KiNativ® In situ kinase profiling Stanford University ChEM-H
confidential @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 2 ATP acyl phosphate probe covalently modifies kinase in the active site
Lysine 2
Lysine 1
3 ATP acyl phosphate probe covalently modifies kinase in the active site
Lysine 2
Lysine 1
4 Samples trypsinized, probe-labeled peptides captured with streptavidin, and analyzed by targeted LC-MS2 Identification Quantitation Explicit determination of peptide Integration of signal from MS2 sequence and probe modification site fragment ions from MS2 spectrum
5 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 6 Profiling compound(s) on the KiNativ platform
Control sample – add probe
Sample: Lysate derived from any cell line or tissue from ANY species Treated sample – add inhibitor followed by probe
Inhibited kinase Green: Kinases Blue: Probe Gray: Non-kinases Red: Inhibitor 7 Profiling compound(s) on the KiNativ platform
Control sample – add probe MS signal MS
Sample: Lysate derived from any cell line or tissue from ANY species Treated sample – add inhibitor followed by probe
Inhibited MS signal MS kinase
Time
8 KiNativ – Displaying processed data
9 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 cell- based 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 10 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 11 Why profile compounds on KiNativ – A study of JAK inhibitors • KiNativ IC50 values for selected JAK inhibitors, PBMC lysate
*Clinically approved compounds 12 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 13 Comparing JAK2 pIC50 to cell- based pEC50 KiNativ Recombinant Ruxolitinib Fedratinib Baricitinib Pacritinib
Filgotinib
Tofacitinib Fedratinib
Ruxolitinib Tofacitinib Filgotinib Baricitinib
Upadacitinib
Upadacitinib Pacritinib Abrocitinib Abrocitinib
IC50 and EC50 values within 3-fold
Cell-based assay: Monitor inhibition of pSTAT5 in HEL cells 14 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) 15 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 myeloproliferative diseases had a surprisingly low Baricitinib* S = 7.8 selectivity score Ruxolitinib* S = 7.0 Off-targets Abrocitinib# S = 6.0 more potently Filgotinib^ S = 5.8 inhibited than Fedratinib* S = 3.7 on-target Pacritinib^ S = 3.0 * Clinically approved compounds # Undergoing clinical trials ^ Failed clinical trials
More potent on-target activity The steeper the slope, the fewer off-targets were observed
Kinases 16 Efficacy of Fedratinib may be due to an off-target
• Cell-killing efficacy was determined for Baricitinib and Baricitinib Fedratinib in either HEL or Jurkat cells • HEL cells are driven by constitutively active JAK2 (V617F) • Jurkat cells are not known to HEL have any aberrations in JAK Jurkat signaling pathways Fedratinib • Baricitinib, a potent JAK2 inhibitor kills HEL cells significantly more potently than Jurkat, while Fedratinib kills HEL and Jurkat cells with similar potencies
17 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 gel- filtered and then probe-labeled
18 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
19 Profiling kinases during cell cycle progression
Confluent cells Profiling kinases in A375 during cell cycle progression (kinase activities compared to 0 h)
Sub-culture
0 h Harvest cells at 1.5X 8 h various times 32 h after sub- 0.67X culturing, analyze
96 h by MS Relative kinase activity kinase Relative
Kinases 20 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 0 h Harvest cells at 8 h various times 32 h after sub- culturing, analyze 96 h by MS
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 21 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
22 Profiling Palbociclib in either sensitive (Colo205) or resistant (MDA-MB-486) cells – live cell format
Colo205 (sensitive) MDA-MB-468 (resistant) 1µM Palbociclib 1µM Palbociclib
1.5X
Direct
Direct Indirect
MS Signal Ratio [(Treated)/(Control)] 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 23 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
24 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 25 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
26 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 G13D WT BRAF KRAS
• Target engagement Direct observed in both sensitive targets and resistant cells
• 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 27 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, Direct LY3009120 binds RAF targets kinases in sensitive and resistant cells
• In contrast to Similar Vemurafenib, pathways pathway effects are observed in effects both V600E BRAF and G13D KRAS driven cell lines Different pathway effects
28 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 Aurora 48 hours Pathway effects
MS Signal Ratio [(Treated)/(Control)] 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 29 Acyl phosphate GTP probe modifies GTPases at a conserved lysine in the phosphate binding loop (P site)
GTP probe Phosphate binding loop
Lysine residue
30 Using the GTP probe to profile RAS mutants
• H,K,N-RAS probe-labeled peptide contains codons 12 and 13 which are mutated in a variety of cancers
• Wild type peptide 6 – LVVVGAGGVGK*SALTIQLIQNHFVDEYDPTIEDSYRK
• Mutant peptides have distinct m/z values and MS2 spectra from the wild-type peptide
• Thus, the GTP probe can be used to detect and quantitate mutant RAS protein in lysates 31 In each cell line profiled, mutant RAS detected with the probe was consistent with the genetic information
• A375: Wild-type • A549: G12S K-Ras • RPMI-8226: G12A K-Ras • PANC1: G12D K-Ras • MDA-MB-231: G13D K-Ras
32 Pharmacological inhibition of RAS
• The role of constitutively active RAS driving tumor growth has been recognized for more than 30 years
• However, due to the high affinity RAS has more nucleotide, the protein was considered undruggable
• A breakthrough was achieved in the development of inhibitors that covalently bind G12C RAS in cells
• Clinical trials underway for three compounds • Amgen – AMG510 • Wellsprings – ARS-1620 • Mirati – MRTX849
33 Pharmacological inhibition of RAS G12C
• Both AMG510 and ARS-1620 potently inhibit probe-labeling of G12C RAS (H358 cells harboring G12C KRAS treated with 10 µM compound for two hours) • No off-targets observed amongst the GTPases profiled (results shown for AMG510
WT RAS
Control AMG510 (10 µM) GTPase Selectivity Profile for AMG510 (10 µM) G12C RAS
Time
34 Pharmacological inhibition of RAS G12C – observation of pathways effects • Significantly, long term treatment of cells (48 hours) resulted in the downregulation of several kinases in H358 cells, driven by G12C KRAS, but not in HCT116, driven by G13D KRAS • The downregulation of these kinases is consistent with cell-killing, observed for several kinase inhibitor drugs Inhibition (%)
Direct targets observed with the GTP probe (2 hours)
Indirect targets/ pathway effects observed with the ATP probe (48 hours)
35 Advantages of the KiNativ platform
KiNativ is a universal assay that can be applied to different stages of drug discovery • Confirm on-target potency and selectivity (lysate format) • Determine in-cell target engagement to confirm compound permeability and MOA (live cell format) • Long-term cell treatment experiments to examine pathway effects (live cell format) • Pre-clinical efficacy and safety studies, same assay applied to different species (live animal format) • Profiling tissues from tumor xenografts - measure both target engagement and detect pathway effects (live animal format) • Confirm target engagement in human clinical samples
@KiNativPlatform 36