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.
Atli.Thorarensen@pfizer.com
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 K
M
of 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 K
M
ATP 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 K
M

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 and
pharmacological 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
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 K
M
producing IC
50,KM values or by describing the intrinsic affinity of the inhibitors as a K
D
. 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 K
D
nor an IC
50
measurement at ATP K
M
can be a good descriptor of
cellular activity for ATP competitive inhibitors. The relationship of activity at various substrate
concentrations 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 determine
correlations 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 K
M
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.
IC
50
= Ki*(1+[ATP]/[K
M
,
ATP
]) (eq. 1)

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 IC
50
values is determined at [ATP]=K
M
.
Shift = IC
50A
/IC
50B
= (1+[ATP
A
]/[K
M
,
ATP
])/ (1+[ATP
B
]/[K
M
,
ATP
]) (eq. 2)
Shift = IC
50A
/IC
50KM = (1+[ATP
A
]/[K
M
,
ATP
])/2 (eq. 3)
Experimentally, we measured the enzyme activity of the catalytic domains of the four JAK
isoforms, determining both K
M
as 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 IC
50
values obtained in assays run at [ATP]=K
M
and 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, IC
50
values above 3 nM were considered relevant
for this discussion. Similarly, while the compound concentrations used allowed for IC
50
measurements of
up to 10 M, we chose to restrict the range of IC
50
values used in this analysis to those below 1 M due
to the lack of solubility at 10 M of some compounds in this broad set. The entire range of determined
IC
50
for 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). When
compared 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 K
M
Assays
1mM ATP
Assays
Theoretical
Shift
Experimental
Shift ATP
(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]=K
M
vs 1mM

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 IC
50
values obtained at [ATP]=K
M
for 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 K
M
or 1mM ATP (Figure 2a-b). In both
instances compounds which are permeable (green) and have high selectivity at 1mM ATP (JAK1
IC
50
/JAK3 IC
50
>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 K
M
, b) enzyme assay with ATP concentration at 1 mM.
2a) 2b)
Legend color by binned RRCK passive permeability AB red <2.5 (low), yellow 2.5-10 (moderate), green
>10 (high), gray data not available. Shape by x = JAK1/JAK3 binned selectivity using 1mM ATP assay IC
50

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
value. Square: x <1, circle: 1<x<50, triangle: 50<x. Lines illustrate shifts between the assay (black unity,
blue 10x, red 100x)
In order to better understand the relationship between inhibitions of JAK1 and JAK3 enzymes to
cellular function, we conducted a more refined analysis using the subset of compounds with good
cellular permeability. The correlations between the individual JAK1 or JAK3 isoforms and the cellular
heterodimeric readout are depicted in Figure 3a-b. It should be noted that these correlations are only
observed when utilizing enzyme and selectivity data obtained at 1 mM ATP. This is due to a very limited
overlapping dynamic range of the cell based assay IC50s and the Km based JAK3 enzyme assay IC50s
that would be expected to translate into a measurable cellular inhibition. Here, compounds with
selectivity greater than one for a specific isoform display an excellent correlation between enzyme
inhibition of this individual JAK isoform at 1mM ATP and the cellular readout. This illustrates that activity
of the more dominantly inhibited JAK isoform has good translation to cellular function. There was no
correlation between inhibition of other JAK isoforms (JAK2 and TYK2) and cellular readout utilizing IL-15
as stimulant (data not shown). An alternative way of illustrating the relationship between JAK1 and JAK3
biochemical potency and IL-15 mediated cell signaling is to plot the cellular activity against the enzyme
activity ratios for both JAK1 and JAK3 (Figure 4). Figure 4A depicts the theoretical correlation one could
expect to observe depending on whether cellular activity would be controlled by JAK1 and/or JAK3. This
plotting approach illustrates that selective compounds for either isoform can lead to complete inhibition
of cellular activity. In conclusion, inhibition of IL-15 signaling can be achieved by sufficiently inhibiting
either one of the members of the JAK1/JAK3 heterodimer and the potency in the enzyme assay
determined with 1 mM ATP is predictive of the cellular readout.

Figure 3. Relationship between IL-15 stimulated STAT5 phosphorylation and JAK3, JAK1 enzymatic
activity only considering highly permeable compounds. a) JAK3 enzyme assay at 1 mM ATP data is only
illustrated for permeable compounds with a JAK1/JAK3 ratio greater than 1, b) JAK1 enzyme assay at 1
mM ATP, data is only illustrated for permeable compounds with a JAK1/JAK3 ratio lower than 1.

3a) 3b)
Legend Shape by x = JAK1/JAK3 binned selectivity using 1 mM ATP assay IC
50
values. Square: x <0.1, star:
0.1<x<1, triangle: 1<x<10, circle: 10<x. Lines illustrate shifts between the assay (black unity, blue 10x,
red 100x)
Figure 4. Relationship between A) predicted and B) experimental cellular and enzyme (1mM ATP)
potency for JAK1 and JAK3
A

B
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

Legend color by binned IL-15 cellular IC
50
, pink: 0.25<x<1 M, blue: 0.05<x<0.25, yellow: 0.01< x <0.05,
green: x <0.01 M . J1: JAK1, J3: JAK3.

There are several reported examples of compounds utilized to investigate the role of the
individual JAK partners in signaling events.(16-20) We decided to evaluate two JAK3 selective inhibitors,
NIBR3049 (28) and WYE-151650 (27), previously characterized in the literature. Previous work with
NIBR3049 led to the conclusion that JAK1 is dominant over JAK3 in cellular signaling. In contrast, studies
conducted with WYE-151650 indicated that inhibiting JAK3 is sufficient to fully inhibit JAK1/JAK3
dependent signaling pathways and to achieve efficacy in the mouse collagen-induced arthritis model
(27). We profiled both compounds in our [ATP]=K
M
and 1mM enzyme assays as well as in a series of
cellular assays (Table 2). For NIBR3049, the half maximal inhibition of JAK3 enzyme activity in the
presence of 1 mM ATP was measured at 131 nM as compared to 1.5 nM at K
M
(4 M) ATP, an IC
50
of 8
nM obtained with 18 M ATP was reported in ref 28. Its IC
50
for JAK1 was approximately 2 M at 1 mM
ATP. This compound therefore only displays 18 fold selectivity for JAK3 over JAK1 at high ATP.
Importantly, the cellular inhibition measured downstream of IL-2 or IL-15 signaling is now comparable to
the observed inhibition of JAK3 enzyme activity. In comparison, WYE-151650 showed a similar ratio of
selectivity for JAK3 over JAK1 at 1 mM ATP with the JAK3 inhibition at 1 mM ATP translating fairly well
into the cellular inhibition observed downstream of IL-15. The additional JAK3 inhibition data obtained
at 1 mM ATP thus bridges the contradiction within the literature on the importance of JAK3 function in
-common chain cytokine receptor signaling. Importantly the PBMC assays displayed little variability
from batch to batch of PBMCs and day to day. For example, in the IL-15 assay IC50 = 535 nM as a
geometric mean of 5 individual data points obtained on three different days with two batches of PBMC
(Batch A2544 IC50 = 437, 439, 534, 648 nM and batch 2687 IC50 = 659 nM). This clearly illustrates that
cellular concentrations of ATP should be taken into account when interpreting enzyme to cell shifts for
kinase targets. When this is done and with a bigger dataset, it becomes clear that JAK3 inhibition is in
fact sufficient for blocking STAT5 phosphorylation in PBMCs stimulated with IL-15.
Table 2. Characterization of NIBR3049 and WYE-151650.
NIBR3049* NIBR3049
#
WYE-151650
JAK1 IC
50
(nM) K
M
/1mM ATP 1,017 211/2,387 (n=4) 16/111 (n=2)
JAK2 IC
50
(nM) K
M
/1mM ATP 2,550 141/5,453 (n=4) 1/28 (n=2)
JAK3 IC
50
(nM) K
M
/1mM ATP 8 1.5/131 (n=4) 0.4/12 (n=2)
TYK2 IC
50
(nM) K
M
/1mM ATP
8,055
2101/>10,000
(n=4)
17/401 (n=2)
P-STAT5/IL-2 (nM) [J1,J3] 1,294 524 (n=5) ND*
P-STAT5 /IL-15 (nM) [J1,J3] 525 535 (n=5) 29 (n=4)
P-STAT3/ IL-10 (nM) [J1,T2] NA ~10000 (n=4) 610 (n=2)
P-STAT3/IFN (nM) [J1,T2] NA 536 (n=6) 41 (n=1)*
*data from reference Error! Bookmark not defined. ,
#
data generated for this paper, IL-2 mediated
signaling occurs through the JAK1, JAK3 heterodimer, IL-15 mediated signaling occurs through the JAK1,
JAK3 heterodimer, IL-10 mediated signaling occurs through the JAK1, TYK2 heterodimer, IFN mediated
signaling occurs through the JAK1, TYK2 heterodimer. Cellular assays were conducted using human PBMCs.
* Testing limited due to compound availability
In summary, we have illustrated that determining enzyme inhibition of the individual JAK1 and
JAK3 isoforms under more physiologically relevant ATP conditions provides a better understanding of
the role that each individual partner plays in the heterodimeric signaling pair. With a series of
compounds displaying appropriate selectivity and high permeability we demonstrated that inhibiting
only one partner of the heterodimeric JAK1/JAK3 pair is sufficient to fully inhibit IL-15 signaling.
Accordingly, the differences in the ATP K
M
of the different JAK enzymes appear to be the major culprit
behind the observed disconnects between enzymatic and cellular assays with regard to inhibition and
selectivity. However, the significant differences observed in IC
50
values for WYE-151650 with different
cytokines (e.g. IL-10 and IFN) signaling through the JAK1/TYK2 heterodimer pair illustrates that
additional work is still required to fully understand the role of JAKs in cytokine receptor signaling.
Nonetheless, the approach described in this paper can be applied to other cytokine receptors and help
decipher the interplay of JAK enzymes in cytokine signaling. Furthermore, this study illustrates the
importance of utilizing biochemical IC
50
values obtained with a physiologically relevant ATP
concentration rather than at ATP K
M
in 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. See
supplementary 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 K
M
for 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 K
M
for ATP using the Caliper JAK Enzyme Assays
Kinase assays were carried out at room temperature in a 384-well polypropylene plate in 80 L 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 1 M 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 were
initiated 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 K
M
for ATP was determined using the Michaelis-Menten model,
Y = V
max
*X/(K
M
+ X). V
max
is the maximum enzyme velocity and K
M
, the Michaelis-Menten constant, is the
substrate concentration needed to achieve a half-maximum enzyme velocity.
Caliper JAK Enzyme Endpoint IC
50
Assays
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 15 L 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 1 M 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 K
M
for 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 15 L 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 (IC
50
) 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 (5 L/well) at various concentrations (0.2% DMSO final)
at 37C for 60 minutes, followed by the challenge with cytokine (5 L/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 800 L per well of staining buffer (0.5% heat inactivated FBS
and 0.001% sodium azide in D-PBS). The washed cell pellets were resuspended with 350 L 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 (800 L/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 was performed 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 CellQuest Pro
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 K
M
vs 1mM [ATP]

Figure S2. Correlation of K
M
enzyme activity to inhibition of IL-15 stimulated STAT5 phosphorylation in
Kit225 cells in relationship to JAK1/JAK3 selectivity derived from enzyme assay at K
M
. a) JAK3 enzyme
activity at K
M
. 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
a)
b)
Legend color by binned RRCK passive permeability AB red <2.5 (low), yellow 2.5-10 (moderate), green
>10 (high). Shape by x = JAK1/JAK3 binned selectivity using K
M
assay value. Square: x <1, circle: 1<x<50,
triangle: 50<x.
Jak1 kinase domain
Invitrogen part Number PV4775
Recombinant Human Protein, Catalytic Domain, GST-tagged, expressed in insect cells
GenBank Accesion Number NP_002218.2
Amino Acids 866 1154

841 dpthf ekrflkrird lgeghfgkve lcrydpegdn
901 tgeqvavksl kpesggnhia dlkkeieilr nlyhenivky kgictedggn giklimeflp
961 sgslkeylpk nknkinlkqq lkyavqickg mdylgsrqyv hrdlaarnvl vesehqvkig
1021 dfgltkaiet dkeyytvkdd rdspvfwyap eclmqskfyi asdvwsfgvt lhelltycds
1081 dsspmalflk migpthgqmt vtrlvntlke gkrlpcppnc pdevyqlmrk cwefqpsnrt
1141 sfqnliegfe allk

Jak2 kinase domain
Invitrogen part Number PV4288
Recombinant Human Protein, Catalytic Domain, GST-tagged, expressed in insect cells
GenBank Accesion Number NP_004963
Amino Acids 808 1132

781 lft pdyelltend mlpnmrigal gfsgafedrd
JAK3 Caliper IC50 (4uM ATP, (uM)
0.005
0.01
0.05
0.1
0.5
1
5
10
20
0.00... 0.00... 0.001 0.005 0.01 0.05 0.1 0.5 1 5 10
JAK1 Caliper IC50 (40 uM, uM)
0.005
0.01
0.05
0.1
0.5
1
5
10
20
0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1 5 10
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 domain
Invitrogen 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 domain
Purification: 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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