Accepted Manuscript Bmcl digest Selective inhibitors of the Janus Kinase Jak3 – are they effective? Gebhard Thoma, Peter Drückes, Hans-Günter Zerwes PII: DOI: Reference:

S0960-894X(14)00890-7 http://dx.doi.org/10.1016/j.bmcl.2014.08.046 BMCL 21944

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

2 August 2014 19 August 2014 20 August 2014

Please cite this article as: Thoma, G., Drückes, P., Zerwes, H-G., Selective inhibitors of the Janus Kinase Jak3 – are they effective?, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl. 2014.08.046

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Selective inhibitors of the Janus Kinase Jak3 – are they effective? Gebhard Thomaa*, Peter Drückesb and Hans-Günter Zerwesc* a

Global Discovery Chemistry, bCenter for Proteomic Chemistry, Screening Sciences and cAutoimmunity, Transplantation and Inflammation Research, Novartis Institutes for Biomedical Research, Forum 1 Novartis Campus, 4056 Basel, Switzerland

*Corresponding authors. E-mail address:

[email protected] (G. Thoma) [email protected] (H.-G. Zerwes)

Keywords: Jak3, Jak1, Janus kinase inhibitors, Selectivity, Immunosuppression

Abstract: Jak3, together with Jak1, is involved in signal transduction initiated by cytokines signaling through the common gamma chain which are important in immune homeostasis and immune pathologies. Based on genetic evidence Jak3 has been considered to be an attractive target for immunosuppression. The Jak inhibitor tofacitinib (CP-690,550) which is an approved drug for rheumatoid arthritis was originally introduced as a selective Jak3 inhibitor, however, also inhibits Jak1 and Jak2. The search for new selective Jak3 inhibitors has yielded several compounds whose profiles will be reviewed here. Implications on Jak3 as a therapeutic target are also discussed.

Signal transduction initiated by multiple cytokines and growth factor receptors is mediated by dedicated non-receptor tyrosine kinases of the Janus kinase (Jak) family (table 1). The four members of this family (Jaks 1-3 and Tyk2) are multi-domain proteins of about 130 kDa and are highly homologous with respect to their domain structure. The catalytic kinase domain located at the C-terminus is preceded by a pseudokinase domain, a Src homology 2 (SH2) domain and the N-terminal FERM (four-point-one, ezrin, radixin and moesin homology) domain. The latter serves to facilitate the interaction between the Jak protein and the cytokine receptor.1–3 According to the canonical signaling pathway ligand binding to its cognate receptor triggers engagement of Jak kinases which, in a series of phosphorylation events targeting the receptor, the Jaks themselves and one or several of the 6 representatives of the STAT (signal transducer and activators of transcription) family members relay the signal into the cells. Phosphorylated STATs dimerize and migrate to the nucleus where they become part of transcriptional regulatory complexes which lead to transcription of responsive genes. The canonical Jak - STAT signaling pathway is evolutionary conserved and is active in multiple cell types where it is utilized by a variety of hormones, growth factors and cytokines and their receptors (table 1). This key signaling pathway has been elucidated over the last 25 years and has been the subject of multiple excellent reviews.1,3–8

Table 1. Cytokine receptors and the involvement of Jak kinases (from Cox and Cools9) Type

Subgroup

Receptors for

Jak Kinase Usage

Type I

Homodimeric

EPO, TPO, GH, G-CSF

Jak2

Type I

Using the common beta chain (CSF2Rβ, CD131)

IL3, IL5, GM-CSF

Jak2

Type I

Using the gp130 chain (IL6ST, CD130)

IL6, IL11, OSM, LIF

Jak1, Jak2, Tyk2

Type I

Using the common gamma chain (γc, IL2Rγ, CD132)

IL2, IL4, IL7, IL9, IL15, IL21

Jak1, Jak3

Type II

—

IFNα, IFNβ, IFNγ, IL10, IL19, IL20, IL22, IL24, IL28, IL29

Jak1, Jak2, Tyk2

The receptors for EPO, TPO, GH, and G-CSF are homodimers; all other cytokine receptors are heterodimers containing a ligand-specific chain and a common chain used by different receptors. Jak3 in conjunction with Jak1 is required for signaling of the type I receptors that use the common gamma chain (C).

Jak3 has been of particular interest because unlike the other Jak family members its expression is restricted to cells of the hematopoietic lineage. Jak3 (together with Jak1) is involved in signaling initiated by 6 cytokines (interleukins 2, 4, 7, 9, 15 and 21) which are important in immune homeostasis and, when de-regulated, in pathologies of the immune system.4,10 These receptors share the common gamma chain (CD132, C) which is paired with an alpha chain (for IL-4, 7, 9 and 21) or a beta chain (IL-2 and 15) as signaling partners and, in the case of IL-2 and 15, a non-signaling alpha chain. The importance of Jak3 in immune homeostasis has been underscored by the observations that loss-of-function mutations in humans result in severe combined immunodeficiency (Jak-3 SCID).3,11,12 A similar phenotype has been observed in mice in which Jak3 was deleted experimentally.13 Deficiency in the C also results in SCID (termed X-SCID because of the location of the C on the x-chromosome).

Based on this essential role of Jak3 in the regulation of immune responses it was considered a “tantalizing target for immunosuppression”14 and consequently, a number of academic and industrial research groups embarked on drug discovery projects aimed at inhibiting the kinase activity of Jak3. As Jak/STAT signaling is involved in a broad range of biological processes, it was deemed important to achieve high selectivity for Jak3 vs. other Jak kinases (as well as other kinases). Because of the high homology between the Jak family members, particularly in the catalytic domain, this was considered to be a challenging task which seemed to have been fulfilled by 1 (CP-690,550, figure 1), a compound which was reported to be a selective Jak3 inhibitor in a series of seminal publications.15,16 However, at closer inspection by the originating labs as well as by others it turned out that the compound, while displaying excellent selectivity over non-Jak kinases, also substantially inhibited Jak1, Jak2 and, to somewhat lesser extent Tyk2.17–22 The inhibition data for the different Jak kinases reported from several labs are shown in table 2.

Figure 1. Structures of Jak inhibitors

Table 2. Inhibition of Jak kinases by CP-690,550 in enzyme assays using the kinase domain Reference Changelian 200315 Jiang 200821 Williams 2009

19

Jak1a

Jak2a

Jak3a

Tyk2a

112

20

1

nd

3

2

0.7

250

1.6

22

6.5

nd

22

3.2

4.1

1.6

34

20

6.1

12

8

176

Soth 201323

1.6

4

2.2

nd

McDonnell 201424

3.8

11

1.4

24

Meyer 2010

Thoma 2011

a

IC50 [nM]

The fact that CP-690,550 (now known as tofacitinib and marketed in the US under the name Xeljanz) acts as a pan-Jak inhibitor may contribute to its broader pharmacological profile than that attributed to

selective ablation of C/Jak3-dependent cytokines as shown in experimental18,25 and in clinical studies.26 Data on CP-690,550 as well as on several Jak inhibitors with different isoform selectivity profiles currently in the clinic or approved have been recently summarized.27 Among them is VX-509 (Decernotinib) which is a potent inhibitor of Jak3 with limited selectivity within the Jak kinases.27,28 So far there is not enough openly accessible information to thoroughly evaluate the cellular selectivity profile of this compound. The quest for selective Jak3 inhibitors without activities on the other Jak kinases thus was still considered to be an important goal of many groups. In addition to being potentially safer immunosuppressive agents such compounds could also be of value to dissect the roles of individual Jak kinases in complex signaling processes involving more than a single Jak kinase. Despite of considerable efforts only a small number of more or less selective Jak3 inhibitors have been reported so far. The properties of the compounds which most closely approach the desired selectivity for Jak3 are discussed below. The definition of selectivity of inhibitors for individual Jak kinases poses challenges as discussed below. Since CP-690,550 is used in many labs as a reference compound, the data on the new agents should be reported in direct comparison to this compound. The IC50 values in Jak enzyme assays reported by several labs are given in table 2. Efficacy and selectivity in cellular assays are best assessed by readouts which are proximal to the Jak kinases. For most compounds discussed below data on STAT phosphorylation induced by various cytokines utilizing Jak kinases are available, IL2-induced STAT5 phosphorylation being the most consistently used readout. More “distal” effects including in vivo pharmacology (which likely involve other kinases as well) are not considered in this overview. We identified maleimide 2 (NIBR3049, figure 1) with an IC50 value of 8 nM for Jak3 which exhibited >120fold selectivity over the other Jak kinases in enzyme assays with IC50 values of 1017, 2550 and 8055 nM for Jak1, Jak2 and Tyk2, respectively.20 This remarkable selectivity has been confirmed by other groups (data shown in table 3).24,29 To our surprise NIBR3049, despite being equipotent to CP-690,550 on Jak3 in the kinase assay, the compound was more than 20-fold less potent in cellular assays interrogating IL-2triggered STAT5 phosphorylation (which depends on both, Jak1 and Jak3). This poor potency could not be explained by poorer cellular penetration because NIBR3049 was active in cellular assays independent of Jak kinases and showed intracellular compound concentrations similar to CP-690,550.20 The latter compound also potently inhibited Jak1 and Jak2 with IC50 values of 6 and 12 nM. Since Jak3 always cooperates with Jak1 in signal transduction through C we hypothesized that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction which is required for strong immunosuppression.

Table 3. Enzymatic and cellular data on NIBR3049 and CP-690,550 Assaya

NIBR304920

NIBR304924

NIBR304929

CP-690,55020

Jak1

1017

311

211

6.1

Jak2

2550

>10000

141

12

Jak3

8.0

0.5

1.5

8.0

Tyk2

8055

1691

2101

176

IL2 / P-STAT5

1294

48

IL15 / P-STAT5

525

24

a

IC50 [nM]

A group from Wyeth disclosed compound 3 (WYE-152050, figure 1) which inhibited Jak3 in an enzyme assay with an IC50 value of 0.9 nM.30 The compound was almost equipotent to CP-690-550 in an IL2induced STAT5 phosphorylation assay (IC50 values of 59 and 31 nM, respectively; table 4). The authors explain the efficacy of WYE-152950 with the selective inhibition of Jak3.30 However, the compound is also a nanomolar inhibitor of Jak1, Jak2 and Tyk2 (IC50 values of 32, 13 and 31 nM, respectively) and these effects could contribute to the efficacy observed in cells. Thus, these data do not necessarily prove that selective inhibition of Jak3 is sufficient to abrogate IL-2 signaling. WYE-152050 was also tested on GMCSF-induced STAT5 phosphorylation (depending on Jak2) as well as on IL6-induced STAT3 phosphorylation (depending on Jak1, Jak2 and Tyk2) and showed IC50 values of 1700 and 610 nM, respectively. The ratios of the IC50 values GM-CSF/IL-2 and IL-6/IL-2 of 29 and 10 seem to indicate functional selectivity within the Jak kinases. However, the cellular selectivity ratios observed for CP690,550 which is equipotent on Jak1, Jak2 and Jak3 (table 1) were 35 and 5, respectively. Thus, based on these data - despite the numerical selectivity for Jak3 in the kinase assay - WYE-152050 did not provide a significant cellular selectivity advantage over the unselective Jak inhibitor CP-690,550. Unfortunately, no data on CP-690,550 from the Wyeth enzymatic Jak assays were provided.

Table 4. Enzymatic and cellular data on WYE-152050 and CP-690,55030 Assaya

WYE-152050

CP-690,550

Jak1

32

nd

Jak2

13

nd

Jak3

0.9

nd

Tyk2

31

nd

IL2 / P-STAT5

59

31

1700

1100

610

160

GM-CSF / P-STAT5 IL6-P / STAT3 a

IC50 [nM]

Soth and colleagues recently published compound 4 which inhibited Jak3 with an IC50 value of 0.26 nM but also showed potent inhibition of Jak1 and Jak2 with IC50 values of 0.8 and 3.2 nM, respectively.23,31 Compound 4 efficiently inhibited IL2-induced STAT5 phosphorylation with an IC50 value of 31 nM (table 5). Interestingly, CP-690,550 which, compared to compound 4, was 2-fold more potent on Jak1 and 8fold less potent in in Jak3 kinase assays, showed the same level of efficacy (IC50 = 28 nM) in the IL2induced STAT5 phosphorylation assay. In STAT5 phosphorylation assays induced by GM-CSF (Jak2) and IFN- (Jak1, Jak2) both compounds showed similar potencies despite CP-690,550 being 5-fold less potent in the enzymatic Jak2 assay. In IL6-induced STAT phosphorylation assays (Jak1, Jak2, Tyk2) compound 4 was found to be 3-4 fold less potent than CP-690,550. However, due to the relatively small effect and the missing data on Tyk2 it is difficult to differentiate compound 4 and CP-690,550 and to rationalize the selectivity of 4 in cellular assays. If at all, the data indicate a slightly superior cellular potency of CP690,550 vs. 4.

Table 5. Enzymatic and cellular data on Compound 4 and CP-690,55023 Assaya

Compound 4

CP-690,550

Jak1

3.2

1.6

Jak2

0.8

4.0

Jak3

0.26

2.2

Tyk2

nd

nd

IL2 / P-STAT5

31

28

GM-CSF / P-STAT5

188

184

IFN / P-STAT1

291

170

IL6 / P-STAT3 (T cells)

1200

380

IL6 / P-STAT3 (monocytes)

864

238

a

IC50 [nM]

A recent Merck patent application on Jak inhibitors claimed pyrrolopyrimidines such as compound 5 (example 5.10, figure 1) with more than 100000 fold selectivity for Jak3 (IC50 = 0.013 nM) over Jak2 (IC50 > 1496 nM).32,33 Unfortunately, neither enzymatic inhibition data on Jak1 and Tyk2 nor data from cellular assays have been reported. As the compounds of interest carry reactive Michael acceptors it is conceivable that they are covalent modifiers reacting with Cys909 in proximity of the ATP-binding pocket of Jak3. As neither Jak2 nor Jak1 or Tyk2 have a cystein in this position the irreversible mode of action would explain the stunning selectivity observed in the biochemical assays. It is important to note that for irreversible inhibitors only “apparent” IC50 values can be determined in enzymatic assays because equilibrium conditions cannot be established. A covalent modifier of Jak3 with a fast “on-rate” will completely and irreversibly inactivate the enzyme. Thus, the apparent IC50 value will be extremely low for highly reactive compounds such as 5.33 If these compounds exhibited similar selectivity for Jak3 over Jak1 they would be perfect tools to investigate the effect of truly selective and complete inhibition of Jak3 on IL2 or IL15 signaling in presence of fully active Jak1.

Thorarensen and colleagues recently claimed to provide “the missing link in understanding the contribution of individual Jak kinase isoforms to cellular signalling”.29 The authors tested the ATPcompetitive Jak inhibitors NIBR3049 and WYE-152950 (along with a large number of Jak inhibitors from the Pfizer compound collection) in their biochemical Jak assays at either ATP concentrations corresponding to their Km values (40, 4, 4, 12 µM for Jak1, Jak2, Jak3 and Tyk2, respectively; table 6) or the high concentration of 1 mM ATP. As expected all IC50 values increased with increasing ATP concentrations (table 7).34 Due to the lower Km for ATP of Jak3, the IC50 values for Jak3 were more affected by the high ATP concentration than those for Jak1. Thus, the selectivity ratio for Jak3 over Jak1

at 1 mM ATP compared to Km for ATP dropped considerably (NIBR3049, 141→18; WYE-152950, 40→9) leading to the conclusion that the originally reported selectivity ratios were overstated. The compounds were also tested in STAT phosphorylation assays dependent on either Jak1/3 or Jak1/Tyk2 (table 7). Both activity and selectivity of NIBR3049 and WYE-152950 for Jak1 and Jak3 obtained at 1 mM ATP translated fairly well into potency and selectivity in the cellular assays. Thus, the authors suggested that Jak kinase assays utilizing 1 mM ATP yield data which are more predictive for the cellular activity and selectivity of compounds compared to assays utilizing ATP concentrations at Km. The authors also concluded that inhibition of either Jak1 or Jak3 is sufficient to fully inhibit STAT5 phosphorylation. However, due to the limited selectivity of WYE-152950 for Jak3 over Jak1 (table 4) the data do not necessarily prove that the effects seen in the Jak1/3 dependent cell assay are indeed solely explained by inhibition of Jak3. It is noteworthy that CP-690,550 had been tested earlier at 1 mM ATP and showed an IC50 value of 55 nM27 which is just 2.4-fold lower compared to NIBR3049 in the same assay (133 nM).29 In Jak1/3-dependent cellular assays CP-690,550 was found to be more than 20-fold more potent (table 3).20,35 If IC50 values for Jak3 at 1 mM ATP were predictive for cellular potency one would have expected similar cellular potencies for both compounds. Thus, the data on NIBR3049 and CP-690,550 do not support the offered hypothesis. Generally, the predictability of cellular potency and selectivity by in vitro kinase assays is limited due to numerous parameters in addition to the ATP concentration. The true “in vivo substrate affinities” of kinases are unknown but have been suggested to be similar to the measured Km values of kinase constructs used in vitro.36 All reported biochemical Jak assays utilize recombinant kinase (JH1) domain with variable tags or amino acid extensions instead of full length kinase. Km values of different JH1 constructs of the same Jak kinase differ (see table 6). Furthermore, Km values of recombinant kinases are sensitive to the specific assay conditions such as choice of protein substrate or buffer composition (in particular cations).36 Performing assays at concentrations of ATP at Km attempts to compensate for these differences. Thus, these assays generally utilize ATP concentrations which are low compared to the cellular ATP concentration estimated to be in the range of 1-5 mM. However, if the same high ATP concentration (1 mM) is utilized across all biochemical Jak assays independent of the Km of the construct29 the IC50 values of an inhibitor depends on the Jak construct and the assay conditions used. The selectivity profile (i.e. the ratio of the IC50 values for two Jak kinases) will be even more affected. As a further complication, in the cellular context the catalytic activity of the full length kinase might be regulated by additional domains. Indeed both the FERM domain as well as the JH2 (pseudokinase) domain have been reported to impact on the catalytic activity of Jak3 in the cellular context.37,38 It is also well established that kinases are part of a multi-protein signaling complexes in which they fulfill important structural roles independent of their catalytic function.39,40 These aspects are not captured in the enzyme assays. Importantly, Keil et al.41 recently reported that Jak2 has kinase independent functions in the context of the IFNGR in vivo. Because of these considerations performing kinase assays at high ATP concentrations does not provide deeper insight compared to the “conventional” assays utilizing ATP concentrations at Km.42

Table 6. Km values for ATP for different batches of Jak enzyme used in biochemical assays Kinasea

Pfizer29

Roche23

Novartisd

Novartise

Knight and Shokat36

40b

20b

70c

45b

15

b

a

nd

Jak1

b

Jak2

4

Jak3

4b

6

c

1.5c

20

18

18c

Tyk2 12b nd 33c a Km [µM] b Enzyme purchased from Invitrogen c Enzyme prepared in house, d, e two different enzyme batches (unpublished)

28c

6

25c

nd

Table 7. Data on NIBR2049 and WYE-152050 from biochemical and cellular assays29 WYE-152050c

NIBR3049 Assaya

Km ATP

1 mM ATP

Shiftb (obs.; exp.)

Km ATP

1 mM ATP

Shiftb (obs.; exp.)

Jak1

211

2387

(11x; 13x)

16

111

(7x; 13x)

Jak2

141

5453

(39x; 125x)

1

28

(28x; 125x)

Jak3

1.5

131

(87x; 125x)

0.4

12

(30x, 125x)

2101

>10000

(>5x, 42x)

17

401

(24x, 42x)

Tyk2 IL-2/P-STAT5

a

524

nd

IL-15/P-STAT5

535

29

IL-10/P-STAT3

10000

610

a

a

IC50 [nM]

b

observed shift when switching from ATP at Km to 1 mM ATP; expected shift based on Cheng-Prusoff equation c

All data n=2 except IL-2/P-STAT5 (n=4)

In summary, none of the current compounds targeting Jak3 exhibits sufficient selectivity within the Jak kinase family to determine the individual contribution of Jak1 and Jak3 to signaling processes through the C. Data on NIBR3049 which appears to be the most selective ATP-competitive Jak3 inhibitor to date (table 3) suggested that the potent and selective inhibition of Jak3 kinase activity is inferior to dual inhibition of Jak1 and Jak3. The data obtained with this compound have, however, limitations. First, the enzymatic data may not be predictive for the cellular behavior of the kinase (see above). Second,

although selective for Jak3, the compound does inhibit Jak1 at higher concentrations and thus, does not allow to unequivocally assess the individual roles of Jak1 and Jak3 in cellular assays. Given these limitations we set out to interrogate the individual roles of Jak1 and Jak3 in signaling through the C in a cellular system in which we reconstituted the essential components of IL2 signaling in U4C cells which lack IL-2 receptor chains as well as Jak1 and Jak3.35 We first generated stable transfectants for IL-2Rβ and IL-2R (C) and stably transfected these cells with one of the nine combinations of wild-type, kinase-deficient, and constitutively active mutants of Jak1 and Jak3. We then assessed basal or IL2-induced STAT5 phosphorylation. The results clearly showed that Jak1 catalytic activity is essential for STAT5 phosphorylation (whether constitutive or IL2-induced) and the role of Jak3 is to phosphorylate Jak1, thereby increasing the Jak1-dependent STAT5 phosphorylation. We also generated “analog sensitive (AS)” mutants of Jak1 and Jak3 by mutating the gatekeeper methionine with glycine in both kinases thereby increasing the volume of the ATP binding pocket in the catalytic domain,35 an elegant “chemical genetics” procedure that has been established in order to assess kinase activity in a cellular context.43 We generated transfectants with combinations of Jak1AS/Jak3WT and Jak1WT/Jak3AS and tested the effect of the specific inhibitor of analog sensitive mutants 1NM-PP1 on IL2-induced STAT5 phosphorylation (figure 2). In keeping with the data discussed above, inhibition of Jak1 but not Jak3 abrogated STAT5 phosphorylation confirming that specific inhibition of Jak3 does not suppress C-dependent signal transduction. We concluded that Jak1 kinase activity is dominant over Jak3 in signal transduction and selective pharmacological inhibition of Jak3 will not yield the immunosuppressive effect required for in vivo efficacy. 35

Figure 2. A) Inhibition of the analog sensitive Jak mutants Jak3AS and Jak1AS with 1NM-PP1, a frequently used specific inhibitor of kinases with AS mutations. Western blots were probed with antibodies against P-STAT5 and STAT5. 34 B) Chemical structure of 1NM-PP1.

The severe phenotype of SCID patients with Jak3 mutations indicates a pivotal role of Jak3 in signal transduction via γC-receptors and consequently in maintaining a functional immune system. Knock-down of Jak3 in cells (which abrogates both, catalytic activity as well as scaffolding functions of Jak3) completely abolished IL2 signaling, however, selective pharmacological inhibition of Jak3 kinase (which only affects the catalytic activity) does not.34 So far, Jak3 mutations leading to a “kinase-dead” but

otherwise intact protein which theoretically, would correspond to blockade of the catalytic activity of Jak3 have not been described in SCID patients.12 In conclusion the currently available data generated with ATP-competitive Jak inhibitors as well as with engineered cells do not provide evidence that selective inhibition of Jak3 leads to the same cellular potency as dual inhibition of Jak1 and Jak3. The data are consistent with a model in which Jak1 has a dominant role over Jak3 in C-dependent signal transduction and indicate that selective Jak3 inhibition is not enough to achieve efficient immunosuppression. However, in the future cellular data on more potent and more selective Jak3 inhibitors, such as covalent modifiers may show if this model needs to be revised.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

18.

Haan, C.; Kreis, S.; Margue, C.; Behrmann, I. Biochem. Pharmacol. 2006, 72, 1538. Ghoreschi, K.; Laurence, A.; O’Shea, J. J. Immunol. Rev. 2009, 228, 273. Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 90. O’Shea, J. J.; Plenge, R. Immunity 2012, 36, 542. Kiu, H.; Nicholson, S. E. Growth Factors 2013, 30, 88. Stark, G. R.; Darnell, J. E. Immunity 2012, 36, 503. Aaronson, D. S.; Horvath, C. M. Science 2002, 296, 1653. Schindler, C. W. J. Clin. Invest. 2002, 109, 1133. Cox, L.; Cools, J. Chem. Biol. 2011, 18, 277. OShea, J. J.; Pesu, M.; Borie, D. C.; Changelian, P. S. Nat. Rev. Drug Discov. 2004, 3, 555. Casanova, J.-L.; Holland, S. M.; Notarangelo, L. D. Immunity 2012, 36, 515. Pesu, M.; Candotti, F.; Husa, M.; Hofmann, S.; Notarangelo, L. D.; O’Shea, J. J. Immunol. Rev. 2005, 203, 127. Thomis, D. C.; Gurniak, C. B.; Tivol, E.; Sharpe, A. H.; Berg, L. J. Science 1995, 270, 794. O’Shea, J. J.; Husa, M.; Li, D.; Hofmann, S. R.; Watford, W.; Roberts, J. L.; Buckley, R. H.; Changelian, P.; Candotti, F. Mol. Immunol. 2004, 41, 727. Changelian, P. S.; Flanagan, M. E.; Ball, D. J.; Kent, C. R.; Magnuson, K. S.; Martin, W. H.; Rizzuti, B. J.; Sawyer, P. S.; Perry, B. D.; Brissette, W. H.; McCurdy, S. P.; Kudlacz, E. M.; Conklyn, M. J.; Elliott, E. a; Koslov, E. R.; Fisher, M. B.; Strelevitz, T. J.; Yoon, K.; Whipple, D. a; Sun, J.; Munchhof, M. J.; Doty, J. L.; Casavant, J. M.; Blumenkopf, T. a; Hines, M.; Brown, M. F.; Lillie, B. M.; Subramanyam, C.; Shang-Poa, C.; Milici, A. J.; Beckius, G. E.; Moyer, J. D.; Su, C.; Woodworth, T. G.; Gaweco, A. S.; Beals, C. R.; Littman, B. H.; Fisher, D. a; Smith, J. F.; Zagouras, P.; Magna, H. a; Saltarelli, M. J.; Johnson, K. S.; Nelms, L. F.; Des Etages, S. G.; Hayes, L. S.; Kawabata, T. T.; Finco-Kent, D.; Baker, D. L.; Larson, M.; Si, M.-S.; Paniagua, R.; Higgins, J.; Holm, B.; Reitz, B.; Zhou, Y.-J.; Morris, R. E.; O’Shea, J. J.; Borie, D. C. Science 2003, 302, 875. Kudlacz, E.; Perry, B.; Sawyer, P.; Conklyn, M.; McCurdy, S.; Brissette, W.; Flanagan and, M.; Changelian, P. Am. J. Transplant. 2004, 4, 51. Clark, M. P.; George, K. M.; Bookland, R. G.; Chen, J.; Laughlin, S. K.; Thakur, K. D.; Lee, W.; Davis, J. R.; Cabrera, E. J.; Brugel, T. A.; Vanrens, J. C.; Laufersweiler, M. J.; Maier, J. A.; Sabat, M. P.; Golebiowski, A.; Easwaran, V.; Webster, M. E.; De, B.; Zhang, G. Bioorg. Med. Chem. Lett. 2007, 17, 1250. Ghoreschi, K.; Jesson, M. I.; Li, X.; Lee, J. L.; Ghosh, S.; Alsup, J. W.; Warner, J. D.; Tanaka, M.; Steward-Tharp, S. M.; Gadina, M.; Thomas, C. J.; Minnerly, J. C.; Storer, C. E.; LaBranche, T. P.; Radi, Z. a; Dowty, M. E.; Head, R. D.; Meyer, D. M.; Kishore, N.; O’Shea, J. J. J. Immunol. 2011, 186, 4234.

19. 20. 21. 22.

23.

24. 25. 26. 27. 28. 29. 30.

31.

32. 33. 34. 35. 36. 37.

38. 39. 40. 41. 42.

Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P. M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. J. Mol. Biol. 2009, 387, 219. Thoma, G.; Nuninger, F.; Falchetto, R.; Hermes, E.; Tavares, G. A.; Vangrevelinghe, E.; Zerwes, H.G. J. Med. Chem. 2011, 54, 284. Jiang, J.; Ghoreschi, K.; Deflorian, F.; Chen, Z.; Perreira, M.; Pesu, M.; Smith, J.; Nguyen, D.-T.; Liu, E. H.; Leister, W.; Costanzi, S.; O’Shea, J. J.; Thomas, C. J. J. Med. Chem. 2008, 51, 8012. Meyer, D. M.; Jesson, M. I.; Li, X.; Elrick, M. M.; Funckes-Shippy, C. L.; Warner, J. D.; Gross, C. J.; Dowty, M. E.; Ramaiah, S. K.; Hirsch, J. L.; Saabye, M. J.; Barks, J. L.; Kishore, N.; Morris, D. L. J. Inflamm. 2010, 7, 41. Soth, M.; Hermann, J. C.; Yee, C.; Alam, M.; Barnett, J. W.; Berry, P.; Browner, M. F.; Frank, K.; Frauchiger, S.; Harris, S.; He, Y.; Hekmat-Nejad, M.; Hendricks, T.; Henningsen, R.; Hilgenkamp, R.; Ho, H.; Hoffman, A.; Hsu, P.-Y.; Hu, D.-Q.; Itano, A.; Jaime-Figueroa, S.; Jahangir, A.; Jin, S.; Kuglstatter, A.; Kutach, A. K.; Liao, C.; Lynch, S.; Menke, J.; Niu, L.; Patel, V.; Railkar, A.; Roy, D.; Shao, A.; Shaw, D.; Steiner, S.; Sun, Y.; Tan, S.-L.; Wang, S.; Vu, M. D. J. Med. Chem. 2013, 56, 345. McDonnell, M. E.; Bian, H.; Wrobel, J.; Smith, G. R.; Liang, S.; Ma, H.; Reitz, A. B. Bioorg. Med. Chem. Lett. 2014, 24, 1116. Migita, K.; Komori, A.; Torigoshi, T.; Maeda, Y.; Izumi, Y.; Jiuchi, Y.; Miyashita, T.; Nakamura, M.; Motokawa, S.; Ishibashi, H. Arthritis Res. Ther. 2011, 13, R72. Berhan, A. BMC Musculoskelet. Disord. 2013, 14, 332. Clark, J. D.; Flanagan, M. E.; Telliez, J.-B. J. Med. Chem. 2014, 57, 5023. Hoock, T.; Hogan, J.; Mahajan, S.; Shlyakhter, D.; Oh, L.; Park, L. Arthritis Rheum. 2011, 63 Suppl. 10, Abstract 1136. Thorarensen, A.; Banker, M. E.; Fensome, A.; Telliez, J.-B.; Juba, B.; Vincent, F.; Czerwinski, R. M.; Casimiro-Garcia, A. ACS Chem. Biol. 2014, 9, 1552. Lin, T. H.; Hegen, M.; Quadros, E.; Nickerson-Nutter, C. L.; Appell, K. C.; Cole, A. G.; Shao, Y.; Tam, S.; Ohlmeyer, M.; Wang, B.; Goodwin, D. G.; Kimble, E. F.; Quintero, J.; Gao, M.; Symanowicz, P.; Wrocklage, C.; Lussier, J.; Schelling, S. H.; Hewet, A. G.; Xuan, D.; Krykbaev, R.; Togias, J.; Xu, X.; Harrison, R.; Mansour, T.; Collins, M.; Clark, J. D.; Webb, M. L.; Seidl, K. J. Arthritis Rheum. 2010, 62, 2283. Lynch, S. M.; DeVicente, J.; Hermann, J.C.; Jaime-Figueroa, S.; Jin, S.; Kuglstatter, A.; Li, H.; Lovey, A.; Menke, J.; Niu, L.; Patel b, V.; Roy, D.; Soth, M.; Steiner, S.; Tivitmahaisoon, P.; Vu, M. D.; Calvin Yee, C. Bioorg Med. Chem. Lett. 2013, 23, 2793. Norman, P. Expert Opin. Ther. Pat. 2014, 24, 121. Ahearn, S.; Christopher, M.; Jung, J.; Pu, Q.; Rivkin, A.; Scott, M.; Wittner, D.; Woo, H.; Cash, B.; Dinsmore, C. J.; Guerin, D. WO Patent 2013085802, 2013. Cheng, Y.; Prusoff, W. Biochem. Pharmacol. 1973, 22, 3099. Haan, C.; Rolvering, C.; Raulf, F.; Kapp, M.; Drückes, P.; Thoma, G.; Behrmann, I.; Zerwes, H.-G. Chem. Biol. 2011, 18, 314. Knight, Z. A.; Shokat, K. M. Chem. Biol. 2005, 12, 621. Zhou, Y. J.; Chen, M.; Cusack, N. A.; Kimmel, L. H.; Magnuson, K. S.; Boyd, J. G.; Lin, W.; Roberts, J. L.; Lengi, A.; Buckley, R. H.; Geahlen, R. L.; Candotti, F.; Gadina, M.; Changelian, P. S.; O’Shea, J. J. Mol. Cell 2001, 8, 959. Saharinen, P.; Silvennoinen, O. J. Biol. Chem. 2002, 277, 47954. Rauch, J.; Volinsky, N.; Romano, D.; Kolch, W. Cell Commun. Signal. 2011, 9, 23. Endicott, J. A.; Noble, M. E. M.; Johnson, L. N. Annu. Rev. Biochem. 2012, 81, 587. Keil, E.; Finkenst, D.; Wufka, C.; Trilling, M.; Liebfried, P.; Strobl, B.; Mathias, M.; Pfeffer, K. Blood 2014, 123, 520. Hafenbradl, D.; Baumann, M.; Neumann, L. In Protein Kinases as Drug Targets; Klebl, B.; Müller, G.; Hamacher, M., Eds.; WILEY-VCH Verlag GmbH & Co. KGaA: Weinheim, 2011, pp. 3–43.

43.

Bishop, A.; Buzko, O.; Shokat, K. M. Trends Cell Biol. 2001, 11, 167.

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