Download Supplementary Tables and Figures Legends (doc 39K)

Titles and Legends to Supplementary Tables and Figures
Supplementary Table S1. CML patient sample details. WBC (G/l): white blood count (grams/liter); Hb
(g/dl): hemoglobin (grams/deciliter); Plt (G/l): platelets (grams/liter); Basos: basophils; pb: peripheral
blood; CRP (mg/dl): c-reactive protein (milligrams/deciliter); HU (mg/d): hydroxyurea (mg/day); n.t. not
tested.
Supplementary Table S2a. All proteins purified on the INNO-406 matrix from K562 lysate. Proteins
are sorted by the number of identified unique peptides (Peptide Count) in pulldown 1 (PD1). Proteins
listed have at least one specific peptide that confirms the presence of the protein in the pulldown.
IPI_IDs are IPI protein database entries to which identified peptides were assigned. PD1 and PD2
refer to the duplicate pulldowns performed for each cell type. Competitive refers to pulldowns where
lysates were pre-treated with 20 µM INNO-406 before incubation with the matrix. Core proteome
refers to the most abundant proteins in the K562 total cell lysate, which were also present in the
INNO-406 pulldowns. Frequent Hitters refers to proteins identified from the corresponding cell type on
matrices of five kinase-inhibitor-unrelated drugs (paroxetine, amphotericin B, ciprofloxacin,
daunorubicin and kanamycin). Potential targets are subtracted from right to left based on 1) lack of
presence in duplicate experiments, 2) presence in the Frequent Hitter experiments, 3) presence in the
Core Proteome and 4) lack of competition. Protein kinases are indicated by bold print. The remaining,
and therefore most confident INNO-406 targets, are marked in red.
Supplementary Table S2b. All proteins purified on the INNO-406 matrix from KU812 lysate. Proteins
are sorted by the number of identified unique peptides (Peptide Count) in pulldown 1 (PD1). Proteins
listed have at least one specific peptide that confirms the presence of the protein in the pulldown.
IPI_IDs are IPI protein database entries to which identified peptides were assigned. PD1 and PD2
refer to the duplicate pulldowns performed for each cell type. Competitive refers to pulldowns where
lysates were pre-treated with 20 µM INNO-406 before incubation with the matrix. Frequent Hitters
refers to proteins identified from the corresponding cell type on matrices of five kinase-inhibitorunrelated drugs (paroxetine, amphotericin B, ciprofloxacin, daunorubicin and kanamycin). Potential
targets are subtracted from right to left based on 1) lack of presence in duplicate experiments, 2)
1
presence in the Frequent Hitter experiments and 3) lack of competition. Protein kinases are indicated
by bold print. The remaining, and therefore most confident INNO-406 targets, are marked in red.
Supplementary Table S2c. All proteins purified on the INNO-406 matrix from CML chronic phase
patient pool lysate. Proteins are sorted by the number of identified unique peptides (Peptide Count) in
pulldown 1 (PD1). Proteins listed have at least one specific peptide that confirms the presence of the
protein in the pulldown. IPI_IDs are IPI protein database entries to which identified peptides were
assigned. PD1 and PD2 refer to the duplicate pulldowns performed for each cell type. Competitive
refers to pulldowns where lysates were pre-treated with 20 µM INNO-406 before incubation with the
matrix. Frequent Hitters refers to proteins identified from the corresponding cell type on a matrix of the
kinase-inhibitor-unrelated drug paroxetine. Potential targets are subtracted from right to left based on
1) lack of presence in duplicate experiments, 2) presence in the Frequent Hitter experiment and 3)
lack of competition. Protein kinases are indicated by bold print. The remaining, and therefore most
confident INNO-406 targets, are marked in red.
Supplementary Table S3. List of frequent hitters identified in K562 or KU812 total cell lysates. Five
structurally kinase inhibitor-unrelated drugs (paroxetine, amphotericin B, ciprofloxacin, daunorubicin
and kanamycin) were directly immobilized via their amino groups on Sepharose beads as previously
described (Rix, 2007). The resulting matrices were exposed either to K562 (Table S3a) or KU812
(Table S3b) total cell lysates. All proteins identified in each experiment are shown in the list. Proteins
are sorted by the number of times identified across the five drugs. The higher the incidence amongst
the experiments, the more likely the protein is to be a non-specific "frequent hitter" and selecting it as a
"real" target of the drug of interest should be done with caution. IPI-IDs are IPI protein database
entries to which identified peptides were assigned. Proteins listed have at least one specific peptide
that confirms the presence of the protein in the pull-down.
Supplementary Table S4. Complete Millipore KinaseProfilerTM panel. For each kinase, the percent
activity remaining at 1 µM and 10 µM of INNO-406 was measured.
2
Supplementary Figure Legends
Supplementary Figure S1. Millipore KinaseProfiler™ Service Assay Protocols. This manual provides
the screening conditions and protocols used in the KinaseProfiler radiometric protein kinase assays
and HTRF® lipid kinase assays.
Supplementary Figure S2. Structures of the five kinase inhibitor-unrelated drugs amphotericin B,
ciprofloxacin, daunorubicin, kanamycin and paroxetine used to identify frequent hitters in K562, KU812
and chronic phase CML patient cells. Amino groups employed to immobilize each drug on the
Sepharose beads (Rix, 2007) are shown in blue. The five compounds, being drugs themselves, were
chosen for their properties such as size and polarity, but also for their lack of known mammalian
targets (e.g. the antibiotic ciprofloxacin) or knowledge that, through coupling to the resin, binding to
known targets would be disrupted (e.g. the serotonin transporter inhibitor paroxetine). The higher the
incidence a certain protein is seen amongst the five experiments, the more likely it is to be a frequent
hitter and selecting it as a potential target of the drug of interest should be done with caution. For the
purpose of this study, we stringently subtracted all proteins identified by the negative control
experiments.
3