Discovery and Development of Janus Kinase (JAK) Inhibitors for

Perspective pubs.acs.org/jmc

Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases Miniperspective James D. Clark,† Mark E. Flanagan,*,‡ and Jean-Baptiste Telliez† †

Pfizer Immunosciences, 200 CambridgePark, Cambridge, Massachusetts 02140, United States Center for Chemistry Innovation and Excellence, Pfizer, Inc., Eastern Point Road, Groton, Connecticut 06340, United States

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‡

ABSTRACT: The Janus kinases (JAKs) are a family of intracellular tyrosine kinases that play an essential role in the signaling of numerous cytokines that have been implicated in the pathogenesis of inflammatory diseases. As a consequence, the JAKs have received significant attention in recent years from the pharmaceutical and biotechnology industries as therapeutic targets. Here, we provide a review of the JAK pathways, the structure, function, and activation of the JAK enzymes followed by a detailed look at the JAK inhibitors currently in the clinic or approved for these indications. Finally, a perspective is provided on what the past decade of research with JAK inhibitors for inflammatory indications has taught along with thoughts on what the future may hold in terms of addressing the opportunities and challenges that remain.

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INTRODUCTION The Janus kinases or JAKs are a family of intracellular tyrosine kinases that function as hubs in the signaling process of many cytokine receptors.1 The JAKs play a critical role in both innate and adaptive immunity as well as hematopoiesis, making them attractive targets for a number of therapeutic indications.2 Consequently, since their discovery in the early 1990s, the JAKs have steadily garnered interest as targets for new medicines.3 There are now multiple chemical entities in the clinic across various indications including myeloproliferative diseases and various inflammatory diseases. Because of the volume of clinical activity in recent years, this review will focus on the inhibition of the JAK enzymes for the treatment of inflammatory diseases. The etiology of many inflammatory and autoimmune diseases is not fully understood but involves a combination of genetic predispositions and environmental and lifestyle triggers. The pathogenic inflammatory state often involves a break in Tand B-cell tolerance against self-antigens leading to undesirable autoimmune responses. When antigens from affected tissues are presented on antigen presenting cells (APCs) to naive T-helper cells (Th0) along with co-stimulatory factors, cytokines, which are low molecular weight (∼30 kDa) protein or glycoprotein signaling molecules, are secreted leading to activation, differentiation and expansion of Th0 cells to distinct T-helper phenotypes (e.g., Th1, Th2, Th17). These activated T-cells themselves secrete cytokines that activate other cells of the immune system, such as macrophages, NK cells, and neutrophils that infiltrate the targeted tissue, causing the damage associated with these diseases. Therefore, by orchestrating the intercellular communication in this process, cytokines play a central role in the inflammatory response. © 2014 American Chemical Society

Cytokines signal through a variety of receptor superfamilies (Figure 1).4 By binding to the extracellular domain of the receptor, the cytokine induces changes that are detected at the intracellular domain, triggering signal transduction events ultimately leading to changes in gene expression.5 Protein kinases are key players in signal transduction pathways for these receptor superfamilies. As a consequence, many of these kinases have been targeted in an effort to modulate the inflammatory response. However, because of the complex network of these signal transduction pathways, which can be redundant, some of these kinases make better drug targets than others. The JAKs associate with the intracellular domain of receptor subunits of the class I and class II receptor superfamily.1a The class I receptors all bind ligands with a common four helical structure and share a common WSXWS motif extracellularly and, although divergent intracellularly, have a conserved membrane proximal region for associating with JAKs. The class II receptors bind interferons and the IL-10 family and are structurally related to the class I receptors.6 The activation step occurs when a cytokine binds to its receptor, inducing a multimerization (dimerization or higher order complexes) of receptor subunits. This brings the JAKs associated with each subunit proximal to one another, triggering a series of phosphorylation events ultimately resulting in the phosphorylation and activation of signal transducers and activators of transcription (STAT) proteins. A phosphorylated STAT dimer then translocates to the nucleus of the cell where it binds to target genes modulating their expression (Figure 2).5 Received: September 25, 2013 Published: January 13, 2014 5023

dx.doi.org/10.1021/jm401490p | J. Med. Chem. 2014, 57, 5023−5038

Journal of Medicinal Chemistry

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Figure 1. Cytokine receptor superfamilies and associated signal transduction pathways. Protein kinases feature prominently as mediators in signal transduction. The complex nature of these signaling pathways lead to redundancies in some cases. However, there are no known compensatory pathways around JAK/STAT. Furthermore, many cytokine receptors lack intrinsic kinase activity, instead relying on associated tyrosine kinases, such as the JAKs to transmit signals from the extracellular environment to the nucleus. Consequently, many cytokines and growth factors important for a variety of immune, inflammatory, and hematopoietic functions signal through JAK/STAT.

receptors are implicated in the pathogenesis of autoimmune diseases, adding to the attraction of the JAKs as drug targets.7 There are currently several biologics available that have clearly demonstrated the benefits of inhibiting cytokine activity in the treatment of inflammatory diseases.8 The majority of these are monoclonal antibodies that derive their activity by binding to and blocking the cytokine receptor or the cytokine itself. For example, biologic tumor necrosis factor (TNF) inhibitors have been available for over 10 years and used to treat patients with rheumatoid arthritis, psoriasis, psoriatic arthritis, and inflammatory bowel disease in which nonbiologic disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX), are insufficient.9 However, RA patients often require therapy for decades, and recent data show that after 2 years, only about half of patients remain on their first TNF inhibitor.10 Thus, additional treatment options are needed. Moreover, many patients are reluctant to self-inject or undergo iv infusions required for administration of biologics. JAK inhibition with an orally available small molecule represents an alternative approach to biologic therapies. Since the JAK enzymes associate with the intracellular domains of the class I and class II cytokine superfamilies, they cannot be readily targeted by biologics with the current state of the art but can be accessed and inhibited with a small molecule possessing the appropriate physicochemical properties.11 Small molecules also offer the potential advantage of oral dosing, adding to the attraction of this approach. In addition, unlike biologics that generally act by binding to individual cytokines or receptors and nearly completely inhibiting an individual cytokine for prolonged periods of time, JAK signaling is downstream of multiple cytokines implicated in the pathogenesis of inflammatory diseases, and therefore, efficacy can be observed at doses that partially and reversibly modulate the signaling of multiple pathways. Higher doses that fully inhibit multiple pathways may drive greater efficacy but would likely be immunosuprpressive.

Figure 2. Cytokine signaling through the JAK/STAT pathway. Cytokine binding to the receptor leads to JAK activation and phosphorylation of the JAKs and associated receptors. Phosphorylation of the receptors in turn initiates recruitment of the STATs via their SH2 domains and subsequent phosphorylation of STAT proteins. The phosphorylated STAT homodimers or heterodimers then translocate to the nucleus where they bind to specific DNA binding sites regulating gene transcription that leads to changes in cellular function.

Importantly, since there are no known compensatory pathways around JAK/STAT, the JAK enzymes are essential in regulating the cytokines that signal through these pathways. Furthermore, many of the cytokines that signal through the class I and class II 5024



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Figure 3. Biological significance of signaling through different JAK combinations. Cytokine signaling is mediated by specific JAK and STAT combinations due to preferential binding to the intracellular domains of the individual cytokine receptor chains. For example, JAK3 only associates with the γ-common chain and therefore only mediates IL-2, -4, -7, -9, -15, and -21 signaling, whereas JAK1 plays a broader role in cytokine signaling.

phenotypically indistinguishable from the γ-common chain deficiency, demonstrating the link between this receptor chain and JAK3.16 In patients with two mutated JAK3 alleles, enzymatic activity is completely abrogated, disrupting the signaling of multiple cytokines that would ordinarily signal through this pathway and that are important for T-cell and NK cell activation, development, and homeostasis. While these patients have a profoundly impaired immune system, they are otherwise normal and can be rescued by bone marrow transplantation, providing good genetic validation for JAK3 as a therapeutic target for selective immune modulation limited to the hematopoietic system. Heterozygous parents of the SCID patients did not exhibit an immunocompromised phenotype, suggesting that partial inhibition could be used to modulate the immune response. This has important implications in a number of clinical settings, such as prevention of transplant rejection and treatment of various autoimmune disorders. Importantly, while complete inhibition of JAK3 and/or JAK1 would result in immunodeficiency, clinical experience has taught that partial and reversible inhibition of multiple JAKs, including JAK3, during the course of a day can provide safe and effective treatment in a number of therapeutic settings. The homology within the JAK family combined with the fact that JAK family crystal structures have only recently become available made it challenging to target JAK3 selectively. As a consequence, early JAK clinical candidates, such as CP-690,550 (1), now known as tofacitinib, inhibit a combination of JAK enzymes, the implications of which will be discussed in subsequent sections.17 To date, there is no evidence of human deficiencies in JAK1 and JAK2. This is consistent with perinatal and embryonic lethality in phenotypes of JAK1 and JAK2 KO mice, respectively.18 JAK2 activity is essential for hematopoiesis, and JAK1 signaling is required across many of the type I and type II cytokine receptors.19 Type I receptors contain conserved Cys residues and a WSXWS extracellular motif. Type

There are four members of the JAK family: JAK1, JAK2, JAK3, and TYK2. The four JAKs possess two kinase domains, a true kinase domain, and a likely inactive pseudo-kinase domain. Thus, they get their name from Janus, the two-faced Roman god of doors and new beginnings. An important element of JAK function is the pairing of the JAK kinases, which are associated with the intracellular domains of different subunits of the receptor. The biological significance of signaling through different JAK combinations is illustrated in Figure 3.12 The pairing of the JAKs with a given cytokine receptor is determined by their association with specific receptor chains. For example, JAK3, being coupled to the γ-common chain, is always paired with JAK1 and in this arrangement controls the signaling for the six known γ-common cytokines IL-2, IL-4, IL7, IL-9, IL-15, and IL-21 primarily associated with adaptive immune functions.13 JAK1, however, also pairs with JAK2 and TYK2, regulating the signaling through a wide array of cytokine receptors and therefore affecting several proinflammatory cytokines associated with the innate immune response, such as IL-6 and the type I interferons. JAK2 is the only member of the JAK family that pairs with itself. In this combination JAK2 controls the signaling of various cytokines and growth factors, such as IL-3, IL-5, granulocyte macrophage colony-stimulating factor (GM-CSF), erythropoietin (EPO), and thrombopoietin (TPO). Consequently, since specific cytokines may be implicated in the pathogenesis of certain diseases, while modulation of others may result in undesired side effects, striking the appropriate selectivity balance for inhibition within the JAK family continues to represent both an opportunity and a challenge for drug development. JAK3 was among the first of the JAKs targeted for therapeutic intervention in this regard.14 This is due to strong validation following characterization of human JAK3 deficiencies.15 Inactivating mutations in JAK3 confer a severe combined immune deficient (SCID) phenotype in humans that is 5025



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Figure 4. Schematic of JAK structural domains. The JAKs all contain seven homology regions (JH) and four structural domains. A characteristic feature of the JAKs is the presence of a pseudo-kinase domain JH2 that exerts regulatory function over the catalytic kinase domain JH1. The SH2 and FERM domains provide structural and regulatory control over the protein.

Figure 5. Schematic of JAK3 structure and site specific inactivating mutations leading to a SCID phenotype or activating mutations leading to hematological malignancies. (A) Many mutations have been identified in JAK3 that lead to a SCID phenotype in humans. This phenotype results from the disruption of normal signaling of the γc-dependent cytokines that are important for T cell function, development, and homeostasis. Mutations have been identified in all of the structural domains of the kinase. Therefore, abrogation of normal catalytic activity can result from abnormalities in structural, regulatory, or catalytic function. (B) Gain of function mutations have also been identified in JAK3. These mutations are associated with lymphoproliferative disorders.

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II receptors are related to the type I receptors but do not contain the WSXWS motif. Like JAK3, TYK2 mediates a smaller number of cytokine pathways. To date, two human patients with TYK2 deficiency have been described with distinct but overlapping phenotypes.20 Both patients appear to have impaired but not abolished resistance to viral and bacterial pathogens consistent with defects in IFNγ, IFNα/β, IL-12, and IL-23 signaling.21 Differences in the phenotype of the two patients, the phenotype of TYK2 deficient mice and inconsistencies between the effect of TYK2 deficiency and cytokine deficiencies thought to be mediated by TYK2 suggest that TYK2 dependent signaling is not yet fully delineated. There are currently at least seven JAK inhibitors in clinical development for inflammatory diseases and potentially many more being profiled preclinically. The following sections will take a deeper look into the nature of the JAK enzymes and a more detailed examination of the entities in the clinic that show potential for the treatment of inflammatory diseases.

JAK ENZYME STRUCTURE AND SIGNALING

The JAK enzymes are relatively large proteins (120−130 kDa) of the tyrosine kinase family.22 There is a high degree of sequence homology across the JAK family with the highest homology observed within the catalytic domain. These enzymes contain seven distinct homologous regions comprising four structural domains (Figure 4). One characteristic feature of the JAKs is the two structurally related domains JH1 and JH2.23 JH1 is the active kinase catalytic domain, while JH2 is termed the pseudo-kinase domain that is not functional catalytically but is thought to play a key regulatory role in concert with JH1.24 Interestingly, recent work has shown that the JH2 domain of JAK2 might negatively regulate JAK2 function by phosphorylating key residues within the JH2 domain.25 The JH3 and JH4 domains are believed to primarily play a structural role in stabilizing the confirmation of the JAKs, while the domains JH5-JH7 (FERM domain) have been shown to interact directly with the intracellular domain of the cytokine receptor as well as 5026



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Figure 6. Homology in JAK family ATP binding site. The JAKs exhibit a high degree of homology in the ATP binding site. This sequence alignment illustrates in color and in three dimensions, where amino acid differences occur across the JAK family.

Figure 7. Cocrystal structure of tofacitinib (purple) with JAK3 showing overlay with ATP (green). This overlay illustrates the ATP competitive behavior of tofacitinib (1). The pyrrolopyrimidine heterocycle of 1 binds to the hinge region of the kinase where the purine of ATP binds. The piperidine headgroup scaffold of 1 positions the cyanoacetamide group toward the glycine-rich loop (P-loop) and the ring methyl into a lipophilic pocket toward the C-terminal lobe at the base of the active site. This binding mode and arrangement of structural features of the inhibitor are believed to impart the high degree of kinome selectivity observed for the JAK family.

interact with the JH1 domain.26 Upon cytokine binding and conformational changes the JAKs are activated and become phosphorylated notably on tyrosine residues in the activation loop of the kinase (JH1) domain. Activated JAKs phosphorylate specific tyrosine residues in the intracellular domain of the receptor, creating recruitment sites for STATs. Recruitment of STATs to the receptor in turn allows JAKs to phosphorylate STATs, leading to their dimerization and subsequent migration to the nucleus where they bind to specific DNA binding sites, regulating gene transcription that leads to changes in cellular function. Mutations in JAKs are associated with various cancers, myeloproliferative disorders (MPD), and autoimmune con-

ditions.27 Mutations associated with a loss of function in JAK3 lead to a SCID phenotype in an autosomal recessive fashion as noted above (Figure 5A),28 but many activating mutations have also been described in JAK1, JAK2, and JAK3 (Figure 5B) consistent with autoinhibitory conformations.29 Importantly, the dysregulation of JAK2 in a number of myeloproliferative neoplasms is the direct consequence of a V617F activating mutation in the JH2 domain which led to the original targeting of JAK2 in myelofibrosis.30 Analogously, activating mutations in JAK3 are observed in adult T-cell leukemia or lymphoma, as well as in T-cell ALL and NK-cell or T-cell lymphoma.31 The sequence homology within the ATP binding site of the JAKs is nearly identical making the discovery of selective, ATP 5027



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Figure 8. Inhibitors exhibiting enhanced selectivity within the JAK family. By targeting of specific amino acid residues and aided by single crystal Xray structures, it is possible to achieve some degree of enhanced selectivity within the JAK family. Compound 2 from Genentech achieves enhanced JAK1 selectivity by specifically targeting an interaction with E966 (JAK1). Roche’s inhibitor (3) exhibits enhanced selectivity for JAK3 through a targeted, lipophilic interaction with C909 of JAK3. Novartis’ JAK3 inhibitor (4) derives its selectivity through specific lipophilic interactions and a key through water hydrogen bond with the backbone of JAK3.

determinations are conducted at more physiologically relevant levels of ATP. This latter point will be discussed in more detail in the next section.

competitive inhibitors within the JAK family difficult (Figure 6). Until 2005 there were no published crystal structures of the JAKs adding to this challenge.32 Today there are published high resolution structures of all four JAK family members.33 However, even guided by structure, developing selective JAK structure−activity relationships (SARs) has been challenging owing to the similarity of these enzymes. In published crystal structures of the JAKs, “compound 6” (CMP6)34 and the Pfizer inhibitor tofacitinib (1) have featured prominently. Both compounds interact similarly across the JAKs, binding deep in the ATP binding site. Both compounds make key hydrogen bond interactions with the hinge region and are capable, in most cases, of interacting directly with the glycine rich loop (Ploop). Additionally, in the case of tofacitinib, the piperidine methyl group binds in a lipophilic pocket in the C-terminal lobe at the base of the active site, making van der Waals interactions with key residues (Figure 7). The high degree of shape complementarity that these structural features provide helps explain the exquisite kinome selectivity observed for 1. However, with only four subtle amino acid differences across the JAK family in the region where 1 binds, it once again underscores the challenge of gaining selectivity within the JAKs. Recent reports have shown that although the differences in the ATP binding sites of the JAKs are subtle, when guided by structure and targeting specific amino acid interactions, higher levels of selectivity within the family are achievable (Figure 8). Genentech’s imidazopyrrolopyridine chemical series is an example of this (2).35 By targeting of a specific hydrogen bond interaction with Glu966 in JAK1, which is an Asp in JAK2, an improvement in selectivity for JAK1 relative to JAK2 (>35 fold) was observed. Additionally, 2 exhibits improved physicochemical properties for oral dosing compared to previously reported analogues within this chemotype.36 Similarly, researchers at Roche have specifically targeted Cys 909 of JAK3 with lipophilic functionality (3), demonstrating improved selectivity relative to the other JAKs (∼20-fold relative to JAK2), which have a Ser at the equivalent position.37 Also, a 2011 report from Novartis describes their JAK inhibitor (4) being selective for JAK3, taking advantage of specific hydrophobic interactions as well as a key, through water, backbone hydrogen bond with Asp967 (4 exhibits ∼12-fold selectivity for JAK3 over JAK2 and even greater selectivity relative to the other JAKs).38 It is important to point out that these selectivity improvements are all evaluated with isolated enzyme catalytic domains and at Km concentrations of ATP. IC50 values and therefore selectivity often change when

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JAK INHIBITORS FOR INFLAMMATORY DISEASES There are currently several JAK inhibitors in development for inflammatory indications (Figure 9). The following sections

Figure 9. Experimental and approved JAK inhibitors. Compounds 1 and 5 are the only JAK inhibitors, to date, to have received regulatory approvals. The other inhibitors are in various stages of clinical development across several inflammatory indications.

provide additional details around the discovery and development of these entities. The authors have made an advised decision not to summarize clinical data for compounds other than tofacitinib nor to try to make any direct comparisons of clinical outcomes for these entities, since head-to-head studies have not yet been conducted. However, we have profiled many of them in our standard enzyme and whole blood assays for comparative purposes. The enzyme IC50 values reported in Table 1 were obtained with the purified JH1 domains of the four respective JAKs using caliper nanofluidics technology. The assays were performed at 1 mM ATP instead of Km for ATP to better mimic the ATP physiological concentration, resulting in a more relevant comparison of inhibitory properties between them. As expected, the observed IC50 values for these ATP competitive inhibitors were higher than that reported at the Km values, and the shifts were greatest for JAK3 which has the lowest Km for ATP. 5028



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Table 1. JAK Kinase and Whole-Cell Assays: Comparison of Enzymatic and Whole-Cell Activity for Experimental and Approved JAK Inhibitors for Inflammatory Indications enzyme assay IC50 (nM)a

human whole blood (HWB) IC50 (nM)84 b

c

compd

JAK1

JAK2

JAK3

TYK2

IL-15 P-stat5

IL-6 P-stat1

IL-12d P-stat4

IFNαe P-stat3

IL-23d P-stat3

CD34+ g cells EPOf P-stat5

1 5 6 7 8

15.1 6.4 4.0 112 363

77.4 8.8 6.6 619 2400

55.0 487.0 787.0 74.4 >10K

489 30.1 61.0 >10K 2600

55.8 1850 259 932 2140

75.4 298 21.1 1870 918

409 1090 149 16400 13362

35.0 194 28.7 1290 1500

229 818 81.9 11200 10123

302 677 87.8 >20K 13200

a

Run in the presence of 1 mM ATP. bSignals through JAK1/JAK3. cSignals though JAK1/JAK2 or TYK2. dSignals through JAK2/TYK2. eSignals through JAK1/TYK2. fSignals through JAK2/JAK2. gCD34+ cells spiked into human whole blood (HWB). Data reported for tofacitinib (1), ruxolitinib (5), baricitinib (6), decernotinib (7), and filgotinib (8).

Figure 10. Evolution of compound structures from HTS lead (9) to tofacitinib (1). Progression of the Pfizer JAK inhibitor from HTS lead to clinical candidate (1) is shown. This schematic of seminal compounds represents an approximately 3-year medicinal chemistry effort wherein greater than 1000 synthetic analogues in this pyrrolopyrimidine series were prepared and evaluated.

is also being developed by Pfizer for other inflammatory indications, including psoriasis (oral formulation, phase 3; topical formulation, phase 2), psoriatic arthritis (phase 3), ankylosing spondylitis (phase 2), ulcerative colitis (phase 3), and Crohn’s disease (phase 2).41 The medicinal chemistry program at Pfizer that culminated in the discovery of tofacitinib began with a high throughput screen, which resulted in the identification of a series of inhibitors represented by 9 that possessed a pyrrolo[2,3d]pyrimidine pharmacophoric subunit (Figure 10).42 Early efforts explored SAR around the pyrrolopyrimidine ring, which was assumed to be the hinge-binding element of the molecule. Attempts to either replace this heterocycle or make additional substitutions around the ring generally led to reduced potency. Substitutions at C-5 were tolerated in terms of JAK3 kinase potency; however, these modifications often resulted in suboptimal metabolic stability as indicated by human liver microsome incubations and/or diminished cell potency. As a consequence of this initial survey, efforts focused on optimization of the amino headgroup in order to achieve program objectives. The first major advance in the evolution of the amino headgroup came as a result of an empirical observation associating structure with improved potency in the IL-2 Tcell blast cellular proliferation assay being used at the time. These data suggested an advantage for compounds processing an N-methylcycloalkyl headgroup motif as illustrated by 10. This improvement in cellular potency was likely associated with a concomitant increase in JAK1 potency observed, which was the first indication that combining inhibition of multiple JAK

A comparison of in vitro cellular inhibitory properties for these compounds was performed in primary cells in human whole blood (HWB) assays by fluorescence-activated cell sorting (FACS) as described previously.39 The phosphorylation state of various STAT proteins was measured as a proximal readout of JAK activity. The data reported with the set of cytokines shown in Table 1 were chosen to assess as much as possible the cellular selectivity against the four JAK isoforms. Although we will not directly compare efficacy and safety, several general themes are worth noting from the results of Table 1. The relative potency within this series of compounds varies greatly, suggesting that significantly different concentrations will be necessary for clinical activity. Potent inhibition of JAK1 is sufficient for inhibition of JAK1/JAK3 dependent IL15 signaling. When assessed in kinase assays using 1 mM ATP, none of the compounds are JAK3 selective and all inhibit both JAK1/JAK3 dependent IL-15 signaling and JAK1/TYK2 dependent interferon α signaling. At higher concentrations, several of the compounds will be expected to inhibit JAK2 dependent EPO signaling, which is consistent with a decrease in hemoglobin levels at higher doses of tofacitinib in phase 2 rheumatoid arthritis (RA) studies. Lastly, several of the compounds are expected to inhibit IL-6 signaling which is consistent with lipid elevations seen for several JAK inhibitors and the anti-IL-6 receptor antibody tocilizumab. Tofacitinib (1). The first compound to enter development for the treatment of inflammatory indications was tofacitinib from Pfizer, which recently received its first regulatory approval for the treatment of moderate to severe RA. Tofacitinib was approved by the U.S. Food and Drug Administration on November 6, 2012, under the trade name Xeljanz.40 Tofacitinib 5029



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understood, it is noteworthy that the receptor signaling mechanisms for EPO and GM-CSF are different. For example, EPO signals through homodimers,44 whereas GM-CSF signals through dimerization of two hexamers.45 Hence, reduction in hemoglobin was observed at higher doses with tofacitinib and other JAK inhibitors. It is more relevant to monitor EPO rather than GM-CSF. While a greater degree of selectivity relative to JAK2 was originally desired because of the role of JAK2 in red blood cell homeostasis, the functional selectivity of tofacitinib in humans has proven to be manageable at the clinical doses tested. Since changes in hemoglobin were monitorable in phase 2 studies, the phase 3 doses were chosen to maximize the probability of achieving efficacy while minimizing the likelihood of hemoglobin reduction of >2 g/dL. The challenge of identifying highly selective, ATP competitive inhibitors within the JAK family stems from the high degree of homology in the active sites across the JAK family. However, outside of the JAKs, exploitable differences do exist. Tofacitinib is highly selective for the JAK family across the human kinome. Results of a broad kinase selectivity assessment have now been corroborated by studies from external groups.46 As alluded to in a previous section, there are a number of key protein−ligand interactions that are believed to contribute to the kinome selectivity observed for tofacitinib. Importantly, this high degree of selectivity across the human kinome may be essential in minimizing unwanted off-target pharmacology. In preclinical studies, tofacitinib demonstrated efficacy in both mouse and rat models of arthritis exhibiting dose dependent improvements in end points.39,47 Tofacitinib was shown to be efficacious in the collagen induced arthritis (CIA) model. Inflammatory biomarkers including cytokines, chemokines, and acute phase proteins (IL-6, SAA, IP-10, G-CSF, and KC) are increased in mice with CIA relative to naive mice. Treatment with efficacious doses of tofacitinib resulted in the rapid reduction of these mediators within 4 h. The expression of many STAT1 responsive genes indicative of inflammation was also rapidly suppressed in the tissue of inflamed paws. With continuing treatment, significant improvements in arthritis scores were observed within 48 h and inflammatory cell infiltrates, including T cells and macrophages, were reduced by 7 days of treatment.48 Analogously, tofacitinib was tested in the rat adjuvant induced arthritis (AIA) model.17,43 Treatment during development of disease (days 14−21 after immunization) results in a dose dependent decrease in paw swelling. Inflammatory cytokines IL-6 and IL-17 are elevated in AIA disease animals, and tofacitinib normalizes the levels of these mediators. Neutrophil levels are also elevated with AIA as in active RA and tofacitinib normalized neutrophil levels. A thorough dose response analysis demonstrated that this was secondary to inhibition of proinflammatory cytokines and not due to inhibition of JAK2 dependent hematopoietic factors which require higher concentrations of tofacitinib to inhibit.49 In additional studies, AIA rats were treated after disease had developed. Elevated plasma IL-6 and IL-17 levels decreased rapidly, and gene expression of cytokines and chemokines decreased in the tissue. Importantly, osteoclast mediated bone resorption, which is common in the AIA model, was reduced with tofacitinib treatment and tofacitinib was shown to decrease the level of receptor activator of nuclear factor κB ligand (RANKL), resulting in a reduction in osteoclasts and the associated bone resorption.49,50

enzymes could present an advantage, at least in certain contexts. With no JAK crystal structures available at the time to guide subsequent work, efforts to expand SAR around this Nmethylcycloalkyl motif were facilitated by new (at the time) high-speed analoging (HSA) technology. The library of compounds produced provided several SAR insights, key of which was the potency advantage associated with a small alkyl substituent at the 2′-position, the cyclohexyl group (11). A methyl group at this position would later also be recognized as contributing to the enhanced kinome selectivity observed with more advanced analogues including tofacitinib. Questions of optimal stereochemistry associated with the complex mixture of isomers produced with 11 were addressed by incorporating natural products as synthetic building blocks. Specifically, the natural terpenoids, “carvones”, possessed the desired substitution around the cyclohexyl ring and were commercially available in both stereochemical antipodes. Analogues prepared from the carvones led to the conclusion that the optimal relative and absolute stereochemistry around the cyclohexane ring was the all-cis configuration derived from (S)-carvone (12). Subsequent analogues prepared from 13 using the isopropenyl group as a synthetic handle taught that a large pocket or perhaps solvent exposed site was accessible from this vector. However, the need to decrease stereochemical complexity and improve the druglike properties of new analogues being prepared inspired a move to the corresponding piperidine scaffold 14. The piperidine also facilitated synthetic access to this active site pocket, which is now known to project toward the glycine rich loop (P-loop) of the kinases. Many analogues were prepared from the piperidine scaffold in an effort to optimize potency, selectivity, and druglike properties. In doing so, a deliberate effort was made to improve upon these attributes while avoiding addition of unnecessary lipophilicity or molecular weight. Eventually, it was the cyanoacetamide side chain of tofacitinib that imparted the best combination of these attributes. In all, greater than 1000 synthetic analogues of the original lead molecule (9) were prepared and profiled over an approximately 3-year period. The citrate salt of tofacitinib was nominated into development in 2000. Original reports of JAK kinase inhibition for tofacitinib using a solid-phase ELISA-based assay system suggested that this compound was primarily a JAK3 inhibitor, exhibiting approximately 20-fold selectivity over JAK2 and 100-fold selectivity over JAK1.14 In more contemporary caliper assays, tofacitinib exhibits nanomolar potency against the JAK enzymes, although somewhat less potent against TYK2 (IC50 of 3.2, 4.1, 1.6, and 34.0 nM, respectively, for JAK1, JAK2, JAK3, and TYK2).17,43 These data, however, were collected at Km concentrations of ATP. As discussed above, when run at 1 mM ATP, these data shift, suggesting that tofacitinib more potently inhibits JAK1 relative to the other JAKs (Table 1). In cellular assays, specificity values derived from original tissue culture assays are in good agreement with phospho-STAT reporter assays used more universally today. In these assays tofacitinib preferentially inhibits cytokines that signal via JAK1 and/or JAK3 with functional selectivity over receptors that signal via JAK2 homodimeric pairs like GM-CSF and EPO. In a comparison of IL-15 which signals via JAK1/3 pairs to GMCSF and EPO, the fold selectivity is 24.7 and 5.4, respectively, based on IC50 values of 55.8 nM for IL-15, 1377 nM for GMCSF, and 302 nM for EPO. Although the reason for the difference in IC50 values between EPO and GM-CSF is not 5030



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Table 2. Phase 3 RA Study Design for Tofacitinib (1)a

population background treatment study duration number of patients a

oral start A3921069

oral standard A3921064

oral scan A3921044

oral sync A3921046

oral solo A3921045

oral step A3921032

MTX naive none 2 years 958

MTX IR MTX 1 year 717

MTX IR MTX 2 years 797

DMARD IR nonbiologic DMARD(s) 1 year 792

DMARD IR none 6 months 610

TNFi IR MTX 6 months 399

DMARD = disease-modifying antirheumatic drug; IR = inadequate responder; MTX= methotrexate; TNFi = tumor necrosis factor inhibitor.

placebo twice daily for 8 weeks. The primary end point was a clinical response at 8 weeks based on the Mayo scoring system. Clinical responses occurred in 32%, 48%, 61%, and 78% of patients at 0.5, 3, 10, and 15 mg of tofacitinib, respectively, compared to 42% for patients receiving placebo. Clinical remission occurred in 13%, 33%, 48%, and 41% of patients, respectively, in these dose groups compared with 10% in the placebo group. These data support the conclusion that patients with moderately to severely active UC receiving tofacitinib were more likely to have a clinical response or remission than those receiving placebo. Tofacitinib has now entered phase 3 studies for the treatment of UC. For the treatment of Crohn’s disease (CD), in a phase 2 double-blinded study, patients with moderate to severe Crohn’s disease were randomized to 1, 5, or 15 mg of tofacitinib or placebo twice daily for 4 weeks. The primary end point was the percentage of patients with a Crohn’s disease activity index (CDAI) reduction of >70 points (response 70) at four weeks. The secondary end points included remission rate (SDAI < 150) and a response 100. In this study response 70 rates were 36.1, 57.6, and 45.7 for the 1, 5, and 15 mg dose groups, respectively, compared to 47.1 for placebo. Tofacitinib, 15 mg b.i.d., resulted in reductions from baseline in C-reactive protein and fecal calprotectin. The conclusion of this study was that although generally well tolerated, tofacitinib had no significant treatment effect within 4 weeks on clinical end points as measured by CDAI in patients with active CD. C-reactive protein and fecal calprotectin reductions observed with tofacitinib 15 mg b.i.d. suggest biological activity. Phase 2b studies are underway to further evaluate these findings. Ruxolitinib (5) and Baricitinib (6). Incyte has two compounds that have been studied in human RA trials.56 Ruxolitinib (5), which was co-developed with Novartis, was evaluated in phase 2 studies of RA. In addition, this entity is approved for the treatment of myelofibrosis under the trade name Jakafi.57 Baricitinib (6), which is being co-developed with Eli Lilly, is currently in recruitment for phase 3 clinical trials for RA and is pursuing a once-a-day (q.d.) therapeutic option. The medicinal chemistry discovery stories for ruxolitinib and baricitinib have not been described in the literature to date. However, like tofacitinib, these two chemical entities also take advantage of a pyrrolopyrimidine hinge binding heterocycle as a pharmacophoric subunit and may make other similar active site interactions with the JAKs.58 As indicated in Table 1, unlike tofacitinib, ruxolitinib and baricitinib exhibit specificity for JAK1 and JAK2 over JAK3 in kinase assays. These data are consistent with previous reports from Incyte for these two compounds. However, since JAK1 is involved in receptor pairing with all the other JAKs, including JAK3, and therefore regulates signaling of the same cytokine subset as JAK3, the potential advantage of this specificity at the kinase level is not obvious and the effect of subtle changes in potency will only be evident if at all clinically.

Encouraged by these results, where tofacitinib normalized inflammatory mediators associated with disease and reversed bone resorption, tofacitinib entered the clinic in 2001. The development program consisted of 22 phase 1 studies, 8 phase 2 studies, 6 phase 3 studies, and 2 ongoing, open-label, longterm extension (LTE) studies. The phase 3 study designs are presented in Table 2. Safety and tolerability were assessed in a demographically diverse RA patient population. As of April 2013 the RA phase 2, phase 3, and LTE studies included approximately 5700 patients across all treatment groups with approximately 13 000 patient-years of tofacitinib exposure at 5 and 10 mg dosed twice daily (b.i.d.). The development program demonstrated that tofacitinib consistently improved the signs and symptoms of RA, physical functioning, and other patient-reported outcomes including health related quality of life, along with inhibition of structural damage across multiple lines of therapy. Safety findings observed in the overall tofacitinib RA program include serious and other important infections, including tuberculosis and herpes zoster; malignancies, including lymphoma; gastrointestinal perforations; decreased neutrophil and lymphocyte counts; liver enzyme elevations; and lipid elevations.51,52 For the treatment of psoriasis, tofacitinib was first administered in patients during a phase 1 multidose study (A3921003).53 In this study patients experienced a 50% improvement in modified PASI scores over 14 days of dosing at 30 mg b.i.d. While this study was not statistically powered for efficacy, this was, however, the first indication of biological activity for tofacitinib and prompted further investigation in psoriatic patients. In a phase 2b proof of concept study (A3921047), tofacitinib was investigated in patients with moderate-to-severe psoriasis.54 This was a placebo-controlled, double blinded study investigating three dose groups of tofacitinib: 2, 5, and 15 mg b.i.d. The end points of the study were a 75% improvement in psoriasis area severity index (PASI) score (PASI75) and a global physicians assessment (PGA). In this study a significantly higher proportion of patients treated with tofacitinib achieved PASI75 and a PGA of “clear” or “almost clear” compared with placebo during 12 weeks of treatment. A dose response was seen for both the PASI75 and PGA end points, with the magnitude of response increasing over time. Tofacitinib at 2, 5, and 15 mg b.i.d. was generally well-tolerated in this patient population. AEs in this study were qualitatively similar to those reported for the RA trials. Data for this study support a role for JAK1/JAK3 inhibition in the pathophysiology of psoriasis and suggest that tofacitinib has the potential to be a new first-in-class therapeutic option for patients with moderate-to-severe chronic plaque psoriasis. For ulcerative colitis (UC), tofacitinib was evaluated in a phase 2, double-blinded, placebo controlled study in patients with moderately to severely active UC.55 Patients were administered tofacitinib at doses of 0.5, 3, 10, or 15 mg or 5031



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swelling and paw weight and improved histological scores in affected paws, with the effects/potency exceeding the reference standard etanercept (Enbrel). In the mouse oxazolone delayed type hypersensitivity (DTH) model, it displayed dose-dependent effects in alleviating the T-cell mediated skin inflammatory response, which was comparable to the reference standard prednisolone. Decernotinib has completed a phase 2a POC study in patients with RA, and results (efficacy and AEs) have been disclosed in papers and abstracts. A larger, longer-duration phase 2b study that will evaluate this entity in combination with methotrexate is ongoing. This study is expected to be 6 months in duration and will evaluate both q.d. and b.i.d. dosing. Filgotinib (8). More recently, Galapagos has entered development with JAK inhibitors;67 Galapagos now has two compounds in development. Filgotinib, also known as GLPG0634 (8), recently completed a phase 2a proof of concept study in RA, and a new entity GSK2586184 (structure not disclosed) is being advanced to phase 2 trials for lupus erythematosus and chronic plaque psoriasis.68 This small molecule inhibitor of the JAK enzymes (8) has only recently been disclosed.69,70 Some details of the discovery of filgotinib have been described as illustrated in Figure 11.71 Lead

Dysregulation of JAK-STAT signaling is a characteristic of certain myeloproliferative neoplasms believed to be caused at least in part by the JAK2 (V617F) mutation, which is observed in approximately 70% of patients. Inhibition of JAK2 has therefore become a viable treatment strategy for this patient population. As a consequence, ruxolitinib has been developed for myeloproliferative neoplasms and represents a promising new therapy for myelofibrosis (MF). Although the activating JAK V617F mutation provides compelling rationale for JAK2 inhibition in myelofibrosis, the population of cells expressing the V167F allele does not decrease with ruxolitinib treatment and it has been suggested that some of the palliative effects of ruxolitinib may be due to the modulation of JAK1 dependent cytokines. Moreover anemia and thrombocytopenia, which are part of the side effects of MF, are more common with ruxolitinib treatment.59 Consistent with this finding, Incyte has reported the testing of a JAK1 inhibitor INCB-039110 (structure not disclosed) in MF.60 Both ruxolitinib and baricitinib have shown efficacy in rodent models of arthritis.61,56a Both compounds have demonstrated the ability to inhibit the signaling and function of pathogenic cytokines such as IL-6 and IL-23 in relevant cells. Once a day dosing of baricitinib improves histological and radiological signs of disease in the rat AIA model, suggesting that fractional inhibition of JAK1 and JAK2 is sufficient to observe significant efficacy in these models. Baricitinib has completed phase 2 studies for RA and is currently in recruitment for phase 3. Details of phase 1 and 2 studies have been disclosed in papers and abstracts.62,63 Decernotinib (7). Vertex is developing a JAK inhibitor for the treatment of RA and is currently in phase 2 clinical development.51 Vertex recently filed a patent for adelatinib (WO2013/006634), which has recently been renamed decernotinib, a JAK inhibitor that we infer is VX-509 based on published USAN information.64,65 However, we will refer to data reported by Vertex as data for this compound. Decernotinib (7) is a pyrrolopyridine-based inhibitor and is a potent inhibitor of JAK3 (see Table 1).66 In cellular assays, Vertex has reported that their inhibitor demonstrated potent inhibition of endogenous JAK1/JAK3 pathways with IC50 values ranging from 50 to 170 nM. It also exhibits very good kinome selectivity. Enzymatic selectivity over the other JAK family members was less than 10-fold; however, in cellular selectivity assays Vertex reported a selectivity window of approximately 25- to 150-fold, depending on the assay comparators (ACR 2011 poster 1136). At Km concentrations of ATP in biochemical assays the IC50 for decernotinib was 13.2 nM for JAK1, 24 nM for JAK2, and 1.2 nM for JAK3, which is consistent with the Ki values described by Vertex with 11, 13, and 2.5 nM for JAK1, JAK2, and JAK3, respectively (ACR 2011 poster 1136). At 1 mM ATP in biochemical assays, decernotinib (Table 1) shows similar activity against JAK3 (IC50 = 74.4 nM) and JAK1 (IC50 = 112 nM). This similar activity against JAK1 and JAK3 is consistent with the HWB data with IC50 values against IL-15 (IC50 = 932 nM), IL-6 (PSTAT1 IC50 = 1870 nM), and IFNα (IC50 = 1290 nM) also being comparable. The data that we obtained with decernotinib are overall consistent with the published data from Vertex (ACR 2011 poster 1136). In preclinical models Vertex reported activity in animal models of aberrant immune/inflammatory function. When dosed orally in a rat model of rheumatoid arthritis (CIA), their compound showed a dose-dependent reduction in ankle

Figure 11. Progression of Galapagos lead to filgotinib (8). Two sites of the original lead molecule 15 were optimized leading to 8. The conserved triazolopyridine cyclopropanecarboxamide moiety is reported to be a key hinge-binding pharmacophoric feature for the series.

identification en route to filgotinib involved screening of an approximately 10 000 compound, kinase focused collection resulting in compound 15. Docking in a JAK2 crystal structure suggested that a ring nitrogen and the hydrogen associated with the adjacent exocyclic amine are involved in binding to the hinge region of the kinase in a donor−acceptor motif. This places the conserved cyclopropyl group in a lipophilic pocket. Lead optimization was conducted through separate manipulation of the two triazolopyridine substituents. These efforts revealed that replacement of the cyclopropylamide moiety was not tolerated, while substitution on the phenyl group, particularly at the para position, was open to a wide range of substitutions. Compounds prepared were driven toward greater JAK1 selectivity and toward improved ADME parameters of solubility and plasma protein binding, eventually leading to filgotinib. A key feature of the filgotinib enzymatic profile is greater selectivity for JAK1 over the other JAKs compared to other experimental JAK inhibitors in the clinic (Table 1). In preclinical studies, filgotinib selectively inhibited JAK1-related pathways, exhibiting approximately 30-fold selectivity for JAK1 over JAK2 in human whole blood (IL-6/pSTAT1 over GMCSF/pSTAT5).72 In biochemical assays shown in Table 1, filgotinib showed a bias toward JAK1 inhibition. The HWB data 5032



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modulate the signaling of JAK-dependent pathways at clinical doses. At the 5 mg b.i.d. dose of tofacitinib, the plasma concentration of inhibitor exceeds the IC50, as measured in whole blood assays, for the JAK1/3 dependent γ-common chain cytokines and JAK1/TYK2 dependent type I interferons and a portion of IL-6 signaling for a portion of each dosing period. Many of these JAK dependent cytokines were previously postulated to play a role in RA,82 but to date, the relative contribution toward efficacy of each cytokine pathway modulated by tofacitinib remains to be fully understood. As suggested in Figure 3 and noted in Table 1, the current inhibitors in development for autoimmune disease have varying levels of selectivity within the JAK family, and as a consequence, the level of relative cytokine inhibition will vary. For example, baricitinib with greater inhibition of JAK2 and TYK2 than tofacitinib is roughly equipotent vs IFNα signaling but more potent vs TYK2/JAK2 dependent IL-12 signaling and modestly less potent vs IL-15. In theory, it may be possible to design JAK inhibitors with distinct selectivity profiles within the JAK family to deliver differentiated results. Although no highly selective inhibitors have been reported yet, current clinical candidates may inform us about the benefits and liabilities of distinct profiles. For example, a JAK1 selective inhibitor as reported by Galapagos could inhibit the γ-common dependent cytokines, the type I and type II interferons and IL-6 without inhibiting JAK2 dependent EPO and TPO signaling or JAK2/TYK2 dependent IL-12 and IL-23 signaling. Conceivably these inhibitors could modulate the immune response without having negative effects on EPO signaling. Alternatively, the JAK1/JAK2 inhibitor baricitinib may derive some of its efficacy from direct inhibition of JAK2/TYK2 dependent IL-12/23 signaling but may be limited in its upper dose by JAK2 dependent inhibition. However, because of a broader panel of cytokine inhibition, baricitinib may achieve efficacy without needing to dose to the point of significant EPO inhibition. Clinical trials testing biologics are revealing that the cytokines driving autoimmune disease will differ with disease, and therefore, a different mix of JAK selectivity may be optimal for each disease. Recent data have shown that whereas an antiIL-17 therapy is highly effective in psoriasis and promising in ankylosing spondylitis, anti-IL-17 therapy was not highly effective in RA and surprisingly poor in inflammatory bowel disease (IBD).83 Thus, it is reasonable to postulate that a JAK inhibitor with a TYK2 bias that would preferentially inhibit the IL-23 and TH17 differentiation and maintenance may have a better benefit risk profile in psoriasis. Tofacitinb has demonstrated efficacy in a full phase 2 psoriasis program with even more robust activity at higher doses. Whether this represents deeper inhibition of a broad panel of cytokines or inhibition of IL-23 is not clear. If a more biased TYK2 inhibitor can be identified and developed, we can test this hypothesis. Tofacitinib has also reported efficacy in ulcerative colitis and inconclusive results in a small Crohn’s study. IL-27 and IL-10, which signal via JAK1 and TYK2, are postulated to be antiinflammatory, particularly in IBD. Therefore, if the positive activity seen for tofacitinib is largely via modulation of the γcommon chain cytokines, a JAK3 selective inhibitor that spares anti-inflammatory cytokines may provide greater efficacy with a more focused inhibitory profile. Finally, much of our understanding of the combinations of JAKs associated with each cytokine pathway comes from mouse or human deficiencies representing complete loss of catalytic

in Table 1 are also consistent with a bias toward the inhibition of JAK1 dependent cytokine signaling (IL-15, IL-6, IFNα) when compared to JAK2 dependent cytokine signaling (IL-12, IL-23, EPO). These data are also consistent with previously reported HWB data for this compound in terms of selectivity and potency.73 The potency observed for filgotinib suggested that higher exposure would be required for clinical efficacy; however, Galapagos has recently reported that filgotinib is metabolized to a JAK1 selective inhibitor that is 10- to 20-fold less potent in human whole blood but has a compensating increase in exposure to drive prolonged inhibition.74,75 To date, Galapagos reports a unique safety profile for an inhibitor that modulates Il-6 signaling, with no consistent increase in LDL cholesterol.76 AC430 (Structure Not Disclosed). Ambit has also been developing a JAK inhibitor with a different JAK isoform selectivity profile. This compound has been shown to inhibit JAK2 in binding assays and TEL-JAK (fusion of TEL dimerization domain with JAK JH1 domain) in cellular assays.77 It also exhibits potent inhibition of pSTAT5 in an ex vivo assay stimulated by TPO, consistent with JAK2 inhibitory activity.77 In preclinical studies Ambit’s compound demonstrated dose dependent improvement in ankle thickness at or above 5 mg b.i.d. in the rat CIA model. Maximum efficacy was observed at 20 and 60 mg/kg q.d. or b.i.d. correlating with histology.77 It also demonstrated efficacy in the murine myelin oligodendrocyte (MOG) induced experimental autoimmune encephalomyelitis (EAE) model for MS exhibiting dose proportional reversal of disease progression in the 20 and 60 mg/kg b.i.d. dose groups, as well as efficacy in the murine DTH model.77 In a phase 1 multidose study this entity was evaluated over 14 days where it was generally well tolerated with predicted dose-dependent PK. Despite alleging to be a specific JAK2 inhibitor, there were no signs of cytopenias in this study. At the same time ex vivo PD biomarkers exhibited both dose- and time-dependent inhibition of relevant cytokine signaling. ASP015K (Structure Not Disclosed). Astellas has entered into an agreement with Janssen Biotech, Inc. to develop their JAK inhibitor for RA following a promising phase 2a outcome in a 6-week study in patients with psoriasis.78 Beyond the new chemical entities (NCEs) described above that have completed clinical studies, there are still others that are listed at clinicaltrials.gov that have entered development recently or that are in the recruitment stage for phase 1 studies. These include Rigel’s compound R348 that is being positioned for dry-eye disease79 and Cell Therapeutic’s macrocyclic JAK2 inhibitor SB1578, which is being studied for the treatment of RA.80 There are also many more compounds in preclinical stages of discovery. The majority of these have been captured in recent patent reviews.67,81

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CONCLUSIONS AND FUTURE DIRECTIONS The approval of tofacitinib represents a milestone as the first kinase inhibitor used for the treatment of RA. Tofacitinib has demonstrated a high degree of selectivity across the kinome, and thus, the effects observed in the clinical studies inform us about the utility of JAK inhibition in the clinic. JAK inhibition represents a change in approach toward autoimmune diseases. Monoclonal antibodies targeting complete inhibition of one to two pathogenic cytokines have shown efficacy in RA for TNF and IL-6 and in psoriasis for IL-23/12 p40 and IL-17. JAK inhibition represents an alternative approach in that inhibitors are dosed to partially inhibit or 5033



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critical review of this manuscript. We also thank Dr. MariaDolores Vazquez-Abad for illustrations in Figure 1. This study was funded by Pfizer, Inc.

activity or, in some cases, loss of proteins. In each case a limited number of cell types and stimuli have been tested, and then receptor pairing has been surmised. In the case of IL-6 signaling, the role of TYK2 has not been consistent across mice and humans with TYK2 playing a more important role in humans. Another indication of our partial understanding is that beyond JAK3 deficiency, which is a phenocopy of the γcommon chain deficiency, only TYK2 deficiencies have been observed in humans, and the consequences were not predictable from existing receptor, cytokine, or STAT deficiencies. Moreover, the consequence of selective partial inhibition cannot be assessed by studying inborn errors of immune deficiency. Therefore, with the advent of selective inhibitors we will begin to be able to more clearly delineate the importance of each JAK in signaling pathways, the target occupancy required for each kinase to see either beneficial or negative effects, and with careful analysis we will be able to determine the consequences of partial inhibition at steady state.

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ABBREVIATIONS USED AE, adverse event; AIA, adjuvant induced arthritis; ALL, acute lymphoblastic leukemia; APC, antigen presenting cell; ATP, adenosine triphosphate; b.i.d., twice daily dosing; CD, Crohn’s disease; CDAI, Crohn’s disease activity index; CIA, collogen induced arthritis; CMP6, compound 6; DMARD, disease modifying antirheumatic drug; DTH, delayed type hypersensitivity reaction; EAE, autoimmune encephalomyelitis; EPO, erythropoietin; FACS, fluorescence-activated cell sorting; GCSF, granulocyte colony stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; HWB, human whole blood; HSA, high-speed analoging; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; IP-10, interferon γ-induced protein 10; JAK, Janus kinase; KC, keratinocyte chemoattractant; LTE, long-term extension study; MPD, myeloproliferative disorder; MTX, methotrexate; NCE, new chemical entity; NK, natural killer; PASI, psoriasis area severity index; PD, pharmacodynamic; PGA, physician global assessment; POC, proof of concept; q.d., once daily dosing; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor κB ligand; SAA, serum amyloid A; SAR, structure− activity relationship; SCID, severe combined immunodeficiency; STAT, signal transducer and activator of transcription; TNF, tumor necrosis factor; TPO, thrombopoietin; TYK2, tyrosine kinase 2; UC, ulcerative colitis

AUTHOR INFORMATION

Corresponding Author

*Telephone: 860-441-0205. E-mail: mark.e.flanagan@pfizer. com. Notes

The authors declare the following competing financial interest(s): Authors are Pfizer Employees and have stock/ shares in Pfizer, Inc. Biographies James D. Clark received his B.A. in Chemistry from the University of Utah in 1982 and his Ph.D. in Organic Chemistry from Harvard University, MA, in 1988, studying mechanistic enzymology under the guidance of Jeremy R. Knowles. Following his Ph.D., he joined Genetics Institute/Wyeth where he led the cPLA2α inhibitor program. He is currently the Head of the Cytokine Signaling Group within Pfizer Immunosciences where one of the key areas of interest is the development of second generation JAK inhibitors.

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REFERENCES

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Mark E. Flanagan is a graduate of New York University. He received his Ph.D. in Organic Chemistry from Colorado State University, working with Professor Robert M. Williams. He then went on to study as an NIH Postdoctoral Fellow for Professor Peter G. Schultz at the University of California, Berkeley. In 1997 he joined the Immunology Group at Pfizer where he worked for 4 years as a medicinal chemist during Pfizer’s early years investigating the Janus kinases as therapeutic targets. In 2001 he transferred to the Antibacterials Group where he worked for several years before moving to his current position where he is an Associate Research Fellow in the Experimental Design Chemistry Group for Pfizer Worldwide Medicinal Chemistry. Jean-Baptiste Telliez received his B.A. in Biochemistry from the University of Lille I, France, in 1988 and his Ph.D. in Molecular Biology from University of Lille I in 1993, studying the transcriptional regulation of the oncogene Ha-ras. Following his Ph.D., he joined the laboratory of Larry Feig at Tufts University, MA, studying the role of GRF in Ras signaling. He then joined Genetics Institute/Wyeth working in the TNF signaling field prior to leading an MK2 inhibitor program. He is currently an Associate Research Fellow leading a second generation JAK inhibitor program.

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ACKNOWLEDGMENTS The authors acknowledge Drs. Birgitta Benda, Mark Bunnage, Jennifer Hodge, Suvit Thaisrivongs, Anthony Wood, and Samuel Zwillich for many helpful conversations and for their 5034



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Systems, catalog no. 219-ILl final concentration of 100 ng/mL) or IL23 (R&D Systems, catalog no. 1290-IL; final concentration of 100 ng/ mL) or D-PBS (unstimulated control), mixed, and incubated for 15 min at 37 °C. The reaction was quenched by adding lyse/fix buffer [BD Phosflow 5× lyse/fix buffer (BD catalog no. 558049)] to all wells at 1000 μL/well, and the samples were incubated for 20 min at 37 °C. After the samples were washed with FACS buffer [D-PBS (Invitrogen catalog no. 14190) containing 0.1% BSA and 0.1% sodium azide], 400 μL of ice cold 90% methanol/water was added to each well and incubated on ice for 30 min. One more wash was done with cold FACS buffer, and all samples were finally resuspended in 250 μL/well of the desired fluorochrome-labeled anti-phospho-STAT antibody (BD) at 1:125 dilution in FACS buffer. After overnight incubation at 4 °C all the samples were transferred into a 96-well polypropylene Ubottom plate (Falcon catalog no. 353077) and STATs phosphorylation were analyzed and quantified using a FACS Canto flow cytometer.

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Discovery and Development of Janus Kinase (JAK) Inhibitors for

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