com 3. CamCamStudio - Desktop Screen Recorder,http://sourceforge.net/projects/camstudio/ 4. Lagarith Lossless Video Codec,http://lags.leetcode.net/codec.html 5. Adobe Premiere Pro

CS3,http://www.adobe.com/products/premiere/?promoid=121DJGSD_P_US_FP2_PR_MN&tt=P_US_FP2_PR_MN

6. The Universal Protein Resource (UniProt) 2009. Nucleic Acids Res. 37, D169-D174 (2009).

7. Pyruvate kinase assay data in PubChem,http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=361&loc=ea_ras

8. Inglese, J. et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc. Natl. Acad. Sci. U. S. A. 103, 11473-11478 (2006).

9. Enzymatic Assay of PYRUVATE KINASE (EC 2.7.1.40), Sigma Prod. No. P-1903, Sigma-Aldrich, 2008,http://www.sigmaaldrich.com/sigma/enzyme%20assay/p1903enz.pdf

10. Enzymatic Assay of L-LACTIC DEHYDROGENASE1 (EC 1.1.1.27), Sigma-Aldrich, 2008,http://www.sigmaaldrich.com/sigma/enzyme%20assay/l1254enz.pdf

11. Olah, M. et al. WOMBAT and WOMBAT-PK: bioactivity databases for lead and drug discovery. Chem. Biol. 2, 760-786 (2007).


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