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KiNativ® In situ kinase profiling

Stanford University ChEM-H

@KiNativPlatform

Principle of the KiNativ platform

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• 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

Desthiobiotin tag ATP

Acyl phosphate

ATP acyl phosphate probe covalently modifies kinase in the active site

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Lysine 2

Lysine 1

ATP acyl phosphate probe covalently modifies kinase in the active site

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Lysine 2

Lysine 1

Samples trypsinized, probe-labeled peptides captured with streptavidin, and analyzed by targeted LC-MS2

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QuantitationIntegration of signal from MS2

fragment ions

IdentificationExplicit determination of peptide

sequence and probe modification site

from MS2 spectrum

Comprehensive Coverage of Protein and Lipid Kinases

Protein kinases Atypical kinases

~80% of known protein and atypical kinases identified on the platform

http://www.kinativ.com/coverage/protein-lipid.html 6

Green: Kinases detected on KiNativ

Red: Kinases not detected on KiNativ

Profiling compound(s) on the KiNativ platform

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Control sample – add probe

Treated sample – add inhibitor followed by probe

Inhibited

kinaseGreen: Kinases Blue: Probe

Gray: Non-kinases Red: Inhibitor

Sample: Lysate

derived from any

cell line or tissue

from ANY species

Profiling compound(s) on the KiNativ platform

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Control sample – add probe

Treated sample – add inhibitor followed by probe

Sample: Lysate

derived from any

cell line or tissue

from ANY species

Inhibited

kinase

MS

sig

na

lM

S s

ign

al

Time

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 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 sample10

Why profile compounds on KiNativ – A study of JAK inhibitors

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Upadacitinib* Tofacitinib*Ruxolitinib* Baricitinib*

Fedratinib* PacritinibAbrocitinibFilgotinib

*Clinically approved compounds

• Compounds profiled in PBMC lysate

JAKs

Why profile compounds on KiNativ – A study of JAK inhibitors• KiNativ IC50 values for selected JAK inhibitors, PBMC lysate

12*Clinically approved compounds

Comparing JAK1/TYK2 pIC50 to cell-based pEC50

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Ruxolitinib

Tofacitinib

Baricitinib

Upadacitinib

Abrocitinib

Filgotinib

Pacritinib

Fedratinib

Ruxolitinib

Baricitinib

Upadacitinib

Tofacitinib

Abrocitinib

Filgotinib

Fedratinib

Pacritinib

IC50 and EC50 values within 3-fold

KiNativ Recombinant

Cell-based assay: Monitor inhibition of INFα dependent pSTAT1 in Jurkat cells

Comparing JAK2 pIC50 to cell-based pEC50

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Baricitinib

Ruxolitinib

Baricitinib

Tofacitinib

Upadacitinib

Fedratinib

Pacritinib

Filgotinib

Abrocitinib

Ruxolitinib

Baricitinib

Tofacitinib

Upadacitinib

Fedratinib

Abrocitinib

Pacritinib

Filgotinib

IC50 and EC50 values within 3-fold

KiNativ Recombinant

Cell-based assay: Monitor inhibition of pSTAT5 in HEL cells

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

16Kinases

Upadacitinib* S = 9.2

Tofacitinib* S = 7.9

Baricitinib* S = 7.8

Ruxolitinib* S = 7.0

Abrocitinib# S = 6.0

Filgotinib^ S = 5.8

Fedratinib* S = 3.7

Pacritinib^ S = 3.0

* Clinically approved compounds# Undergoing clinical trials

^ Failed clinical trials

The steeper the slope,

the fewer off-targets

were observed

More potent

on-target

activity

Off-targets

more potently

inhibited than

on-target

• Approved JAK inhibitors had good selectivity scores

• Fedratinib, an approved JAK2 inhibitor for

myeloproliferative diseases had a surprisingly low

selectivity score

Efficacy of Fedratinib may be due to an off-target

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HEL

Jurkat

• Cell-killing efficacy was

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

• Baricitinib, a potent JAK2

inhibitor kills HEL cells

significantly more potently than

Jurkat, while Fedratinib kills

HEL and Jurkat cells with

similar potencies

Baricitinib

Fedratinib

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

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Profiling Ibrutinib in Ramos cells

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• 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

Profiling kinases during cell cycle progression

Confluent cells

Sub-culture

0 h

8 h

32 h

96 h

Harvest

cells at

various

times

after sub-

culturing,

analyze

by MS

Profiling kinases in A375 during cell cycle

progression (kinase activities compared to 0 h)

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0.67X

1.5XR

ela

tive

kin

as

e a

cti

vit

y

Kinases

Profiling kinases during cell cycle progression

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Increases

Decreases

Profiling kinases in A375 during cell cycle

progression (kinase activities compared to 0 h)

Confluent cells

Sub-culture

0 h

8 h

32 h

96 h

Harvest

cells at

various

times

after sub-

culturing,

analyze

by MS

• 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

MS Signal ratio

Profiling the CDK4/CDK6 inhibitor Palbociclib, in Colo-205 cells

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Palbociclib

• 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

Profiling Palbociclib in either sensitive (Colo205) or resistant (MDA-MB-486) cells – live cell format

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Colo205 (sensitive)

1µM Palbociclib

MDA-MB-468 (resistant)

1µM Palbociclib

Direct Indirect

Direct

MS

Sig

nal

Rati

o [

(Tre

ate

d)/

(Co

ntr

ol)

]

Kinases

Chemoproteomic Evaluation of Target Engagement by the Cyclin-Dependent

Kinase 4 and 6 Inhibitor Palbociclib Correlates with Cancer Cell ResponseDOI: 10.1021/acs.biochem.6b00629

Kinases

1.5X

Profiling Palbociclib in either sensitive (Colo205) or resistant (MDA-MB-486) cells – live cell format

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Direct

targets

• CDK4 is not inhibited in MDA-MB-468 cells, although the other palbociclib

targets are inhibited to the same extent

Pathway

effects

MS Signal ratio [(Treated)/(Control)]

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

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Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor DrugsDOI: 10.1158/1535-7163.MCT-18-0755

MCF7 cells were transfected with either GFP or

various CDKN proteins, lysates were then

generated, probe-labeled and analyzed by KiNativ

Vemurafenib, selective BRAF inhibitor versus LY3009210, pan-RAF inhibitor

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• 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

Profiling Vemurafenib pathway effects in A375 (V600E BRAF), HCT116 (G13D K-RAS), and PC3 (WT RAF, WT RAS) cells, 48 hours

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Direct

targets

Pathway

effects

• Target engagement

observed in both sensitive

and resistant cells

• Pathway effects

predominantly observed

in sensitive cells (V600E

BRAF)

• Hyper phosphorylation of

MEK and ERK in RAS-

driven cells may result in

HCT116 cells being less

sensitive to Vemurafenib

than PC3

V600E

BRAF

G13D

KRASWT

MS Signal ratio [(Treated)/(Control)]

Profiling LY3009120 pathway effects in A375 (V600E BRAF), HCT116 (G13D K-RAS), and PC3 (WT RAF, WT RAS) cells, 48 hours

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Direct

targets

Similar

pathway

effects

• Similar to Vemurafenib,

LY3009120 binds RAF

kinases in sensitive

and resistant cells

• In contrast to

Vemurafenib, pathways

effects are observed in

both V600E BRAF and

G13D KRAS driven cell

linesDifferent

pathway

effects

V600E BRAF G13D KRAS WT

MS Signal ratio [(Treated)/(Control)]

Profiling the Aurora kinase inhibitor Alisertib – G2/M arrest

• 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

Direct

targets

Pathway

effects

MS Signal ratio [(Treated)/(Control)]

Kinases

1 hour

24 hours

48 hoursAurora

EphR

PLK1, CDK9,

ROCK, FAM20B

MS

Sig

nal

Rati

o [

(Tre

ate

d)/

(Co

ntr

ol)

]

Acyl phosphate GTP probe modifies GTPases at a conserved lysine in the phosphate binding loop (P site)

Lysine residue

GTP probePhosphate

binding loop

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• 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

Using the GTP probe to profile RAS mutants

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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

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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

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Pharmacological inhibition of RAS G12C

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Time

WT RAS

G12C RAS

Control

AMG510 (10 µM)

• 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

GTPase Selectivity

Profile for AMG510

(10 µM)

Pharmacological inhibition of RAS G12C – observation of pathways effects

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Direct targets

observed with

the GTP probe

(2 hours)

• 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

Indirect targets/

pathway effects

observed with

the ATP probe

(48 hours)

Inhibition (%)

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

36@KiNativPlatform