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 2.0 3 , 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
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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-
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
Nature Chemical Biology: doi:10.1038/nchembio.187
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
Nature Chemical Biology: doi:10.1038/nchembio.187
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.
Nature Chemical Biology: doi:10.1038/nchembio.187
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
Nature Chemical Biology: doi:10.1038/nchembio.187
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).
Nature Chemical Biology: doi:10.1038/nchembio.187
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.
Nature Chemical Biology: doi:10.1038/nchembio.187
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)
O75116 ROC2_HUMAN Rho-associated protein
kinase 2 644 Dose-response biochemical assay of inhibitors of Rho kinase 2 (Rock2)
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
Dose Response assay for agonists of 5-Hydroxytryptamine (Serotonin) Receptor Subtype 1A (5HT1A)
P08908 5HT1A_HUMAN 5-hydroxytryptamine
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
P09871 C1S_HUMAN Complement C1s subcomponent
787 Complement factor C1s IC50 from mixture screen
P09871 C1S_HUMAN Complement C1s subcomponent
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
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 628
Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Primary Screen
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 677
Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Confirmation Screen
Nature Chemical Biology: doi:10.1038/nchembio.187
UniProt Accession Code
Uni Prot Name UniProt Description PubChem Assay ID
PubChem Assay Name
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 678
Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Secondary Assay 1
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 859
Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Assay
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 860
Discovery of novel allosteric modulators of the M1 muscarinic receptor: Antagonist Dose-Response Counterscreen
P11229 ACM1_HUMAN Muscarinic acetylcholine
receptor M1 943
qHTS Assay for Antagonists of Acetylcholine Muscarinic M1 Receptor: Kinetic Measurement of Intracellular Calcium Response
P11229 ACM1_HUMAN Muscarinic acetylcholine
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
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)
Nature Chemical Biology: doi:10.1038/nchembio.187
UniProt Accession Code
Uni Prot Name UniProt Description PubChem Assay ID
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)
P21453 EDG1_HUMAN Sphingosine 1-phosphate
receptor 1 1248
Dose Response Cell-Based Assay for Agonists of the Sphingosine 1-Phosphate Receptor 1 (S1P1): Purchased Analogues
P21453 EDG1_HUMAN Sphingosine 1-phosphate
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
P21462 FPR1_HUMAN formylpeptide receptor
(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
P21462 FPR1_HUMAN formylpeptide receptor
(FPR) family 699
Secondary Calcium Response Assay for Formylpeptide Receptor (FPR) Antagonists
Concentration Response Redox Cycling H2O2 Generation assay to characterize small molecule inhibitors identified in the Polo box domain (PBD) of Plk1 Primary HTS.
P53350 PLK1_HUMAN Serine/threonine-protein
kinase PLK1 877
Concentration Response fluorescence polarization-based assay to confirm small molecule inhibitors identified in the Polo box domain (PBD) of Plk1
Nature Chemical Biology: doi:10.1038/nchembio.187
UniProt Accession Code
Uni Prot Name UniProt Description PubChem Assay ID
PubChem Assay Name
Primary HTS.
P53350 PLK1_HUMAN Serine/threonine-protein
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.
P53350 PLK1_HUMAN Serine/threonine-protein
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.
P53350 PLK1_HUMAN Serine/threonine-protein
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.
P53350 PLK1_HUMAN Serine/threonine-protein
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.
P53350 PLK1_HUMAN Serine/threonine-protein
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 1383 PLK1 counterscreening assay for identified PKD inhibitors
P53779 MK10_HUMAN Mitogen-activated protein
kinase 10 530 JNK3 AlphaScreen Assay
P53779 MK10_HUMAN Mitogen-activated protein
kinase 10 746
Primary biochemical high-throughput screening assay for inhibitors of the c-Jun N-Terminal Kinase 3 (JNK3)
P53779 MK10_HUMAN Mitogen-activated protein
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
Nature Chemical Biology: doi:10.1038/nchembio.187
UniProt Accession Code
Uni Prot Name UniProt Description PubChem Assay ID
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
Q07820 MCL1_HUMAN Induced myeloid leukemia cell differentiation protein
Mcl-1 1021 uHTS of Mcl-1/Bid interaction inhibitors
Q07820 MCL1_HUMAN Induced myeloid leukemia cell differentiation protein
Mcl-1 1022 uHTS of Mcl-1/Noxa interaction inhibitors
Q07820 MCL1_HUMAN Induced myeloid leukemia cell differentiation protein
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
Q15139 KPCM_HUMAN Serine/threonine-protein
kinase D1 1286
Fluorescence polarization assay for PKD inhibitors - repeat of primary screen on hits identified from 140K library primary screen
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).