ATP mediated kinome selectivity – The missing link in understanding the contribution of ...

8/12/2019 ATP mediated kinome selectivity The missing link in understanding the contribution of individual JAK kinases isof

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ATP mediated kinome selectivity The missing link in understanding the contribution of

individual JAK kinases isoforms to cellular signaling.

Atli Thorarensen*, Mary Ellen Banker, Andrew Fensome, Jean-Baptiste Telliez, Brian Juba, Fabien

Vincent, Robert M Czerwinski, Agustin Casimiro-Garcia

Pfizer Worldwide Research, 200 Cambridgepark Drive, Cambridge, Massachusetts 02140, USA.

[email protected]

Abstract: Kinases constitute an important class of therapeutic targets being explored by both academia

and the pharmaceutical industry. The major focus of this effort has been directed towards the

identification of ATP-competitive inhibitors. Although it has long been recognized that the intracellular

concentration of ATP is very different from the concentrations utilized in biochemical enzyme assays,

little thought has been devoted to incorporating this discrepancy into our understanding of translation

from enzyme inhibition to cellular function. Significant work has been dedicated to the discovery of JAK

kinase inhibitors; however, a disconnect between enzyme and cellular function is prominently displayed

in the literature for this class of inhibitors. Herein we demonstrate utilizing the four JAK family members

that the difference in the ATP KMof each individual kinase has a significant impact on the enzyme to cell

inhibition translation. We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either

1 mM ATP or KMATP for the four isoforms as well as in primary cell assays. This dataset provided the

opportunity to examine individual kinase contribution to the heterodimeric kinase complexes mediating

cellular signaling. In contrast to a recent study, we demonstrate that for IL-15 cytokine signaling it is

sufficient to inhibit either JAK1 or JAK3 to fully inhibit downstream STAT5 phosphorylation. This

additional data thus provides a critical piece of information explaining why JAK1 has incorrectly been

thought as having a dominant role over JAK3. Beyond enabling a deeper understanding of JAK signaling,

conducting similar analyses for other kinases by taking into account potency at high ATP rather than KM

ATP may provide crucial insights into a compounds activity and selectivity in cellular contexts.

Kinases constitute a large family within the human genome with individual members playing key

roles in numerous cellular signaling pathways. To date, it is estimated that only a fraction of the kinome

has been mined as a source of therapeutic targets.(1) Multiple kinases inhibitors have been approved by

the FDA In recent years. While a large majority of these were directed at oncology indications, recent

approval of Xeljanz for rheumatoid arthritis illustrates the potential importance of this class of targets

for other disease indications.(2) These successes led to explosive growth in the field of kinase drug

discovery and significant effort has been devoted towards the identification, optimization andpharmacological characterization of novel inhibitors. While historically a majority of the work has

focused on the design of ATP competitive inhibitors (type I), kinase inhibitor design started to include

the discovery of alternative inhibition mechanisms (type II-IV) as the field matured.(3) Significant effort

is being devoted to understanding the structural requirements that place inhibitors in the appropriate

mechanistic class. While structural elements likely to result in type I or II inhibitors are known, the other

classes are less well understood.(4,5) Selectivity is another key component in kinase inhibitor design


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and great attention has been devoted to kinase selection for inclusion in selectivity panels.(6)

Substantial selectivity datasets containing either a large number of compounds tested against a select

panel of kinases(7) or comprehensive selectivity assessments across the kinome for a more limited

number of compounds have been published in recent years.(8,9) This work resulted in the need to

quantify selectivity to allow comparisons between compounds and prompted the development of

methods such as Gini or thermodynamic partition index.(10,11)

Janus kinases (JAK) are non-receptor tyrosine kinases required for signaling through type I/II

cytokine receptors.(12) There are four JAK family members, JAK1, JAK2, JAK3 and TYK2. JAK3 and TYK2

are primarily involved in immune related functions while JAK1 and JAK2 also play critical roles related to

hematopoiesis, growth and neuronal functions amongst others. A notable feature of JAK signaling is the

requirement for a dimer of JAK kinases at the cytokine receptor complex level. Cytokine receptors

signaling through JAK1/JAK3, JAK1/JAK2, JAK1/TYK2, JAK2/TYK2 and JAK2/JAK2 have been described.

The importance of the various individual JAKs in cytokine signaling has resulted in significant

effort to identify selective inhibitors for each family member.(13,14) Inhibitor design has been guided

by the availability of crystal structures for each isoform.(15) These inhibitors have served an important

role in elucidating the role of JAKs in cell signaling.(16,17) The selectivity characterization of these

inhibitors has exclusively utilized enzymatic assays performed with an ATP concentration equal to

enzyme KMproducing IC50,KMvalues or by describing the intrinsic affinity of the inhibitors as a KD. This

data was then supplemented by various cellular assays to confirm the selectivity profile.(18,19)

Discrepancies between enzyme and cellular data for JAK kinases has been recognized for some time but

only recently have potential explanations supported by experimentation been published to address

these inconsistencies.(20) It has long been recognized that the cellular concentration of ATP is in the 1

to 5 mM range. Therefore, neither a KDnor an IC50measurement at ATP KMcan be a good descriptor of

cellular activity for ATP competitive inhibitors. The relationship of activity at various substrateconcentrations was described by Cheng-Prusoff in the early 1970s (eq 1) for competitive

compounds.(21) The importance of that relationship did not escape the drug discovery community and

it has been invoked to explain disconnects between biochemical and cellular compound activity,(22,23)

as presented in a seminal article by Shokat et al.(24) It should be noted that these concerns are only

relevant in the context of ATP competitive, type I kinase inhibitors. Nonetheless, the literature is very

sparse in case studies where this concern is clearly articulated or addressed experimentally. An example

of incorporating this thinking in our understanding of kinase function was described for the

identification of inhibitors for interleukin-2 inducible T cell kinase (ITK).(25) In recent years the

importance of JAK3 in the JAK1/JAK3 heterodimeric pair has been the source of significant discussion

and conflicting conclusions.(26, 27, 28) There is a challenge for a dual JAK3/1 compound, to determinecorrelations utilizing enzyme data to cell data and the individual kinase contribution to cellular signaling.

In this report we describe the relationships of enzyme inhibition measured at of both KM and

physiological ATP concentrations and how these relate to cellular activity. We find these considerations

to be critical in understanding the role of individual JAK kinases in their heterodimeric signaling

complexes.

IC50= Ki*(1+[ATP]/[KM,ATP]) (eq. 1)


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Results and discussion

Starting from the Cheng-Prusoff relationship (eq.1), the predicted potency shift for a compound

tested at two different ATP concentrations (for example [A] and [B]) is described by eq.2. This equation

can be further simplified to eq.3 when one of the IC50values is determined at [ATP]=KM.

Shift = IC50A/IC50B= (1+[ATPA]/[KM,ATP])/ (1+[ATPB]/[KM,ATP]) (eq. 2)

Shift = IC50A/IC50KM= (1+[ATPA]/[KM,ATP])/2 (eq. 3)

Experimentally, we measured the enzyme activity of the catalytic domains of the four JAK

isoforms, determining both KMas well as the fraction of active protein through active site titration (Table

1). The assays were also optimized to measure enzyme inhibition in the presence of 1 mM ATP with all

isoforms producing linear correlations between IC50values obtained in assays run at [ATP]=KMand 1 mM

ATP. In order to understand in greater detail the interplay of the various JAK heterodimers on cellular

function, we mined the Pfizer database for compounds having been evaluated in both enzymatic and

cellular JAK assays. We then evaluated these compounds against the four JAK isoforms utilizing a 1 mM

ATP concentration. The enzyme concentration coupled with its active fraction determines the lower

limit of accuracy for enzymatic assays. Consequently, IC50values above 3 nM were considered relevant

for this discussion. Similarly, while the compound concentrations used allowed for IC50measurements of

up to 10 M, we chose to restrict the range of IC50values used in this analysis to those below 1 M due

to the lack of solubility at 10M of some compounds in this broad set. The entire range of determined

IC50for all compounds is nonetheless illustrated in each graph. The measured shift was largest for JAK3

(approximately 100 fold, Figure 1), while being smallest for JAK1 (approximately 10 fold, figure S1). In

the case of TYK2 and JAK2 the shift was around 25 and 50 fold, respectively (data not shown). Whencompared to the shifts predicted by eq.3 there was good agreement between measured and theoretical

shifts.

Table 1. Characterization of the four JAK isoforms.

Enzyme% Active

Enzyme

ATP KMAssays1mM ATP

AssaysTheoretical

Shift

Experimental

ShiftATP

(M)Total

Enzyme

(nM)

Total

Enzyme

(nM)

JAK1 12 40 20 30 12 ~10

JAK2 60 4 1 2 125 ~50

JAK3 40 4 1 2 125 ~100

TYK2 40 12 1 2 42 ~25

Figure 1. Correlation of JAK3 compound potency at [ATP]=KMvs 1mM


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Six cytokines (IL-2, -4, -7, -9, -15, -21) signal through the JAK1/JAK3 heterodimeric pair. In this

study we elected to study IL-15 and IL-21 signaling through JAK1/JAK3 by measuring STAT5

phosphorylation in peripheral blood mononuclear cells (PBMC) and in Kit225 cells. When we compared

enzyme IC50values obtained at [ATP]=KMfor JAK1 and JAK3 isoforms and cellular activities, JAK3 enzyme

potency displayed little correlation with Kit225 cellular activity, even when cellular permeability was

taken into account (Figure S2a-b). Following this finding, we focused our analysis on data generated with

human PBMCs to avoid any artificial bias introduced by using immortalized cells. Using this more

physiologically relevant cellular system, a different picture emerges irrespective of whether the

correlation is being performed with JAK3 enzyme data obtained at KMor 1mM ATP (Figure 2a-b). In both

instances compounds which are permeable (green) and have high selectivity at 1mM ATP (JAK1

IC50/JAK3 IC50>50, triangles) display a good correlation between enzymatic and cellular activities.

Figure 2. Relationship between IL-15 stimulated STAT5 phosphorylation and JAK3 enzymatic activity. a)

Enzyme assay with ATP concentration at KM, b) enzyme assay with ATP concentration at 1 mM.

2a) 2b)

Legend color by binned RRCK passive permeability AB red 10 (high), gray data not available. Shape by x = JAK1/JAK3 binned selectivity using 1mM ATP assay IC50

100x

100x

10x

10x

JAK3 CaliperIC50 (4uM ATP, uM)

0.005

0.01

0.05

0.1

0.5

1

5

10

0.00... 0 .00... 0.001 0.005 0.01 0.05 0.1 0.5 1 5 10

JAK3 Caliper IC50 (1mM ATP, uM)

0.01

0.05

0.1

0.5

1

5

10

20

0.005 0.01 0.05 0.1 0.5 1 5 10


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value. Square: x


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3a) 3b)

Legend Shape by x = JAK1/JAK3 binned selectivity using 1 mM ATP assay IC50 values. Square: x


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Legend color by binned IL-15 cellular IC50, pink: 0.25


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concentration rather than at ATP KMin order to properly assess compound selectivity and better predict

cellular function.

Acknowledgements

We would like to thank the numerous members of Pfizers JAK research project team who designed,

prepared and evaluated JAK compounds thus enabling the data mining and additional evaluation of

these compounds.

Materials and Methods

JAK Enzymes

GST-tagged recombinant human kinase domains of JAK1, JAK2 and JAK3 were purchased from

Invitrogen. His-tagged recombinant human TYK2 kinase domain was expressed in SF21/Baculovirus and

purified using a 2-step affinity (Ni-NTA) and size exclusion (SEC S200) purification method. Seesupplementary information for commercial and sequence information.

JAK Caliper Assays

The human Janus Kinase (JAK) activity was determined by using a microfluidic assay to monitor

phosphorylation of a synthetic peptide by the recombinant human kinase domain of each of the four

members of the JAK family, JAK1, JAK2, JAK3 and TYK2. Reaction mixtures contained 1 M of a

fluorescently labeled synthetic peptide, and ATP at either a level equal to the apparent KMfor ATP or at

1 mM ATP. Each assay condition was optimized for enzyme concentration and room temperature

incubation time to obtain a conversion rate of 20% - 30% phosphorylated peptide product. Reactions

were terminated by the addition of stop buffer containing EDTA. Utilizing the LabChip 3000 mobility

shift technology (Caliper Life Science), each assay reaction was sampled to determine the level of

phosphorylation. This technology is separation-based, allowing direct detection of fluorescently labeled

substrates and products with separations controlled by a combination of vacuum pressure and electric

field strength optimized for the peptide substrate.

Determination of apparent KMfor ATP using the Caliper JAK Enzyme Assays

Kinase assays were carried out at room temperature in a 384-well polypropylene plate in 80L of

reaction buffer containing 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum

albumin (BSA), 0.0005% Tween 20, 1mM DTT and 2% DMSO. Reaction mixtures contained 1M of a

fluorescently labeled synthetic peptide (5FAM-KKSRGDYMTMQID for JAK1 and TYK2 and FITC-

KGGEEEEYFELVKK for JAK2 and JAK3) and various concentrations of ATP. The kinase reactions wereinitiated by the addition of JAK enzymes and were sampled at various time points to determine the level

of peptide phosphorylation. The percent product converted was determined for each sample based on

peak height (percent product = product/(product+substrate)). The enzyme velocities were determined

for each concentration of ATP and a KMfor ATP was determined using the Michaelis-Menten model,

Y = Vmax*X/(KM+ X). Vmaxis the maximum enzyme velocity and KM, the Michaelis-Menten constant, is the

substrate concentration needed to achieve a half-maximum enzyme velocity.


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Caliper JAK Enzyme Endpoint IC50Assays

Test compounds were solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM.

Compounds were diluted in DMSO to create an 11-point half log dilution series with a top concentration

of 600 M. The test compound plate also contained positive control wells with a known inhibitor to

define 100% inhibition and negative control wells with DMSO to define no inhibition. The compound

plates were diluted 1 to 60 in the assay, resulting in a final assay compound concentration range of

10 M to 100 pM and a final assay concentration of 1.7% DMSO. 250 nL of test compounds and controls

solubilized in 100% DMSO were added to a 384 well polypropylene plate (Matrical) using an non contact

acoustic dispenser. Kinase assays were carried out at room temperature in a 15L reaction buffer

containing 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA),

0.0005% Tween 20 and 1mM DTT. Reaction mixtures contained 1M of a fluorescently labeled

synthetic peptide, a concentration less than the apparent KM (5FAM-KKSRGDYMTMQID for JAK1 and

TYK2 and FITC-KGGEEEEYFELVKK for JAK2 and JAK3). Reaction mixtures contained ATP at either a level

equal to the apparent KMfor ATP (40 M for JAK1, 4 M for JAK2, 4 M for JAK3 and 12 M for TYK2) or

at 1 mM ATP. The assays were stopped with 15L of a buffer containing 180 mM HEPES, pH=7.4, 20 mM

EDTA, 0.2% Coating Reagent, resulting in a final concentration of 10 mM EDTA, 0.1% Coating Reagent

and 100 mM HEPES, pH=7.4. Each assay reaction was then sampled to determine the level of

phosphorylation. The data output used for calculations was percent product converted and was was

determined for each sample and control well based on peak height (percent product =

product/(product+substrate)). The percent effect at each concentration of test compound was

calculated based on the positive and negative control well contained within each assay plate using the

formula % effect = 100*((sample well negative control)/(positive control-negative control)). The

percent effect was plotted against the compound concentration compound. An unconstrained sigmoid

curve was fitted using a 4 parameter logistic model and the concentration of test compound required

for 50% inhibition (IC50) was determined for each test compound.Preparation of peripheral blood mononuclear cells (PBMC)

Cryopreserved human PBMCs (Catalog No. PB005F), which were used in the IL-2, IL-10, IL-15, and IFN

assays, were purchased from Allcells (Emeryville, CA). Frozen PBMCs were thawed in a water bath

(37C), and washed once with RPMI1640 medium (Catalog No. 72400, Invitrogen, Grand Island, NY).

Cells were resuspended in RPMI medium containing 10% fetal bovine serum (FBS) and incubated at 37C

for 1.5 hours for resting.

Cytokine Stimulation and FACS Sample Preparation

Serum was removed from resting PBMCs by washing once with D-PBS. PBMCs were resuspended in

RPMI1640 medium. Ninety (90) L of resuspended PBMCs (5 million cells/mL) were aliquoted in 96-

well, deep well, V-bottom plates (Catalog No. 82007-292; VWR, Radnor, PA) and incubated at 37C for

75 minutes. Cells were treated with compound (5L/well) at various concentrations (0.2% DMSO final)

at 37C for 60 minutes, followed by the challenge with cytokine (5L/well; final concentration of 41

ng/mL IL-2, 41 ng/mL IL-15, 5000 U/mL IFN, 15 ng/mL IL-10) for 15 minutes. Cells were treated with

warm Lyse/Fix buffer (700 L/well; Catalog No. 558049; BD Biosciences) to terminate activation and

further incubated at 37C for 15 minutes. Plates were centrifuged at 300 x g for 5 minutes, supernatant

was aspirated, and cells were washed with 800L per well of staining buffer (0.5% heat inactivated FBS


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and 0.001% sodium azide in D-PBS). The washed cell pellets were resuspended with 350L per well of

cold 90% methanol (-20C), and incubated on ice for 30 minutes. After the removal of 90% methanol,

cells were washed once with staining buffer (800L/well). Cell pellets were resuspended in staining

buffer containing anti-phospho-STAT-AlexaFluor647 conjugated antibodies (1 to 150 dilution, 150

L/well), and incubated at room temperature in the dark overnight. Anti-phospho STAT3-AlexaFluor647

(Catalog No. 557815; BD Biosciences) was used for IL-10 and IFN stimulated cells, and anti-phospho

STAT5-AlexaFluor647 (Catalog No. 612599; BD Biosciences) was used for Il-2 and IL-15 stimulated cells.

Flow Cytometry

Samples were transferred to 96-well U-bottom plates and flow cytometric analysis wasperformed on a

FACSCalibur, FACSCanto or LSRFortessa equipped with a HTS plate loader(BD Biosciences). Lymphocyte

population was gated for the pSTAT3 or pSTAT5 (APC channel) histogram analysis. Background

fluorescence was defined using unstimulated cells and a gate (M1) was placed at the foot of the peak to

include ~0.5% gated population. The histogram statistical analysis was performed using CellQuestPro

version 5.2.1 (BD Biosciences), FACSDiva version 6.2 (BD Biosciences) or FlowJo version 7.6.1 (Ashland,OR) software. Relative fluorescence unit (RFU), which measures the level of pSTAT, was calculated by

multiplying the percent positive population (M1) and its mean fluorescence. IC50 values were

determined using the Prism version 5 software (GraphPad, La Jolla, CA).

Supporting information

Figure S1. Correlation of JAK1 enzyme activity at KMvs 1mM [ATP]

Figure S2. Correlation of KMenzyme activity to inhibition of IL-15 stimulated STAT5 phosphorylation inKit225 cells in relationship to JAK1/JAK3 selectivity derived from enzyme assay at KM. a) JAK3 enzyme

activity at KM. b) JAK3 enzyme activity at 1 mM ATP.

JAK1 Caliper IC50 (40 uM ATP, uM)

0.0005

0.001

0.005

0.01

0.05

0.1

0.5

1

5

10

0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1 5 10

100x

100x

10x

10x


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a)

b)

Legend color by binned RRCK passive permeability AB red 10 (high). Shape by x = JAK1/JAK3 binned selectivity using KMassay value. Square: x


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841 ptqfeerhlk flqqlgkgnf gsvemcrydp lqdntgevva vkklqhstee hlrdfereie

901 ilkslqhdni vkykgvcysa grrnlklime ylpygslrdy lqkhkeridh ikllqytsqi

961 ckgmeylgtk ryihrdlatr nilvenenrv kigdfgltkv lpqdkeyykv kepgespifw

1021 yapesltesk fsvasdvwsf gvvlyelfty ieksksppae fmrmigndkq gqmivfhlie

1081 llknngrlpr pdgcpdeiym imtecwnnnv nqrpsfrdla lrvdqirdnm ag

Jak3 kinase domainInvitrogen part Number PV4080

Recombinant Human Protein, Catalytic Domain, GST-tagged, expressed in insect cells

GenBank Accesion Number NP_000206

Amino Acids 781 1124

781 issdyellsd ptpgalaprd glwngaqlya cqdptifeer hlkyisqlgk gnfgsvelcr

841 ydplgdntga lvavkqlqhs gpdqqrdfqr eiqilkalhs dfivkyrgvs ygpgrqslrl

901 vmeylpsgcl rdflqrhrar ldasrlllys sqickgmeyl gsrrcvhrdl aarnilvese

961 ahvkiadfgl akllpldkdy yvvrepgqsp ifwyapesls dnifsrqsdv wsfgvvlyel

1021 ftycdkscsp saeflrmmgc erdvpalcrl lelleegqrl pappacpaev helmklcwap

1081 spqdrpsfsa lgpqldmlws gsrgcethaf tahpegkhhs lsfs

Tyk2 kinase domainPurification: in house

Recombinant Human Protein, Catalytic Domain, His-tagged, expressed in insect cells

Amino Acid sequence of construct: human His-TEV-tagged TYK2 Wild Type

1 MAHHHHHHHH HHGALEVLFQ GPGDPTVFHK RYLKKIRDLG EGHFGKVSLY

51 CYDPTNDGTG EMVAVKALKA DAGPQHRSGW KQEIDILRTL YHEHIIKYKG

101 CCEDAGAASL QLVMEYVPLG SLRDYLPRHS IGLAQLLLFA QQICEGMAYL

151 HSQHYIHRDL AARNVLLDND RLVKIGDFGL AKAVPEGHEY YRVREDGDSP

201 VFWYAPECLK EYKFYYASDV WSFGVTLYEL LTHCDSSQSP PTKFLELIGI

251 AQGQMTVLRL TELLERGERL PRPDKCPAEV YHLMKNCWET EASFRPTFEN

301 LIPILKTVHE KYQGQAPS

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