JAK2 JH2 Fluorescence Polarization Assay and Crystal Structures for Complexes with Three Small Molecules

Article abstract of DOI:10.1021/acsmedchemlett.7b00154

A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe molecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are reported as well as K_d results for 10 compounds, including JNJ7706621, NVP-BSK805, and filgotinib (GLPG0634). X-ray crystal structures of JAK2 JH2 in complex with NVP-BSK805, filgotinib, and diaminopyrimidine 8 elucidate the binding poses.

Full text of DOI:10.1021/acsmedchemlett.7b00154

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Letter
JAK2 JH2 Fluorescence Polarization Assay and Crystal
Structures for Complexes with Three Small Molecules
Ana S. Newton, Luca Deiana, David Puleo, Jose A. Cisneros,
Kara J Cutrona, Joseph Schlessinger, and William L Jorgensen
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 17 May 2017
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JAK2 JH2 Fluorescence Polarization Assay and Crystal Strucꢀ
tures for Complexes with Three Small Molecules
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Ana S. Newton,†,§ Luca Deiana,†,§ David E. Puleo,‡ José A. Cisneros,† Kara J. Cutrona,† Joseph
Schlessinger,‡,* and William L. Jorgensen†,*
†Department of Chemistry, Yale University, New Haven, Connecticut 06520-8107, ‡Department of Pharmacology, Yale
University School of Medicine, New Haven, CT 06520-8066.
§: Both authors contributed equally to this work.
KEYWORDS: Janus Kinase 2, JAK2, JAK2 JH2, Pseudokinase Domain, Fluorescence Polarization, FP, JNJ7706621, NVP-
BSK805, filgotinib (GLPG0634), protein crystallography.
ABSTRACT: A competitive fluorescence polarization (FP) assay is reported for determining binding affinities of probe mol-
ecules with the pseudokinase JAK2 JH2 allosteric site. The syntheses of the fluorescent 5 and 6 used in the assay are report-
ed as well as Kd results for 10 compounds including JNJ7706621, NVP-BSK805 and filgotinib (GLPG0634). X-ray crystal
structures of JAK2 JH2 in complexes with NVP-BSK805, filgotinib, and a diaminopyrimidine 8 elucidate the binding poses.
Janus kinases (JAKs) are non-receptor tyrosine kinases
involved in the regulation of hematopoiesis, the immune
system, and cellular metabolism. In mammals, the family
has four members: JAK1-3 and tyrosine kinase 2 (TYK2).
Each protein contains an N-terminal FERM domain, an
SH2-like domain, a pseudo-kinase domain (JAK homology
2 or JH2) and the C-terminal kinase domain (JH1).1
Though JH2 domains are similar in structure to kinase
domains and include an ATP binding site, they lack or pos-
sess negligible catalytic activity and serve a primarily regu-
latory role on JH1 kinase activity.2-5 Notably, multiple dis-
eases are associated with mutations in JAK2 JH2 domain;
in particular, V617F has been linked to polycythemia vera
(PV), essential thrombocythemia (ET) and myelofibrosis
structures were obtained to elucidate the binding poses of
(PMF).6-10 Moreover, though disruption of ATP binding in
JH2 complexes.
JH2 showed only minor effects on JAK2 wild-type (WT)
A prerequisite for FP is a fluorescent probe with high
activity, it did inhibit the hyperactivity of the pathogenic
affinity for the binding site. For the JAK2 JH2 domain, the
V617F mutant.11 This suggests that small molecules that
initial choice was BODIPY-ATP 4 (Invitrogen, ThermoFish-
er Scientific supplier), a fluorophore attached to a ribose
bind at the JAK2 JH2 ATP site have potential for therapeu-
tic drug development.
ring.22 However, the affinity of tracer 4 towards JAK2 JH2
was weak (Kd = 7 ꢀM) and therefore could not be used to
determine accurately affinities in the nanomolar range. In
addition, the concentration of protein (7 ꢀM, Table 1)
needed for the assay is not desirable for high-throughput
screening.
Although potent inhibitors of Janus kinases have been
reported in the literature,12-13 drug discovery efforts have
not been able to address diseases caused by mutated
JAK2.11 The need for binders that selectively target JAK2
JH2 domain is therefore pressing. Since the JH2 domain is
not catalytic, an accurate and rapid direct binding assay is
needed for gauging potency. To this end, we report here
such an assay using fluorescence polarization (FP). This
binding assay contrasts conventional JAK assays (e.g., au-
tophosphorylation14-16 and proliferation17) by providing
quantitative measurements of binding constants, Kd.18 The
use of standard microplate readers, the ability to reanalyze
assay plates, and the fact that substrates or radiolabeled
reagents are not needed makes FP attractive for drug dis-
covery. Ten compounds, including the well-known multi-
kinase inhibitors JNJ7706621 1,19 NVP-BSK805 2,20 and
filgotinib 3,21 were tested, and three X-ray crystal
To overcome these problems, we designed and synthe-
sized tracers 5 and 6 (Schemes 1 and 2) by attaching a
fluorescein isothiocyanate (FITC) to two amino-analogs of
1, namely 18 and 24. A detailed description can be found
in the Supporting Information. Compound 1 was found
through an in vitro screen at the Yale Small Molecule Dis-
covery Center and determined to have a Kd of 106 nM by
isothermal titration calorimetry (ITC) with JAK2 JH2.22 The
minimum tracer concentration that retained a satisfactory
signal-to-noise ratio with 5 was found to be 1.5 pM.
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Table 1. Concentration of tracer and JAK2 JH2 protein
needed when using tracers 4 and 5 in FP assays.
carbonyl group of Glu627, and one between its terminal
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hydroxyl group and the sidechain of Asn678. The lower Kd
for 2 than 8 suggests that replacement of the hy-
droxymethylpyrazole with larger, more hydrophobic sub-
stituents could lead to enhanced binding.
Tracer 4
5 nM
7 μM
Tracer 5
1.5 pM
200 nM
[tracer]
[protein]
Scheme 1. Synthesis of tracer 5
Saturation experiments, which involved adding incremen-
tal amounts of JAK2 JH2 (0 to 4.8 ꢀM) to the tracer solu-
tion, were then carried out with measurements over 90
minutes and showed stable Kd values over time. The disso-
ciation constants of tracers 5 and 6 were determined to be
roughly equivalent near 0.2 ꢀM (Figure 1B), a significant
improvement over tracer 4. The lower Kd values translate
to correspondently lower protein concentrations needed
in the competitive assay (Table 1). Interestingly, tracer 5
showed a greater ∆FP over the range of protein concentra-
tions than tracer 6 (2.5-fold vs 1.5-fold, Figure 1A). This
suggests that the fluorescent properties of tracer 6 are
affected by its binding to the protein, so 5 was selected for
the subsequent competitive assays.
In view of the structure of 1, exploratory studies led to
preparation of potential JH2 binders containing diamino-
substituted heterocycles with terminal 4-cyanophenyl
substituents such as 7-13 (synthetic schemes are in the
Supporting Information). These compounds along with 1 –
3 were studied using the optimized FP assay (Table 2). The
most active compound, 1, has a Kd of 0.8 µM in the FP as-
say. This value is 8-fold weaker than obtained from ITC,
reflecting differences in conditions including use of 50 mM
Hepes vs 20 mM Tris-Cl buffer, and less glycerol (10% vs.
20%) and more protein (ca. 5 µM vs. 6 nM) for ITC.22 Filgo-
tinib (3), 7, 9, 10, and 13 showed less than ca. 10% bind-
ing in an initial screen at a concentration of 50 µM, so pre-
cise Kd values were not determined. Though the simple
pyrimidine 7 was inactive, expansion at C6 did provide
active compounds with 8 showing the lowest Kd.
To complement these studies and provide a solid basis
for structure-based design, X-ray crystal structures were
pursued for complexes of the small molecules with JAK2
JH2. Success was obtained for several compounds includ-
ing 2, 3, and 8 at 2.0, 1.9, and 1.6 Å resolution, respectively.
Full details are provided in the Supporting Information.
The binding poses are shown in Figure 2 and confirm the
expected positioning in the hinge region of the JH2 ATP
site. 2 only has one hydrogen bond (yellow dashed line)
with the backbone NH of Val629, while 3 adds one with the
carbonyl group. However, the complex with 8 appears
well-packed and has three additional hydrogen bonds with
the side-chain carbonyl group of Gln626, the backbone
Reagents and conditions: (a) (Boc)2O, THF, 23 °C, 20 h; (b)
THF, 65 °C, 20 h; (c) Hydrazine, THF, 70 °C, 2h; (d) Py, 18 h;
(e) THF, TFA, 50 °C, 3h; (f) Fluorescein-NCS, DIPEA, DMF, 23
°C, 1 h.
Scheme 2. Synthesis of tracer 6.
Reagents and conditions: (a) (Boc)2O, DCM, rt, 20 h; (b) Py-
BOP, HOBt, Et3N, THF, rt, 20 h; (c) THF, 65 °C, 20 h; (d) hydra-
zine, THF, 70 °C, 2 h; (e) Py, rt, 18 h; (f) THF, TFA, 50 °C, 3h;
(g) Fluorescein-NCS, DIPEA, DMF, rt, 1 h.
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A
Tracer 5
Tracer 6
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ꢀ2
ꢀ1
0
1
log [JAK2ꢀJH2 WT], ꢁM
B
1.0
0.5
0.0
5, Kd = 0.205 ± 0.010 µM
6, Kd = 0.227 ± 0.018 µM
0
2
4
[JAK2ꢀJH2 WT] ꢁM
Figure 1. Determination of binding affinities for tracers 5 and
6 (1.5 pM) through saturation experiments. (A) Variation of
FP values as a function of JAK2 JH2 WT concentration. (B) Kd
determination for tracers 5 and 6. Lb/Lt = ratio of ligand
bound to the total. Data from quadruplicate experiments in
three independent assays. Mean ± SEM plotted for all data.
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Table 2. Binding affinity values (Kd, µM) from the FP Assay.
Kd (µM)a
0.80 ± 0.05
Compd
1 JNJ7706621
2 NVP-BSK805
3 filgotinib (GLPG0634)
Figure 2. Renderings from the crystal structures of NVP-
BSK805 2 (A), filgotinib 3 (B), and 8 with wild type JAK2 JH2.
Carbon atoms of 2, 3, and 8 are colored yellow. The PDB codes
are 5UT4, 5UT5, and 5UT6.
42.0 ± 3.5
9% (50
0% (50
57.3 ± 2.8
3% (50 M)
11% (50 M)
µ
M)
µM)
7
8
9
10
11
12
13
µ
µ
In summary, the desire to discover small molecules that
selectively target the JH2 domain of JAK2 led us to pursue
a fluorescence polarization assay. This competitive binding
assay allows fast and accurate measurements of Kd values,
suitable for high-throughput screening. To this end, two
fluorescein-labeled ligands (5, 6) were synthesized. The
high affinity of tracer 5 (Kd = 0.2 µM) allowed for low con-
122.3 ± 18.5
106.0 ±18.8
3% (50 µM)
a
Kd or % bound at indicated concentration in parentheses.
Data shown from quadruplicate experiments in three inde-
pendent assays. Mean ± SEM.
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(6) Baxter, E. J.; Scott, L. M.; Campbell, P. J.; East, C.; Fourouclas,
centrations of both tracer and protein in the FP assay. The
FP assay was then applied to three well-known JAK inhibi-
tors (1-3) and seven synthesized compounds (7-13). In
conjunction with the reported crystal structures for com-
plexes with JAK2 JH2, application of structure-based and
computer-aided design to the discovery of potent JH2
binding molecules has a firm foundation.
N.; Swanton, S.; Vassiliou, G. S.; Bench, A. J.; Boyd, E. M.; Curtin, N.;
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Adelsperger, J.; Koo, S.; Lee, J. C.; Gabriel, S.; Mercher, T.; D’Andrea,
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ASSOCIATED CONTENT
9
Supporting Information. Full synthetic procedures and spec-
tral characterization data for all intermediates and final com-
pounds 5-13; crystallographic data for complexes 2, 3, and 8
with JAK2 JH2 have been deposited in the RCSB Protein Data
Bank with the PDB codes 5UT4, 5UT5, and 5UT6; experi-
mental details of FP assays. This information is available free
of charge via the Internet at http://pubs.acs.org .
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AUTHOR INFORMATION
Corresponding Authors
* joseph.schlessinger@yale.edu
* william.jorgensen@yale.edu
(10) Zhao, R. X.; Xing, S.; Li, Z.; Fu, X. Q.; Li, Q. S.; Krantz, S. B.;
Zhao, Z. H. J. Identification of an acquires JAK2 mutation in Polycy-
themia vera. J. Biol. Chem. 2005, 280, 22788-22792.
(11) Hammaren, H. M.; Ungureanu, D.; Grisouard, J.; Skoda, R.
C.; Hubbard, S. R.; Silvennoinen, O. ATP binding to the pseudoki-
nase domain of JAK2 is critical for pathogenic activation. Proc.
Natl. Acad. Sci. U.S.A. 2015, 112, 4642-4647.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
Gratitude is expressed to the Yale-Gilead collaboration for
support, to the staff of the Advanced Photon Source for assis-
tance, and to the NIH for training support of D.E.P.
(GM007324) and for support of the Yale Medical School X-ray
generator and detector (Shared Instrumentation Grant
1S10OD018007).
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Nat. Rev. Drug Discov. 2011, 10, 717-718.
(14) Changelian, P. S.; Flanagan, M. E.; Ball, D. J.; Kent, C. R.;
Magnuson, K. S.; Martin, W. H.; Rizzuti, B. J.; Sawyer, P. S. Preven-
tion of organ allograft rejection by a specific Janus Kinase 3 inhibi-
tor. Science 2003, 302, 875-878.
(15) Yang, S. M.; Malaviya, R.; Wilson, L. J.; Argentieri, R.; Chen,
X.; Yang, C. M.; Wang, B. B.; Cavender, D.; Murray, W. V. Simplified
staurosporine analogs as potent JAK3 inhibitors. Bioorg. Med.
Chem. Letter 2007, 17, 326-331.
(16) Li, Z.; Xing, S.; Wang, S. F.; Ho, W. T.; Zhao, Z. Z. J. Autoinhi-
bition of JAK2 tyrosine kinase is dependent on specific regions in
its pseudokinase domain. Exp. Hematol. 2007, 35, 1624-1632.
(17) Chen, J. J.; Thakur, K. D.; Clark, M. P.; Laughlin, S. K.;
George, K. M.; Bookland, R. G.; Davis, J. R.; Cabrera, E. J.; Easwaran,
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in small molecule screening. Expert. Opin. Drug Discovery 2011, 6,
17-32.
(19) Lin, R. H.; Connolly, P. J.; Huang, S. L.; Wetter, S. K.; Lu, Y.
H.; Murray, W. V.; Emanuel, S. L.; Gruninger, R. H.; Fuentes-
Pesquera, A. R.; Rugg, C. A.; Middleton, S. A.; Jolliffe, L. K. 1-acyl-
1H-[1,2,4]triazole-3,5-diamine analogues as novel and potent
anticancer cyclin-dependent kinase inhibitors: Synthesis and
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(20) Baffert, F.; Régnier, C. H.; De Pover, A.; Pissot-Soldermann,
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ABBREVIATIONS
JAK, Janus Kinases; JH2, pseudo-kinase domain; FP, Fluores-
cence Polarization; Boc, tert-Butyloxycarbonyl protecting
group; DCM, dichloromethane; THF, tetrahydrofuran; DMF,
dimethylformamide; Py, pyridine; TFA, trifluoroacetic acid;
DIPEA, N,N-diisopropylethylamine, PyBop, benzotriazol-1-yl-
oxytripyrrolidinophosphonium hexafluorophosphate; HOBt,
hydroxybenzotriazole.
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