KiNativ® In situ kinase profiling
Stanford University ChEM-H
@KiNativPlatform
Principle of the KiNativ platform
• ATP (or ADP) acyl phosphate binds to, and covalently modifies
Lysine residues in the active site
• Thus, ATP acyl phosphate with a desthiobiotin tag can be used capture and quantitate kinases in a complex lysate
Acyl phosphate
- Desthiobiotin tag
- ATP
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ATP acyl phosphate probe covalently modifies kinase in the active site
Lysine 2
Lysine 1
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ATP acyl phosphate probe covalently modifies kinase in the active site
Lysine 2
Lysine 1
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Samples trypsinized, probe-labeled peptides captured with streptavidin, and analyzed by targeted LC-MS2
- Identification
- Quantitation
Explicit determination of peptide sequence and probe modification site from MS2 spectrum
Integration of signal from MS2 fragment ions
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Comprehensive Coverage of Protein and Lipid Kinases
Protein kinases
Atypical kinases
Green: Kinases detected on KiNativ Red: Kinases not detected on KiNativ
~80% of known protein and atypical kinases identified on the platform
http://www.kinativ.com/coverage/protein-lipid.html
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Profiling compound(s) on the KiNativ platform
Control sample – add probe
Sample: Lysate
derived from any
cell line or tissue
Treated sample – add inhibitor followed by probe
from ANY species
Inhibited
kinase
- Green: Kinases
- Blue: Probe
Gray: Non-kinases Red: Inhibitor
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Profiling compound(s) on the KiNativ platform
Control sample – add probe
Sample: Lysate
derived from any
cell line or tissue
Treated sample – add inhibitor followed by probe
from ANY species
Inhibited kinase
Time
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KiNativ – Displaying processed data
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KiNativ profiling formats
• Lysate
• Compound(s) added to lysate prepared from relevant cell line/tissue
followed by probe
• Efficient approach to determine on-target potency and selectivity
• Live cell
• Compound(s) added to cells for a period of time, after which cells are harvested, washed, lysed and probe-labeled
• Confirm cell permeability and compound MOA, i.e., how well does
on-target potency (KiNativ) compare to EC50 values from a cellbased assay
• Note: 10X dilution during lysis prior to probe addition, reversible compounds may re-equilibrate with target(s)
• Live animal
• Animals treated with compound, after which they are sacrificed, relevant tissues harvested, snap-frozen and sent out for profiling on KiNativ
• Recommended 100 mg tissue per sample
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Why profile compounds on KiNativ – A study of JAK inhibitors
• Compounds profiled in PBMC lysate
- Upadacitinib*
- Ruxolitinib*
- Baricitinib*
- Tofacitinib*
JAKs
Fedratinib*
- Filgotinib
- Abrocitinib
- Pacritinib
*Clinically approved compounds
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Why profile compounds on KiNativ – A study of JAK inhibitors
• KiNativ IC50 values for selected JAK inhibitors, PBMC lysate
*Clinically approved compounds
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Comparing JAK1/TYK2 pIC50 to cell-based pEC50
- KiNativ
- Recombinant
Abrocitinib
Ruxolitinib
Filgotinib
Tofacitinib
Pacritinib
Abrocitinib
Ruxolitinib
Baricitinib
Baricitinib
Pacritinib
- Upadacitinib
- Upadacitinib
Tofacitinib
Fedratinib
Filgotinib Fedratinib
IC50 and EC50 values within 3-fold
Cell-based assay: Monitor inhibition of INFα dependent pSTAT1 in Jurkat cells
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Comparing JAK2 pIC50 to cell- based pEC50
- KiNativ
- Recombinant
Ruxolitinib
Fedratinib
Pacritinib
Filgotinib
Baricitinib
Tofacitinib
Fedratinib
Tofacitinib
Ruxolitinib
Baricitinib
Filgotinib
Baricitinib
Upadacitinib
Upadacitinib
Pacritinib
Abrocitinib
Abrocitinib
IC50 and EC50 values within 3-fold
Cell-based assay: Monitor inhibition of pSTAT5 in HEL cells
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Quantifying the selectivity of kinase inhibitors
• One of the main reasons for profiling inhibitors against a panel of kinases is to assess selectivity
• Assessing selectivity can be arbitrary!
Method to quantify selectivity – Selectivity score (S)
• Based on the method described by Piotr Grazcyk “Gini
Coefficient:ꢀ A New Way To Express Selectivity of Kinase
Inhibitors against a Family of Kinases”
doi.org/10.1021/jm070562u
• Profile compound at 2-4 doses, estimate IC50s • Convert IC50s to pIC50s and normalize to target pIC50, i.e., on-
target normalized pIC50 = 1
• Plot the normalized IC50s against the targets and determine area under the curve
• Reciprocal of the area under the curve = Selectivity score (S)
(higher the score, more selective the compound)
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Quantifying Selectivity – JAK inhibitors
• Approved JAK inhibitors had good selectivity scores
Upadacitinib* S = 9.2
• Fedratinib, an approved JAK2 inhibitor for
Tofacitinib*
S = 7.9
Baricitinib*
S = 7.8
myeloproliferative diseases had a surprisingly low
Ruxolitinib* S = 7.0
selectivity score
Abrocitinib# S = 6.0
Off-targets
Filgotinib^
Fedratinib*
Pacritinib^
S = 5.8
S = 3.7
S = 3.0
more potently inhibited than on-target
* Clinically approved compounds # Undergoing clinical trials ^ Failed clinical trials
More potent on-target activity
The steeper the slope,
the fewer off-targets were observed
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Kinases
Efficacy of Fedratinib may be due to an off-target
• Cell-killing efficacy was
Baricitinib
determined for Baricitinib and Fedratinib in either HEL or Jurkat cells
• HEL cells are driven by constitutively active JAK2 (V617F)
• Jurkat cells are not known to have any aberrations in JAK signaling pathways
HEL Jurkat
Fedratinib
• Baricitinib, a potent JAK2
inhibitor kills HEL cells significantly more potently than Jurkat, while Fedratinib kills HEL and Jurkat cells with similar potencies
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Profiling the covalent BTK inhibitor Ibrutinib
• Ramos cells were treated with Ibrutinib (10 and 1 µM, and no-inhibitor control) for one hour
• Cells were then washed, harvested and lysed
• Lysate was divided into two parts and either probe-labeled as is, or gelfiltered and then probe-labeled
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Profiling Ibrutinib in Ramos cells
• Ibrutinib modifies BTK on Cys-481 (highlighted)
• For all kinases that appear to be covalently modified by Ibrutinib, there is a
Cys residue either precisely aligned with BTK Cys-481, or in close proximity
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Profiling kinases during cell cycle progression
Confluent cells
Profiling kinases in A375 during cell cycle progression (kinase activities compared to 0 h)
Sub-culture
Harvest cells at
various
times after subculturing, analyze by MS
0 h
8 h
32 h
1.5X 0.67X
96 h
Kinases
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Profiling kinases during cell cycle progression
Confluent cells
Profiling kinases in A375 during cell cycle progression (kinase activities compared to 0 h)
MS Signal ratio
Sub-culture
Increases
Harvest cells at
various
times after subculturing, analyze by MS
0 h
8 h
32 h
96 h
Decreases
• These dynamic changes in the kinome may be modulated by kinase inhibitors
• Long-term treatment of cells with inhibitors might result in the observation of changes in the kinome due to pathway effects, in addition to the direct targets
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Profiling the CDK4/CDK6 inhibitor Palbociclib, in Colo-205 cells
• Palbociclib is an FDA approved CDK4/CDK6 inhibitor for the treatment of ER-positive and HER2-negative breast cancer
• The compound exhibits a wide-range of potencies in cell-killing assays • The molecular basis determining sensitivity or resistance to the compound is not fully understood
Palbociclib
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Profiling Palbociclib in either sensitive (Colo205) or resistant (MDA-MB-486) cells – live cell format
Colo205 (sensitive)
1µM Palbociclib
MDA-MB-468 (resistant)
1µM Palbociclib
1.5X
Direct
Direct Indirect
- Kinases
- Kinases
Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell Response
DOI: 10.1021/acs.biochem.6b00629
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Profiling Palbociclib in either sensitive (Colo205) or resistant (MDA-MB-486) cells – live cell format
• CDK4 is not inhibited in MDA-MB-468 cells, although the other palbociclib targets are inhibited to the same extent
MS Signal ratio [(Treated)/(Control)]
Direct targets
Pathway effects
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Mechanistic basis for sensitivity/resistance of cells to CDK4/CDK6 inhibitors
• Elevated levels of CDKN2
proteins, but not CDKN1 proteins inhibit both the binding of ATP probe and inhibitor to CDK4 and CDK6
• Observation is consistent with
the fact that CDKN2 proteins bind CDK4 and CDK6, while CDKN1 proteins bind CDK2
MCF7 cells were transfected with either GFP or
various CDKN proteins, lysates were then generated, probe-labeled and analyzed by KiNativ
Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs
DOI: 10.1158/1535-7163.MCT-18-0755
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Vemurafenib, selective BRAF inhibitor versus LY3009210, pan-RAF inhibitor
• Vemurafenib is a potent BRAF inhibitor while LY3009120 also inhibits
ARAF and RAF1
• Vemurafenib is only efficacious in cells driven by V600E BRAF while
LY3009120 is efficacious in both V600E BRAF cells, as well as in cells driven by activating RAS mutants
- Vemurafenib
- LY3009120
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Profiling Vemurafenib pathway effects in A375 (V600E BRAF), HCT116 (G13D K-RAS), and PC3 (WT RAF, WT RAS) cells, 48 hours
MS Signal ratio [(Treated)/(Control)]
V600E BRAF
G13D KRAS
WT
• Target engagement observed in both sensitive and resistant cells
Direct
targets
• Pathway effects predominantly observed in sensitive cells (V600E BRAF)
Pathway effects
• Hyper phosphorylation of
MEK and ERK in RAS- driven cells may result in HCT116 cells being less sensitive to Vemurafenib than PC3
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Profiling LY3009120 pathway effects in A375
(V600E BRAF), HCT116 (G13D K-RAS), and PC3 (WT RAF, WT RAS) cells, 48 hours
MS Signal ratio [(Treated)/(Control)]
- V600E BRAF G13D KRAS
- WT
• Similar to Vemurafenib,
LY3009120 binds RAF
kinases in sensitive
Direct targets
and resistant cells
• In contrast to
Similar
pathway effects
Vemurafenib, pathways
effects are observed in
both V600E BRAF and G13D KRAS driven cell lines
Different
pathway
effects
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Profiling the Aurora kinase inhibitor Alisertib – G2/M arrest
MS Signal ratio [(Treated)/(Control)]
PLK1, CDK9, ROCK, FAM20B
Direct targets
EphR
1 hour 24 hours 48 hours
Aurora
Pathway effects
Kinases
• Aurora kinase inhibitors induce G2/M arrest in contrast to CDK and RAF inhibitors which induce G0/G1 arrest
• PLK1 is downregulated during G0/G1 arrest, but upregulated at G2/M arrest
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Acyl phosphate GTP probe modifies GTPases at a conserved lysine in the phosphate binding loop (P site)
GTP probe
Phosphate
binding loop
Lysine residue
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