Stanford Chem-H Presentation (PDF)

KiNativ® In situ kinase profiling Stanford University ChEM-H c<a href="/goto?url=https://twitter.com/KiNativPlatform" target="_blank">onfidential </a>@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 <ul style="display: flex;"><li style="flex:1">Desthiobiotin tag </li><li style="flex:1">ATP </li></ul>2ATP acyl phosphate probe covalently modifies kinase in the active site Lysine 2 Lysine 1 3ATP acyl phosphate probe covalently modifies kinase in the active site Lysine 2 Lysine 1 4Samples trypsinized, probe-labeled peptides captured with streptavidin, and analyzed by targeted LC-MS2 <ul style="display: flex;"><li style="flex:1">Identification </li><li style="flex:1">Quantitation </li></ul>Explicit determination of peptide sequence and probe modification site from MS2 spectrum Integration of signal from MS2 fragment ions 5Comprehensive 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 <a href="/goto?url=http://www.kinativ.com/coverage/protein-lipid.html" target="_blank">http://www.kinativ.com/coverage/protein-lipid.html </a>6Profiling 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 <ul style="display: flex;"><li style="flex:1">Green: Kinases </li><li style="flex:1">Blue: Probe </li></ul>Gray: Non-kinases Red: Inhibitor 7Profiling 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 8KiNativ – Displaying processed data 9KiNativ 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 10 Why profile compounds on KiNativ – A study of JAK inhibitors • Compounds profiled in PBMC lysate <ul style="display: flex;"><li style="flex:1">Upadacitinib* </li><li style="flex:1">Ruxolitinib* </li><li style="flex:1">Baricitinib* </li><li style="flex:1">Tofacitinib* </li></ul>JAKs Fedratinib* <ul style="display: flex;"><li style="flex:1">Filgotinib </li><li style="flex:1">Abrocitinib </li><li style="flex:1">Pacritinib </li></ul>*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 <ul style="display: flex;"><li style="flex:1">KiNativ </li><li style="flex:1">Recombinant </li></ul>Abrocitinib Ruxolitinib Filgotinib Tofacitinib Pacritinib Abrocitinib Ruxolitinib Baricitinib Baricitinib Pacritinib <ul style="display: flex;"><li style="flex:1">Upadacitinib </li><li style="flex:1">Upadacitinib </li></ul>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 cellbased pEC50 <ul style="display: flex;"><li style="flex:1">KiNativ </li><li style="flex:1">Recombinant </li></ul>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 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 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 16 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 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 gelfiltered 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 Harvest cells at various times after subculturing, analyze by MS 0 h 8 h 32 h 1.5X 0.67X 96 h 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 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 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) 1µM Palbociclib MDA-MB-468 (resistant) 1µM Palbociclib 1.5X Direct Direct Indirect <ul style="display: flex;"><li style="flex:1">Kinases </li><li style="flex:1">Kinases </li></ul>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 <ul style="display: flex;"><li style="flex:1">Vemurafenib </li><li style="flex:1">LY3009120 </li></ul>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 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 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)] <ul style="display: flex;"><li style="flex:1">V600E BRAF G13D KRAS </li><li style="flex:1">WT </li></ul>• 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 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 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 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

Stanford Chem-H Presentation (PDF)

Details

Download

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

Support