Kinase Inhibitors for the Treatment of ... - ACS Publications

Perspective Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Kinase Inhibitors for the Treatment of Immunological Disorders: Recent Advances Marian C. Bryan* and Naomi S. Rajapaksa

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Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States

ABSTRACT: Small molecule inhibitors targeting autoimmune and inflammatory processes have been an area of intense focus within academia and industry. Much of this work has been aimed at key kinases operating as central nodes in inflammatory signaling pathways. While this focus has led to over 30 FDA-approved small molecule kinase inhibitors, only one is currently approved for autoimmune and inflammatory diseases. Despite this lack of success, there remains tremendous reason for excitement. Our growing understanding of the biology involved in the inflammatory response, the factors that lead to safer small molecule kinase inhibitors, and the availability of selective tool molecules for interrogating specific nodes and pathways are all pushing the field forward. This article focuses on recent developments requiring novel approaches to create safe and effective small molecule kinase inhibitors and where further work is needed to realize the promise of small molecule kinase inhibitors for patient benefit.

1. INTRODUCTION “Immunological disorders” include allergies, autoimmune diseases, autoinflammatory syndromes, and immunological deficiencies that result from dysfunctions of the immune system. With normal immune system function, self-antigens are presented to naive T cells leading to T-cell activation, differentiation, and expansion.1−6 Activation is controlled by co-stimulatory molecules and soluble factors, such as cytokines. Upon activation, T cells can secrete additional cytokines to attract and activate other cells of the immune system, including macrophages, natural killer (NK) cells, and neutrophils. When this immune tolerance or unresponsiveness to self-antigens is broken, a chronic inflammatory state can persist that can also contribute to further tissue damage.1−6 In some cases (particularly in the inherited genetic disorders) the causative pathways are known. In most cases however causations are multifactorial and remain elusive. While these diseases and disorders are detrimental and potentially life-threatening, the past decade has seen tremendous progress in the advancement of therapies for their treatment. Approval of both large and small molecules targeting diseases such as rheumatoid arthritis (RA) and psoriatic arthritis (PA), as well as the first new approved treatment for systemic lupus erythematosus (SLE) in over 50 years, the monoclonal antibody belimumab (Benlysta)7−9 have all made significant inroads in patient care. Of these, it is the large molecules in particular that have made tremendous strides in © XXXX American Chemical Society

targeting autoimmune and inflammatory processes. Antibodies capable of neutralizing TNF-α, a major cytokine involved in various aspects of innate and adaptive immunity, in particular have made significant impact on RA, PA, Crohn’s disease, and ulcerative colitis.7−9 Like belimumab, which binds B cell activating factor (BAFF), the anti-TNF-α antibodies and decoy receptors work by sequestering inflammation signals away from their immune receptors, thereby preventing or dampening the inflammatory signaling cascades.10 Novel inhibitors remain highly desirable with small molecules enabling inhibition of intracellular targets for improved efficacy. In addition, small molecule inhibitors provide an alternative for patients who have failed on large molecule therapies or who would benefit from combination therapies where multiple pathways are involved or where synergy leads to improved efficacy.11 Over 30 FDA-approved small molecule kinase inhibitors are available for use outside of inflammatory diseases. In particular, inhibition of kinase enzymatic activity has become a powerful tool for disease treatment in the oncology setting.8 These include both reversible and irreversible inhibitors targeting receptor and nonreceptor tyrosine kinases as well as serine/ threonine kinases.12 Numerous clinical trials have been executed hailing only one approved drug demonstrating clear patient benefit for autoimmune diseases: tofacitinib (Xeljanz).13 Received: April 26, 2018 Published: June 5, 2018 A

DOI: 10.1021/acs.jmedchem.8b00667 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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of type I and type II cytokine receptors activate JAK family members upon cytokine binding to the receptor leading to JAK phosphorylation and subsequent downstream signaling (Figure 1).17,18 Interleukins, interferons, colony-stimulating factors, and

The reasons behind these failures include both efficacy and safety concerns. A majority failed to show adequate safety in preclinical or phase I clinical trials. For those that advanced to phase II, insufficient efficacy was observed such as those targeting kinases IκB kinase (IKK) and p38α. In these cases, the kinase inhibitors failed in the clinic despite encouraging animal preclinical models or proof-of-concept studies.14,15 Extended exposure to inhibitors of the Janus kinase (JAK) family led to loss in response in animal models which was recapitulated in patients.16 Factors leading to this escape mechanism are not fully understood, but functional adaptation and reactivation of signaling from chronic JAK1 inhibition have been reported.16 Higher doses that would fully inhibit multiple pathways for highly heterogeneous inflammatory diseases may drive greater efficacy but may also lead to broad immunosuppression.5 Uncovering the right balance here has been largely empirical and varies widely depending on the drug, target pathway, halflife, and disease indication. A number of cases described below will highlight the safety challenges of targeting such critical nodes of the innate and/or adaptive immune response. There is still much hope for the field, though. Growing understanding over the past 10 years within medicinal chemistry in targeting immunologically relevant kinases, achieving ever-higher kinase selectivity, and applying emerging biological tool and techniques continues to drive excitement. Even concern around the risk of malignancy and lymphoma from TNF inhibitors and other disease-modifying antirheumatic drug treatments (DMARDs, e.g., methotrexate) in RA have not come to pass with the rate of lymphomas and other lymphoproliferative disorders being consistent between small molecule kinase inhibitor tofacitinib and other DMARDs.8 Despite this, the safety bar is still very high as patients with these conditions may require lifelong treatment. The focus of this review will center on recent advances in small molecule kinase inhibitors for autoinflammatory and immunological diseases, seeking to highlight the “good” (where medicinal chemistry has led to safe and effective small molecule kinase inhibitors), the “bad” (where chemistry has not been successful for a variety of reasons), and the “ugly” (where chemistry may need to go beyond the standard rule of five and competitive inhibitor paradigm to significantly benefit patients). In an effort to limit redundancy, the goal of this Perspective is not to comprehensively review all targeted kinases for autoimmune and inflammatory disease but to summarize recent advances in the field including clinical candidates and recently disclosed preclinical molecules by highlighting the progress of drug development in several (but not all) key immune signaling kinases. Given our ever-advancing knowledge of crosstalk in signaling cascades within immunology, the authors have divided sections among kinase families with the multiple and varied immunological disorders potentially impacted covered within each section.

Figure 1. JAK/STAT pathway. Step 1: Cytokine binding, complex formation, activation, and phosphorylation of JAK. Step 2: Recruitment and phosphorylation of STAT. Step 3: Phosphorylated STAT (p-STAT) dimerization. Step 4: Nuclear translocation and DNA binding of p-STAT dimer. Step 5: Gene transcription.

hormone-like cytokines all exert their effects through the activation of JAK family members. Dysregulation can lead to hematologic malignancies, autoimmune disorders, and immunodeficiencies.16,18 JAK family members include Janus kinases 1, 2, and 3 (JAK1, JAK2, and JAK3, respectively) and tyrosine kinase 2 (TYK2).5,19 JAK1 and JAK2 and TYK2 are broadly activated by different cytokines across cell types, while JAK3 is limited to primarily hematopoietic cells.8,18,19 Named for the two-faced Roman God, Janus, the JAK family members all have two protein kinase domains within a single polypeptide chain, a true kinase domain and a pseudokinase domain. As the diverse roles of family members have been well reviewed, only a brief introduction to their mechanism of action will be discussed here in order to give context to recent efforts to identify selective family member inhibitors. A simplified version of the signaling cascade is shown in Figure 1. Type I and type II cytokine receptors bind cytokines and form activated and phosphorylated homo- or heteropolymers with their JAK partners (step 1). Cytosolic DNA-binding STAT (signal transducers and activators of transcription) proteins then bind the receptor:JAK complex and are themselves phosphorylated by JAK family members (step 2). Upon phosphorylation, phosphorylated STAT (p-STAT) proteins form homo- and heteropolymers (step 3) which translocate to the nucleus (step 4). p-STAT dimer transcription factors bind specific DNA binding sites regulating gene transcription and cellular function (step 5).5,8,17,18,20 While Figure 1 represents the basic molecular mechanism underlying cytokine signaling through the JAK/STAT pathway, the reality is that JAK/STAT signaling is much more complex. Combinatorial association of JAK family members with different cytokine receptors followed by activation of specific STAT family members can involve JAK1, JAK2, JAK3, and/or TYK2 assembled into homo- or heterodimeric or multimeric complexes, with this association determined by specific receptor chains.16,20 2.A. Challenges of Targeting the JAK Family. Underlying the importance of the JAK family to biological processes are the dramatic effects seen when they are mutated, dysregulated or lost.19 Significant effort has been expended into understanding the impact of inhibiting specific JAK family

2. JAK FAMILY AND THE JAK/STAT PATHWAY Interfering in the JAK-STAT pathway has yielded the only approved small molecule kinase inhibitor for immunological indications. This pathway forms a critical node affecting multiple signaling pathways in both the innate and adaptive immune responses.5 The JAK family is an example of nonreceptor tyrosine kinases (or receptor-associated signaling kinases) that were first discovered in the early 1990s.5 In contrast to transmembrane receptors whose intracellular component contains kinase activity, the cytoplasmic domain B



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Figure 2. Example inhibitors of the JAK/STAT pathway in the clinic for the treatment of immunological diseases or approved by the FDA. Atoms participating in binding to the hinge region of the active site are shown in red with dashed lines for putative hydrogen bonds.

immune cell types involved. It was therefore of critical importance to understand the implications of small molecule inhibition of this pathway in a clinical setting. 2.B. First-Generation JAK Inhibitors. While other family members are ubiquitously expressed and play key roles throughout the innate and adaptive immune system, JAK3 was an attractive preliminary target due to its restriction to hematopoeitic cells, its selective association with type I cytokine receptors with the common γ chain (γc),8,18 and its nonredundant function in lymphocytes.20 Research targeting JAK3 culminated in the first JAK family inhibitors evaluated in the clinic and later approved as tofacitinb (1, Figure 2), the first and currently only approved JAK inhibitor for immunological indications.20 In order to frame discussions around how JAK family inhibitors build on and depart from this first-generation inhibitor, the cocrystal structure of tofacitinib is highlighted briefly in this paragraph and shown in Figure 3. Structural features of the JAK family as well as the discovery of tofacitinib and first-generation JAK inhibitiors have been reviewed previously.5 Briefly, hinge binding is through a two-pronged

members. Significant safety concerns for targeting JAK family members for inhibition arose early with reported lethality in two JAK1 and JAK2 rodent knockout studies. Perinatal lethality upon JAK1 knockout was due to the kinase’s critical roles in IFN-α/β and IFN-γ signaling, mostly through heterodimer formation with either JAK2 or TYK2 and in IL-2 receptor signaling through heterodimer formation with JAK3.16,20 Loss of JAK2 leads to embryonic lethality via loss of definitive erythropoiesis16 due to dysregulated hormone-like cytokine receptor signaling.18 While lethality is a dramatic response to loss of a family member, mutations and deficiencies can also have broad impact. JAK1 or JAK2-deficient humans have not been described, most likely due to immune deficiency, neurological defects (JAK1 deficient), or defective erythropoiesis (JAK2 deficient).18 Mutations in JAK3 were the first mutations reported in the JAK family. Loss-of-function mutations of JAK3 and TYK2 profoundly inhibit immune response in both mice and humans. Inactivating JAK3 mutations give rise to severe combined immunodeficiency caused by poor lymphocyte development and proliferation through JAK3 deficiency.5,8,16,18 TYK2 deficiency affects type I and type II interferons through its association with JAK1 and JAK2. This leads to increased susceptibility to viral and intracellular infection in mouse knockouts.16,18 This is recapitulated in humans with the single patient reported with TYK2 deficiency experiencing multiple opportunistic infections of various organs from viruses, bacteria, and fungi.18 In addition, mutations also led to atopic dermatitis and elevated serum immunoglobulin E (IgE). JAK2 gain-offunction mutations are also a predominant trait in polycythemia vera patients and those suffering from myeloproliferative diseases.8 Finally, mutations in the signaling proteins downstream of TYK2 and JAK1 are responsible for the immune defects seen in autosomal dominant hyper-IgE syndrome (also known as Job syndrome) characterized by abnormally high levels of IgE in the blood. All of this cross-talk highlights how JAK/STAT signaling forms a complex web critical for cellular function and cannot be segregated between innate and adaptive immunity due to the multiple different components and

Figure 3. Tofacitinib (1) cocrystallized with JAK3 (3LXK).21 Hydrogen bonds to hinge residues Tyr904 and Glu903 and a conserved water are presented as black dashes. In addition, gatekeeper residue Met902 is highlighted. C



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interaction between the pyrole NH and the pyrimidine N1 with the backbone of hinge residues Tyr904 and Glu903 in JAK3.21,22 This is a common motif among JAK inhibitors. The C6 hydrogen points back toward gatekeeper residue Met902 and a Met for all members of the JAK family. The aminopiperidine presents the acyl nitrile under the P-loop cleft while the piperidine methyl groups helps maximize interaction with a hydrophobic pocket. Much of the interest in this first generation inhibitor was due to its impressive efficacy in preclinical models. Tofacitinib was advanced into clinical development based on the prevention of cartilage damage in both collagen-induced and adjuvantinduced arthritis models in rodents.23 Once in RA patients, tofacitinib was effective as a monotherapy or in combination with the standard of care, methotrexate. It was tofacitinib’s efficacy in distinct patient populations such as methotrexate nonresponders that generated the most excitement.20 In 2012, tofacitinib was approved by the FDA for the treatment of RA20 and remains in the clinic for additional indications including psoriatic arthritis (NCT01976364), SLE (NCT02535689), and alopecia (NCT02812342). Further exploration in RA is ongoing (NCT02092467, NCT02831855, NCT02321930). As the JAK family functions at critical nodes in the immune response, there was significant interest in the safety and tolerability of tofacitinib in patients. Safety signals including overall risk of infection were similar for patients receiving either monotherapy or in combination with nonbiologics for RA.24 Some of the safety concerns, including increased incidence of infection as well as dose-dependent neutropenia and significant lymphopenia, could be related to both JAK1 and inhibition of other kinases.18,20,25 While touted as a JAK3 inhibitor, tofacitinib, like many first generation kinase inhibitors, is not a single-kinase inhibitor. The molecule exhibits a high degree of broad kinome selectivity but is in fact a pan-JAK-inhibitor with kinase enzymatic inhibition values ranging from equipotent among JAK family members to a small degree of TYK2 selectivity (Table 1).8,23 Potency reported for cellular assays appear more consistent among reports, and tofacitinib is active in multiple cellular readouts for p-STAT signaling through various JAK1- and JAK3-mediated heterodimers.5,23 The lack of JAK3 specificity is not surprising given the high sequence homology among JAK family members in the ATP binding domain (Figure 4).5,21 The crystal structures have only

Figure 4. X-ray cocrystal structures with sequence alignment for JAK1 (green, 3EYG), JAK2 (magenta, 3FUP), JAK3 (blue, 3LXK), and TYK2 (orange, 5WAL).5,21,32,33

recently become available, highlighting only a small handful of subtle amino acid differences across the JAK family in the region where tofactinib binds.5 Most larger changes do not face into the binding pocket and are therefore not involved in tofacitinib binding (e.g., Asn832 and Arg830 in JAK3 versus His and Glu in JAK1 and TYK) or are understated (Ala966 in JAK3 versus Gly in other members, Ile960 in TYK2 versus Val in others, etc.). Tofacitinib shows greater inhibitory effect against JAK1/3dependent processes like IL-2-dependent T cell proliferation in a cellular setting.20,25 Inhibition of other JAK family members, particularly JAK2, is most likely the cause of the neutropenia. Inhibition of JAK2 may lead to anemia and other hematologic toxicities due to its role in hematopoiesis.16 The severity of these findings can limit dose escalation.16 These safety concerns, including increased incidence of infection and dosedependent neutropenia and significant lymphopenia, may lessen tofacitinib’s applicability to certain indications requiring additional target coverage which may be related to kinase selectivity.18,20,25 Treatment in some indications is not being pursued further, such as in Crohn’s disease and psoriasis where either lack of efficacy or potential safety concerns hamper future developmennt.20 The other JAK3 inhibitor currently in the clinic for immunological disorders, peficitinib (2) from Astellas, has a similar selectivity profile to tofacitinib34 and is currently in phase 3 for RA (NCT01638013). In addition to tofacitinib, there is one other JAK inhibitor approved for patient treatment by the FDA, though not for immunological disorders: ruxolitinib (3, Figure 2). This inhibitor of JAK1 and JAK2 is currently approved for myelofibrosis and polycythemia vera, where JAK2 gain-offunction mutations are common.8 Others, though, are in development.4,35 Pyrolopyrimidine baricitinib (4) is similar in structure to 3, both replacing the aminopiperidine of 1 with a pyrazole functionalized with a cyanoethyl moiety. In a phase III study, patients with RA and inadequate response to biologic DMARDs showed clinical improvement upon receiving baricitinib.36 In the cases of ruxolitinib and baricitinib, the number of safety concerns reported including thrombocytopenia are potentially related to their JAK2 inhibition.37 Fedratinib (5), a selective JAK2 inhibitor,27 was placed under a clinical hold for safety concerns related to Wernicke’s encephalopathy.38

Table 1. Reported Enzymatic Potency for JAK Family Members for Disclosed Molecules enzyme assay IC50 (nM) compd 5

1 226 35 45 527 628 75 85 929 1130 12a,31

JAK1

JAK2

JAK3

TYK2

15 4 6 4 105b 360 112 363 29 43 2

77 5 9 7 3 1 619 2400 803 120 68b

55 10k >10k 2300 280

489 5 30 61 405b 66 >10K 2600 1253 4700 12

a

ki values reported. bEstimated ki using reported JAK1 selectivity index. D



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Vertex has not listed further development in their more recent pipeline disclosures. More selective JAK1 molecules recently reported include triazolopyridine filgotinib (8, Figure 2).47 This molecule is described as JAK2-sparing by design with modest selectivity over other family members that increases in a cellular setting.5,34,47,48 While filgotinib may be less potent than other JAK1 inhibitors with reported IC50 values ranging from 10 nM to >300 nM depending on ATP concentration in the assay,5,47 the compound nonetheless showed efficacy in a rat collageninduced arthritis (CIA) model with significant reduction in clinical score seen at levels well below the measured whole blood IC50. Additional coverage is provided by a JAK1-selective metabolite 13 wherein the amide bond is cleaved by a carboxylesterase to unmask the free amine (Figure 5). While

Development in the oncology space is still being pursued for JAK2 inhibitors including ruxolitinib and BMS-911543 (6), a recently disclosed and highly selective JAK2 inhibitor for myeloproliferative neoplasms.28 Ruxolitinib remains under clinical investigation for a number of oncologic and bone marrow disorders as well as for atopic dermatitis and alopecia areata but only as a topical cream for these immunological disorders (NCT03011892, NCT03257644, NCT02553330). Topical treatment may be ideal for ruxolitinib in the immunology setting as this is less likely to cause systemic adverse effects.39 TYK2 is another JAK family kinase involved in regulating the immune response. TYK2 function has been associated with Th1 and Th17 cell differentiation and activation through its essential role in IL-12 and IL-23 signaling. The Th1 and Th17 pathways have been implicated in the pathogenesis of psoriasis and IBD and an IL-12/IL-23 antibody (ustekinumab) is already approved for psoriasis and is in clinical development for other indications. As such TYK2 is a promising target for therapeutic intervention.40,41 While several research programs have reported efforts toward selectively targeting TYK2 including multiple patent filings by Takeda, Roche/Genentech, and Array Biopharma, no highly selective TYK2 molecules have advanced into the clinic.42 Pfizer’s PF-06700841 (structure undisclosed), which targets TYK2 and JAK1, is currently under investigation for a number of indications in the clinic including chronic plaque psoriasis (NCT02969018), alopecia areata (NCT02974868), and ulcerative colitis (UC) (NCT02958865), all in phase II.35 2.C. Advances in Selective JAK Inhibitors. As a result of our increasing knowledge of the importance of kinase selectivity for safety along with the desire to understand the roles and impact of the individual JAK family members on disease, a new wave of more selective JAK inhibitors have been recently reported.43 These include inhibitors of both JAK1 and JAK3. Decernotinib (7, Figure 2) was recently disclosed as a more JAK3 selective molecule.35 The pyrolopyridine is a potent inhibitor of JAK3 with mild selectivity over other JAK family members in enzymatic assays but improved selectivity over other JAK family members in cellular assays relative to 1 (7, JAK1/2/TYK2-mediated IL-6 p-STAT3 cell IC50 > 11 200 nM vs JAK3/1 mediated IL-2 p-STAT3 cell IC50 = 99 nM (113×); 1, JAK1/2/TYK2-mediated IL-6 p-STAT3 cell IC50 = 666 nM vs JAK3/1-mediated IL-2 p-STAT3 cell IC50 = 30 nM (22×)).5,44 The differences in magnitude of cellular potency shift compared to biochemical potency are well-known across JAK family members and have only recently been exploited to drive our understanding of selectivity.45 Members display different affinities for ATP with KM values ranging from 4 to 40 μM. This has a significant impact on the enzyme to cell inhibition translation. Elegant work from Pfizer compared assay results for several JAK inhibitors across biochemical assays in the presence of ATP at 1 mM or the family member’s KM for ATP and in their primary cellular assays. The resulting analysis illustrated the importance of determining biochemical IC50 values under a physiologically relevant ATP concentration in order to properly assess compound selectivity and to better predict cellular function.45 Decernotinib progressed through phase II where it reduced the signs and symptoms of RA in patients in combination with a DMARD at 12 weeks (NCT01784938).46 Further RA studies have been completed (NCT01590459), but results have not been disclosed and

Figure 5. Metabolism of JAK1-selective molecule filgotinib by carboxylesterase leads to active metabolite 13. IC50 values correspond to inhibition of cytokine-induced STAT phosphorylation in CD4+ cells or CD33+ cells in human whole blood.49

less potent than the parent, amine 13 exhibits higher exposure leading to relatively long duration of JAK1 inhibition following filgotinib dosing.49 Compound 8 is currently recruiting for multiple phase II and phase III studies across indications including Crohn’s disease (NCT02914600, NCT02914561), UC (NCT03201445, NCT02914535, NCT02914522), RA (NCT02889796, NCT03025308, NCT02886728, NCT02873936), cutaneous lupus erythematosus (CLE, NCT03134222), and psoriatic arthritis (NCT03101670). Speaking to the benefit of selective JAK inhibition, early side effects observed with other less selective JAK inhibitors such as anemia (presumedly JAK2-related) or neutropenia were not observed.50 Whether this is due to its improved JAK1 selectivity or a result of weakened JAK activity in general is unclear. Other JAK1-selective molecules reported in the literature include PF-04965842 (9), itacitinib from Incyte (10), imidazopyrrolopyridines 11 from Abbvie, and 12 from Genentech (Figure 2).30,31,43,51 PF-04965842, under development by Pfizer, is undergoing clinical investigation in two phase III studies in atopic dermatitis (NCT03422822, NCT03349060) as well as phase I (NCT03386279). Cyclobutane sulfonamide 9 had previously terminated a phase II study in moderate to severe psoriasis (NCT02201524) for portfolio reasons despite results suggesting the molecule was well tolerated and improved patient symptoms.52 While noting selectivity of >20× relative to JAK2 and >100× relative to other family members, the discovery story of itacitinib has yet to be publicly disclosed and currently there are no trials described for immunological disorders.51 Lastly, two imidazopyrrolopyridines have been reported: upadacitinib (11, ABT-494) and compound 12 from Genentech.30,31 E



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Compound 11 was covered extensively in a patent from Abbvie in 2015 where they profiled the molecule in various cellular and in vivo settings including PK, efficacy, and preliminary phase I study results.30 While 11 is reported as 43 nM in the biochemical assay when performed with 0.1 mM ATP, the potency shifts greater than an order of magnitude to 60 kinases was also noted though the data were not included.30 It is interesting to note that compound 11 potency and JAK family member selectivity were improved when analyzed in various JAK-mediated cellular assays. These include 74-fold selectivity over JAK2 (8 nM vs 608 nM for phospho-STAT6 in TF1 cells) compared to tofacitinib (1) which showed 24-fold selectivity over JAK2 (44 nM vs 1110 nM for phospho-STAT6 in TF1 cells).30 Upadacitinib has been reported as well as tolerated by patients under clinical evaluation and continues to be evaluated in phase II and phase III for a range of autoinflammatory and immune diseases including Crohn’s disease (NCT02782663, NCT03345849, NCT03345823, NCT03345836), atopic dermatitis (NCT02925117), ankylosing spondylitis (NCT03178487), ulcerative colitis (NCT02819635, NCT03006068), psoriatic arthritis (NCT03104374, NCT03104400), and RA (NCT02720523, NCT02955212, NCT02706873, NCT02706951, NCT02049138, NCT02629159, NCT02706847, NCT02675426). Genentech reported imidazopyrrolopyridine 12 with >30× selectivity over JAK2 (Table 1).31 This enhanced selectivity is attributed to a hydrogen bond between the hydroxyl moiety on 12 and glutamic acid E966 which is an aspartic acid in JAK2 and hypothesized to make a weaker water-mediated interaction. Tricycle 12 was highly efficacious in a rat CIA efficacy model though no further development has been reported.31 2.D. Alternative JAK Modalities. Given the difficulties in generating exquisitely selective inhibitors for individual JAK family members, a number of strategies are being explored. One potential is to exploit modalities to avoid systemic safety concerns, such as tissue-targeted formulations as mentioned above which would be useful for alopecia, psoriasis, IBD, and asthma. Due to the importance of JAK family members in the signaling of multiple cytokines involved in asthma, another route being explored is inhaled administration.53 In murine chronic house dust mite models of asthma, the pan-JAK inhibitor VR58822 has demonstrated potent anti-inflammatory activity. A proof-of-concept phase I trial evaluating the molecule (structure not disclosed) on airway epithelial cells of patients with severe asthma has been completed (NCT02740049), but results have not been disclosed. Pfizer has also reported on their pan-JAK inhibitor PF-06263276 for inhaled and topical delivery (Figure 6).54 In this report, the in vitro and in vivo potency, pharmacokinetic, and safety profile demonstrated that compound 14 is potentially well suited for use as an inhaled or topical therapy, through direct administration to the lungs or skin, while avoiding any systemic-based JAK inhibition.54 There is also a particularly interesting case for targeting covalent inhibition of JAK3 due to the presence of Cys909, which is a Ser in other family members (Figure 4).55 Reported irreversible inhibitors have been able to sufficiently block the development of inflammation in rodent RA models without effects on hematopoiesis.55 These contain PF-06651600 (15), whose discovery was previously disclosed.56 The pyrrolopyrimidine acrylamide showed superior PK properties to

Figure 6. Chemical and cocrystal structures of pan-JAK inhibitor PF06263276 (14) and JAK3 covalent inhibitor PF-06651600 (15).54,56

previously described JAK3 covalent inhibitors and exhibited strong anti-inflammatory properties in two rodent models of RA and multiple sclerosis (MS), suggesting that inhibition of JAK3 alone is sufficient for intervention in inflammatory and autoimmunes diseases. As such, this compound is currently being investigated in phase II for UC (NCT02958865), alopecia areata (NCT02974868), and RA (NCT02969044). Finally, targeting either the pseudokinase domain (JH2) or other allosteric sites is an approach that could yield inhibitors with improved selectivity and overcome resistance to JAK inhibitors due to target-specific mutations in the ATP-binding site.16,18,40,57 As previously noted, all JAK family members contain both a kinase and pseudokinase domain. Targeting the pseudokinase domain provides an alternative enticing area for small molecule inhibition through the pseudokinase domain’s role in inhibiting the functional protein kinase domain. The structures of the pseudokinase and kinase domains of several JAK family members have been reported57,58 along with small molecule binders. These include JNJ-7706621 (16), which targets the pseudokinase domain for JAK2,59 and imidazolopyridazine 17 was recently described by BMS, which targets the pseudokinase domain of TYK2 (Figure 7).40 Potential

Figure 7. Reported pseudokinase inhibitors of JAK family members.

development of covalent allosteric inhibitors to the pseudokinase domain has also been hypothesized around indole 18 where a covalent warhead is attached to the piperidine for the selective targeting of TYK2 cysteine C736.60 Whether these alternative modalities will suffer from the loss in response that has been seen with chronic exposure to JAK inhibitors in animal models and recapitulated in patients remains to be seen.16 F



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3. SYK, BTK, AND ITK: T-CELLS, B-CELLS, AND BEYOND T cell receptors (TCRs), B cell receptors (BCRs), and Fc receptors (FcRs), which are collectively termed classical immunoreceptors, signal by a conceptually similar mechanism. Like the JAK-STAT pathway for TCRs, B cells rely on signaling cascades through nonreceptor tyrosine kinases to mediate inflammatory responses in their role as key players in adaptive immunity. Three main protein families, the Src family of kinases like Lyn, spleen tyrosine kinase (SYK), and the TECfamily kinase Bruton’s tyrosine kinase (BTK; Figure 8), have

hematopoietic cells, such as B cells, monocytes, mast cells, and neutrophils, SYK is involved in several immunoreceptor signaling pathways.62 It is also expressed in nonimmune cells such as osteoclasts, where it plays a role in bone resorption. Two examples of signaling pathways involving SYK, one with BCRs and the other with FcRs, are summarized in Figure 8. Ligand engagement of cell surface immunoreceptors induces phosphorylation of two tyrosine residues on the short peptide sequences known as immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytosolic portion of the receptor or associated transmembrane adaptor protein. This phosphorylation step is generally carried out by a Src family kinase (e.g., Lyn in Figure 8). SYK binding to the phosphorylated ITAMs leads to SYK being phosphorylated by Lyn. SYK has several phosphorylation sites and, through selective phosphorylation, has the flexibility to interact with a large diversity of effector molecules and trigger inflammatory events in multiple cell types.62,63 ZAP70, a homolog of SYK expressed in T cells, plays a similar role in TCR signaling (Figure 13). Because SYK is a key component in various signaling cascades, it is considered a “master regulator” of inflammatory responses.65 As such, there is potentially a greater advantage to inhibiting SYK, which is upstream in cell signaling pathways.64 As described earlier in the JAK family, there is a concern with inhibiting such crucial and ubiquitous messengers. A tremendous amount of research has studied the impact of SYK inhibition with murine genetic SYK knockouts initially leading to safety concerns. Homozygous syk‑/syk‑ knockout mice were perinatal lethal due to vascular lymphatic shunting.66 Generation of SYK-deficient bone marrow chimeric mice by transplanting syk‑/syk‑ fetal liver cells to lethally irradiated wild type mice resulted in viable animal models.67 The chimeric mice did not have the developmental defects in the lymphatic system and were found to effectively ablate SYK expression in the hematopoietic compartment.65,67 Importantly, these mice showed no signs of arthritis upon treatment with arthritogenic

Figure 8. Role of SYK in BCR and FcγR1 signaling.

been identified and targeted for both their immune and nonimmune functions.13,61,62 Despite the significant interest in all of these three protein families, we will only focus on those currently being pursued clinically for immunologic intervention: SYK and BTK. 3.A. Spleen Tyrosine Kinase (SYK). SYK is a member of the Src family that has attracted significant attention in the context of immunological disorders.63,64 Primarily expressed in

Figure 9. Select clinical SYK inhibitors. Areas of the molecule that participate in binding the hinge region are shown in red with dashed lines for putative hydrogen bonds. G



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serum in a passive transfer model.65,67 Additional mouse studies demonstrated that SYK is involved in a variety of disease processes, including RA, allergic asthma and rhinitis, SLE and immune thrombocytopenic purpura.68 Together, these studies have provided ample evidence that SYK inhibition may be a safe and effective means of treating several immunological diseases. With such critical importance, it is unsurprising that the discovery and development of SYK inhibitors have been a very active area of research. While a SYK-selective inhibitor has yet to be approved, a number of them have entered clinical evaluation. Rigel Pharmaceuticals has brought forward multiple compounds centered on a diaminopyrimidine scaffold (19−21, Figure 9).63 R-343 (19) was one of the earliest clinical candidates and, while promising phase I clinical trials, did not achieve expected end points upon further evaluation and was discontinued.63 R-406 (21) is the metabolite of prodrug fostamatinib (20), which was approved by the FDA for adult patients with chronic immune thrombocytopenia in April 2018. In this example, the phosphate is rapidly and extensively cleaved to give 21, an ATP-competitive inhibitor that binds to the receptor in a U-shape conformation and interacts with the hinge through the pyrimidine N1 and N2 on the linker.69 While the prodrug discovery efforts leading to 20 have not been disclosed, its PK profile has been reported.70 Phosphate 20 overcomes the challenge of low aqueous solubility of 21, while the PK profile of the active metabolite potentially allows for once daily or twice daily oral administration of fostamatinib.70 Both 20 and 21 have been evaluated in several animal models where they suppressed joint swelling and bone erosion in preclinical arthritic rat models.69 On the basis of these findings, both were advanced into clinical trials for multiple indications. Two phase 2 trials showed 20 afforded clinically significant improvements in RA patients over a DMARD background (NCT00326339 and NCT00665925).71 Despite these promising results, several subsequent studies demonstrated inferior response or limited/no efficacy with fostamatinib.71−73 While fostamatinib displayed insufficient efficacy in patients, it was well tolerated in phase I and II trials. The most common treatment-related adverse events were gastrointestinal intolerance, neutropenia, and elevated liver transaminases, resolving upon discontinuation of treatment. This may be due to offtarget kinase inhibition by 20, 21, or both. Although initially presented as SYK-selective inhibitors, many subsequent reports have demonstrated that they are actually promiscuous and inhibit some kinases to an even greater extent than SYK.74,75 For example, it is known that 21 has inhibitory potency against the JAK pathway.69 This poor kinase selectivity may be both beneficial and problematic toward therapeutic benefit as well as impacted dose-limiting adverse events in clinical trials.74,75 On the basis of the hypothesis that more selective molecule will be advantageous, many other discovery programs have prioritized identification of SYK inhibitors with improved kinase selectivity. One such program culminated in entospletinib (22, Figure 9) from Gilead Sciences, which showed significantly improved kinase selectivity.74 Crystallographic studies reveal similar binding poses for both 21 and 22. The authors hypothesized that pyrimidine 21 has a greater degree of rotational freedom allowing it to adopt alternative conformations that can interact with several kinases, and these are not accessible to the more conformationally restricted 22 (Figure 10).74 Compound 22 is currently in

Figure 10. Overlay of first generation SYK inhibitor R406 (21, magenta, 3FQS) and entospletinib (22, blue, 4PUZ).

clinical trials for several hematological malignancies and in the recruiting stage for a phase 2 trial in chronic graft versus host disease (NCT02701634). Another SYK inhibitor, MK-8457 (23, Figure 9), was also evaluated in two phase II RA trials (NCT01651936, NCT01569152).76 Both were terminated in 2013 and 2014 due to serious infections. Other SYK inhibitors are being evaluated in the oncology setting including PRT062607 and cerdulatinib (24 and 25, Figure 9) which is reported to be a dual SYK/JAK inhibitor (NCT01994382).77 In addition to those described above, multiple SYK inhibitors with largely undisclosed structures have recently undergone clinical evaluation in patients for immunological disorders. These include GlaxoSmithKline’s GSK2646264, Hutchison MediPharma Limited’s HMPL-523, Gilead’s GS-9876, and Ascena Biopharmaceuticals’ ASN002 (26, Figure 9). GSK2646264 is currently under recruitment for a phase I trial in CLE (NCT02927457) using topical application after completion of a phase I for urticaria (NCT02424799), also as a topical cream. Neither the discovery stories or the results from this or SYK inhibitor HMPL-523’s phase I trial in RA (NCT02105129) have been disclosed. A recent presentation of preclinical data on GS-9876 suggests that the compound is a highly selective SYK inhibitor and demonstrates efficacy in rat collagen-induced arthritis models.78 Clinical trials are currently planned or recruiting in adults with impaired renal function (NCT02959138), lupus membranous neuropathy (NCT03285711), Sjorgren’s syndrome (NCT03100942), and CLE (NCT03134222). In a number of these cases, GS-9876 is being investigated in combination with JAK and/or BTK inhibitors. Lastly, pyrimidopyridazinone 26, a dual JAK/SYK inhibitor, was being evaluated in a phase I trial in subjects with atopic dermatitis (NCT03139981).79 The study has been recently completed, and we look forward to the results. Early clinical and preclinical successes of 20/21 have validated SYK as a target for inhibition in the context of immunological diseases. However, the revelation that they are poorly selective has made the interpretation of those results challenging. Additionally, while the treatment-associated adverse events associated with fostamatanib have been suggested to arise from off-target kinase inhibition, the serious infections resulting from treatment with 23 call into question the selectivity profile of that compound along with the safety of inhibiting SYK. These observations have compelled some to develop more highly selective SYK inhibitors and others to intentionally tune broader inhibitory activity against multiple kinases. Both the combination approach being investigated with H



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Figure 11. Overlay of ATP-binding site inhibitors of BTK: (A) 27 (purple, 5P9J) and 36 (orange, 5VFI); (B) 27 (purple, 5P9J) and 37 (blue, 5T18).61,80

Figure 12. Representative examples of BTK inhibitors including those disclosed and under clinical evaluation. Areas of the molecule that participate in binding the hinge region of the active site are shown in red with dashed lines for putative hydrogen bonds where crystallographic data are available.

marrow kinase (Bmx), interleukin-2 inducible T cell kinase (ITK), resting lymphocyte kinase (RLK), tyrosine kinase expressed in hepatocellular carcinoma (TEC), and Txk. Tec kinases are mainly expressed in hematopoietic cells, and BTK expression has been found in B cells, marrow-derived stem cells, mast cells, monocytes/macrophages, neutrophils, dendritic cells, erythroid cells, platelets, plasmablasts, hematopoietic stem cells, multipotent progenitors, and developing myeloid cells. However, it is not expressed or downregulated in plasma cells, natural killer cells, or T cells.81,82 As shown in Figure 8, BTK acts downstream of SYK where it amplifies signals from ITAMs to phospholipase C γ 2 (PLCγ2), which in turn mobilizes Ca2+ and activates NF-κB. BTK also

GS-9876 and the dual JAK/SYK approach of compound 26 are very interesting and speak to the antithesis of single kinase inhibition for disease modulation. Indeed, the assumption that improved selectivity will lead to higher likelihood of safety and efficacy is being challenged with JAK family inhibitors.17,19 With a rich diversity of SYK inhibitors entering the clinic representing varying degrees of kinase selectivity, the results of these trials will have very interesting implications far beyond SYK, likely bringing some clarity and further questions. 3.B. Bruton’s Tyrosine Kinase (BTK). The key kinase in the BCR signaling cascade is Bruton’s tyrosine kinase (BTK), a nonreceptor cytoplasmic tyrosine kinase in the Tec family.61,80,81 In addition to BTK the Tec family includes bone I



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plays a role in TLR and FcR signaling in myeloid cells.19,81−83 In so doing, BTK regulates several phases of the cell life cycle including development, differentiation, proliferation, and apoptosis of B-lineage cells. As a consequence of its distribution and key signaling roles, alterations in BTK synthesis, expression, or activation are related to different human diseases. BTK is overexpressed or hyperactivated in a number of autoimmune diseases and cancers.84,85 Studies using BTK deficient and transgenic mice as well as BTK inhibitors demonstrate the central role BTK plays in these disease associated pathways and cellular responses. BTK-deficient dendritic cells show impaired secretion of inflammatory cytokines, and BTK-deficient mast cells are impaired in degranulation in response to FcR activation.19 Transgenic animal models have shown overexpression of BTK is related to spontaneous germinal center formation in lymph nodes, autoantibody production, and SLE-like autoimmune pathology. Inhibitors of BTK have shown activity in both BCR and FcγRdriven models of autoimmune disease, and BTK-deficient mice are also protected from SLE and autoimmune arthritis, even if associated inflammatory responses are not blocked.80,81,83,85 The role of BCRs and B cell signaling in autoimmune disease and the knowledge that BTK-deficient patients can live relatively normal lives with regular immunoglobin transfusions suggest that BTK inhibitors may be useful in treating a variety of immunological disorders including RA, SLE, MS as well as B cell lymphomas.19,61,83,85 BTK inhibitors are currently in the clinic for a number of these disorders. With the strong interest in BTK as a therapeutic target, there has been much interest in both its structure and activation. Structural elucidation reveals five domains: the amino-terminal pleckstrin homology domain, a Tec homology domain, two Src homology domains (SH2 and SH3), and a C-terminal catalytic domain containing tyrosine kinase activity.83−85 The first domain contains sites for interaction with transcription factors and with phosphatidylinositol 3,4,5-trisphosphate (PIP3), allowing for BTK to associate with the plasma membrane, while the Tec homology domain contains conserved BTK motifs.83−85 Activation happens first at the catalytic domain, also referred to as SH1 with further activation in the SH2 and SH3 domains. Both covalent and noncovalent inhibitors of SH1 have been targeted and show a broad range of shapes and properties for the ATP binding region, as highlighted in Figure 11, despite similar interactions with hinge residues Met477 and Glu475.83 Some inhibitors have been shown to bind to the active state conformation where the DFG sequence is in the “in” position, while others bind and stabilize the DFG “out” conformation. Furthermore, induced pockets have also been noted where helix C is stabilized by salt bridges.83 Two general approaches have been used to take advantage of these structural topographies to design selective BTK inhibitors: target amino acid residues found in BTK but rare in the kinome with covalent inhibitors or target more uncommon binding pockets of inactive BTK conformations with noncovalent inhibitors.83 There is currently only one approved BTK inhibitor to date, the covalent modifier ibrutinib (27, Figures 11 and 12). To date, ibrutinib is approved in only nonimmunological disease settings for certain lymphoproliferative malignancies.80 Molecule 27 features an α,β-unsaturated amide electrophile, in this case an acrylamide, as a Michael acceptor to irreversibly covalently modify an active-site cysteine, Cys481 (Figure 11), in the ATP-binding pocket in SH1.86 With the approval of ibrutinib, there has been increased

excitement for the target with close to 70 active clinical trials for inhibitors of BTK as of February 2018 (www.clinicaltrials.gov), many of which are in oncology settings. Several of the inhibitors that have been and/or continue to be assessed in both oncological and autoimmune diseases are covalent inhibitors of BTK, all of which rely on covalent interaction with Cys481.80 Those being pursued for autoinflammatory and immunological disorders are shown in the top box in Figure 12. These include olmutinib (HM71224, 28), acalabrutinib (29), spebrutinib (30), evobrutinib (31), PRN1008 (32), and BMS-986195 (33).61,81,87−89 With the exception of alkynes 29 and 33, all contain an acrylamide for covalent modification. Olmutinib shows comparable potency to ibrutinib (27 IC50 = 0.7 nM vs 27 IC50 = 2 nM) and a slight improvement in kinase selectivity versus other Tec kinase family members which carry a conserved cysteine in the binding pocket.61,81,88 Compound 28 has completed a phase I study in RA that completed in 2015 (NCT01765478), but the results have not been made public though they have disclosed activity in experimental mouse models of arthritis.85 Reported potency values for acalabrutinib (ACP-196, 29) appear more modest, with biochemical IC50 values ranging from 3 nM to 18 nM. Molecule 29 exhibits superior kinase selectivity when compared to ibrutinib across a 456-member kinase panel, and an in vivo study showed that oral administration of ACP-196 led to superior inhibition of BCR-induced CD69 expression in murine splenic lymphocytes when compared to ibrutinib.81,85,90 Compound 30 is currently undergoing several clinical trial evaluations, though largely in the oncology setting. One phase II clinical trial in RA (NCT02387762) was completed in 2016, though results have not been disclosed.85 Spebrutinib (CC-292, 30) and evobrutinib (M2951, 31) both feature a monocyclic pyrimidine core for putative hinge binding. Pyrimidine 30 bears a similar architecture to thienopyrimidine 28 with a few changes including the replacement of an ether linkage with an aniline and the thiophene with a fluorine atom. Spebrutinib completed a phase II clinical trial in RA in July 2017 (NCT01975610) though results have not been disclosed. Evobrutinib 31 contains a similar substitution pattern to ibrutinib off the central pyrimidine core with the exception of the methylamino chain to the acrylamide. The amine is tied into a pyrazole in the case of 27.85 Evobrutinib is currently recruiting for a phase II clinical trial in RA (NCT03233230). Other covalent inhibitors either have recently been evaluated in the clinic or are currently being evaluated for immunological disorders including BIIB068 (NCT02829541) and PRN1008 (32, NCT03395210, NCT02704429) and BMS-986195 (33) whose clinical candidate structures were just disclosed.87,89 All of the inhibitors for BTK discussed thus far and shown in the top box in Figure 12 are either reversible or irreversible covalent inhibitors relying on covalent bond formation with Cys481. PRN1008, for example, is a reversible covalent inhibitor while ibrutinib is an irreversible covalent inhibitor.91,92 Application of an irreversible versus reversible covalent strategy may impact both the target engagement and off-target kinase selectivity.93 Irreversible inhibition can impact PD through target engagement where the level and frequency of dosing relate to the restoration of pharmacological activity via protein target resynthesis in the case of a true irreversible inhibitor. This uncoupling of response from PK has the ability to enable less frequent dosing and potentially lower dosing.94 J



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Ser543, Val546, and Tyr551 which only occur as such in BTK.85 Despite promising preclinical efficacy, when GDC-0834 was advanced into the clinic, hydrolysis of the amide between the thiophene and the phenyl ring was seen in healthy volunteers.84 GDC-0853 prevents this by cyclizing the amide into a piperazinone reminiscent of the piperidinone seen in compound 34.61 Overlay of the cocrystal structure of 36 with 27 in Figure 11 shows occupation of the deep lipophilic pocket by the piperazinone tricycle in orange.61 This pocket is absent in the ibrutinib cocrystal structure. Optimization of the tricycle, linker pyridine, and solvent front piperazine then allowed for superior potency and in vivo PK when evaluated in the clinic.61 GDC-0853 showed preclinical efficacy in both B and myeloid cell-dependent inflammatory arthritis models and in mouse models of SLE.61 GDC-0853 is currently undergoing clinical evaluation in phase II for SLE (NCT03407482, NCT02908100) and RA (NCT02983227, NCT02833350) after successfully showing suitable safety, PK and PD profiles in phase I. Bristol-Myers Squibb has also disclosed a clinical candidate, BMS-986142 (blue in Figure 11, 37 in Figure 12), that builds on earlier work identifying the carbazole motif as potent inhibitors of BTK and JAK2.80 In this series, the carbazole amide binds to the hinge region with the carbazole NH hydrogen-bonded to the backbone carbonyl of Met477. Much optimization of the phenyl ring and substituents was then undertaken to improve both oral exposure and kinase selectivity of the initial lead and then restrict the conformational flexibility to provide a single stable atropisomer, compound 37.80,95 By overlaying this structure with ibrutinib in Figure 11, one can see the quinazolinone extending past the covalent warhead of ibrutinib in the active site that interacts with Cys481. Furthermore, the back pocket occupied by the phenyl ether of ibrutinib is not occupied by BMS-986142. BMS-986142 is now recruiting volunteers for a phase II study in RA (NCT02638948) after completing multiple phase I studies including three in RA or arthritis (NCT02456844, NCT02762123, NCT02880670). The search for selective small molecule inhibitors of BTK is well underway. A number of potent kinase inhibitors have now been identified with a range of off-target kinase selectivity in both covalent and noncovalent scaffolds. Whether it is a covalent or noncovalent inhibitor that provides the best route for BTK signaling inhibition remains to be seen. It is possible that one method of inhibition will be better suited for different patients, co-dosing strategies, and/or dosing regimens. With selective covalent and noncovalent small molecule inhibitors of BTK kinase activity, these questions will be soon answered. 3.C. Interleukin-2-Inducible T Cell Kinase (ITK). ITK has been primarily associated with the regulation of T helper (Th) 2-driven immunological diseases as well as development of Th17 cells and their production of the proinflammatory cytokine IL17A.96−100 Both knockout and gene expression studies suggest that ITK has a dominant role in mediating TCR signaling, and there is potential therapeutic benefit to its inhibition in inflammatory and immunological disorders. Knockout studies with individual or combined T cell-expressed Tec family kinases have been generated. Loss of ITK leads to pronounced effects on the production of IL-2 and Th2 cytokines, while RLK knockout mice have a more mild phenotype, with loss predominantly affecting Th1 cytokines.101 In vivo studies have shown mice lacking ITK have diminished

In terms of off-target activity, as mentioned earlier, covalent modification can be very useful in promoting kinase selectivity where an uncommon amino acid in the active site can be targeted. In the case of BTK, there are 10 known additional human kinases in the vast kinome that possess Cys residues equivalent to Cys481 in their active site.61,91 Ibrutinib is reported to inhibit all of these and the other covalent members mentioned inhibit some if not all of the Tec family where kinase information is available.81,84,85,90 In addition, a collaboration between Scripps Research Institute and Pfizer has called into question the claims of exceptional kinase selectivity that covalent inhibitors have enjoyed, suggesting that a window of proteome-wide selectivity is a more accurate description.86 Furthermore, these covalent inhibitors can also inhibit other kinases through noncovalent binding given their ATP-like binding mode, further increasing safety concerns due to selectivity.81 Whether those other kinases are inhibited in a reversible or irreversible manner will factor into their off-target potency. Of potentially greater impact to the long-term use of covalent inhibitors in chronic indications such as those in autoimmune and inflammatory conditions is treatment resistance through mutation. This is indeed the case for 27 with C481S being the most common mechanism for ibrutinib-acquired resistance.61,81,83,85 Ibrutinib-resistant patients have also presented with C481R, C481F, and C481Y, and mutations of the gatekeeper Thr (T474I, T474S) have also been disclosed.81 These mutations lead to loss in activity for many covalent inhibitors that were tested against the mutants in biochemical assays performed by Johnson and colleagues at Genentech.81 Second-generation, potentially more selective BTK inhibitors including reversible covalent and noncovalent inhibitors are now being evaluated clinically after showing efficacy in various RA models with three such being recently reported. Examples of noncovalent inhibitors of BTK kinase activity are shown in the lower box of Figure 12 and include RN486 (34), GDC-0834 (35), GDC-0853 (36), and BMS-986142 (37). Pyridone 34 is comparably potent to ibrutinib in a competitive biochemical assay (IC50 = 0.3 nM) with a high degree of selectivity (>100×) in a broad kinome panel.83 Excitingly, 34 demonstrated activity in a variety of cellular and in vivo models of inflammation and immunomodulation.83−85 These comprise BCR and FcR signaling blockade and inhibition of several disease-relevant cytokines in a cellular setting.83 In both mouse CIA and mannan-induced collagen antibody induced arthritis (mCAIA) models, 34 was able to inhibit inflammation and bone erosion as well as show additive effects upon co-dosing with methotrexate in a rat AIA model.83 Furthermore, the molecule inhibited progression of glomerular nephritis and SLE-prone NZB/W mouse model.84 Despite these promising results, no entry for clinical evaluation has been reported. Genentech has reported on multiple noncovalent BTK inhibitors including GDC-0834 (35) and the recently disclosed GDC-0853 (Figure 11 and Figure 12 in orange, 36). Like compound 34, they feature a core 3-aminopyrazinone (35) or 3-aminopyridinone (36) for interaction with the hinge.61,84,85 The molecules also do not attempt to access the pocket filled by ibrutinib’s phenyl ether (Figure 11 in pink). Like 34, molecules 35 and 36 stabilize the inactive conformation resulting in the formation of a decidedly specific lipophilic pocket, referred to as the H3 pocket, leading to broad selectivity across the kinome.61,81,85 The pocket is flanked by K



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leukocyte protein of 76 kDa (SLP-76, in navy in Figure 13) by ZAP-70. Activation of this complex allows for ITK (in green) recruitment to the membrane where it becomes phosphorylated by Lck.96,100 Phosphorylation of ITK activates the kinase, and upon autophosphorylation of Tyr180 in the SH3 domain, ITK is primed for interaction with and phosphorylation of PLCγ1 lipase. PLCγ1 then hydrolyzes phosphoinositol diphosphate (PIP2) into inositol triphosphate (IP3) and diacyl glycerol (DAG), leading to stimulation of nuclear factor of activated T cells (NFAT) and NF-κB, respectively. Stimulation of these factors leads to multiple responses including production and release of proinflammatory cytokines, Erk activation, Ca2+ flux, actin reorganization, and stimulation of T cell transcription factors. These in turn lead to cytokine production and T cell proliferation and differentiation in T cells, NK cells, and mast cells where ITK is predominantly expressed.96,99 Several noncovalent ITK inhibitors have been described in the patent and scientific literature, but only a few select molecules are mentioned here as the ITK inhibitor space has been well-covered in other reviews.97,98,104 Beginning with BMS-509744 (38), whose discovery predates public disclosure of the ITK structure, the aminothiazole was identified as a potent ATP-competitive inhibitor of ITK with >50-fold selectivity over a panel of 18 other protein kinases and >200fold selectivity when compared to other Tec family tyrosine kinases (Figure 14, parts A and B).105 In the first preclinical demonstration of ITK inhibitor efficacy, this compound showed dose-dependent reduction in lung inflammation in a murine ovalbumin allergic asthma model.105 The discovery and cocrystal structures of two other noncovalent inhibitors followed suit with thienopyrazole 39 and tetrahydroindazole 40 (GNE-4997) (Figure 14A).104,106,107 These two molecules are interesting in their similarity to each other and other known

Th2 responses to various infectious agents while retaining Th1 responses. ITK-deficient mice challenged with ovalbumin exhibited drastically reduced airway hyper-responsiveness and lung inflammation in allergic asthma models, and T cells from these mice showed reduced IL-5 and IL-13 secretion.102 ITK gene expression is also upregulated in patients with atopic dermatitis.103 Together these studies suggest potential therapeutic benefits of inhibiting ITK in Th2-driven diseases like asthma and atopic dermatitis, and several organizations have set out to discover potent small molecule inhibitors of its kinase activity.96,100,102,103 TCR activation triggers a signaling cascade involving three kinases: Zap-70, Lck, and ITK, with a simplified representation of that pathway shown in Figure 13.96,100 Activation of Src

Figure 13. Role of ITK in TCR signaling.

kinase Lck, shown in orange in Figure 13 leads to phosphorylation of the CD3 ITAM, resulting in ZAP-70 activation, phosphorylation, and subsequent phosphorylation of a complex comprising linker of activated T-cells (LAT, in brown in Figure 13) and Src homology 2 domain containing

Figure 14. Noncovalent inhibitors of ITK. (A) Structures of noncovalent ITK inhibitors 38−42. Areas of the molecule that participate in binding the hinge region are shown in red. (B) Cocrystal structure of BMS-509744, 38, 3MJ2.108 (C) Cocrystal structure of 39, 3V8T. (D) Cocrystal structure of GNE-4997, 40, 4rfm. L



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ITK inhibitors as well as for their differences from BMS509744.104 As shown in Figure 14parts B, C, and D, all three molecules bind to the hinge region of the active site through Glu439 and Met438. Thiazole 38 and pyrazoles 39 and 40 adopt a hydrogen bond with the NH of Met438. The amide NH of 38 and 40 then forms a hydrogen bond with the backbone carbonyl of Glu439. The pyrrole moiety of 39 then mimics this same interaction. Diversion is then seen when the molecules approach gatekeeper residue Phe435. For thienopyrazole 39 and hexahydrocyclopropylindazole 40, the molecules do not extend past the gatekeeper but either interact with residues near the gatekeeper or fill nearby hydrophobic pockets (Figure 14, parts C and D). The difluoro moiety of 39 points away from the Phe toward the side chain of Lys391 (Figure 14C).107 The two fluorine atoms of GNE-4997 40 occupy a hydrophobic pocket within the active site adjacent to Phe435 (Figure 14D). BMS-509744 (38) takes a strikingly different approach with Phe435 (Figure 14B) by constructing hydrophobic contacts with the gatekeeper via the sulfur atom and then progressing down the side of the active site with the adjacent methylbenzamide moiety. The benzamide is in-plane to the aminothiazole, presumably through stabilizing interactions between the thiazole sulfur and the benzamide oxygen. Molecule binding leads to stabilization of the activation loop. Cocrystallization studies reveal the activation loop folded into a short helix blocking the protein substrate-binding site, giving an autoinhibited inactive conformation and improved kinase selectivity relative to other kinases outside the Src family.108 Molecules 38, 39, and 41 follow a similar path toward solvent along the hinge. Other recent scaffolds such as benzthiazole 41 and aminopyrazole 42 use elements of these core scaffolds while also filling the ribose pocket with a (1R,4R)-4-aminocyclohexanol moiety (Figure 14A).109,110 Pyrazole 42 also makes a modified hinge interaction through Glu436 as opposed to Glu439. The branched alkyl chains of 38 and 39 are solvent exposed as are the phenyl and sulfone moieties of 40.104,106−108 Both BMS-509744 and GNE-4997 extend past the opening to the active site with the alkane of 38 pointing perpendicular to the ligand core in a similar region to the phenyl of 40.106,108 The cyclic sulfone helps to orient the phenyl ring for an edge-to-face interaction with Phe437.106 Thiazole 38 progressed into in vivo studies where it reduced anti-CD3-induced IL-2 production and ovalbumin-induced lung inflammation.104 Molecules related to pyrazolocyclohexane 40 have been reported to show inhibition of IL-2 and IL-13 upon dosing in an ITK PD model as well.106 Despite promising potency and selectivity, no clinical progress has been reported for BMS-509744 or GNE-4997 with both potentially hampered by metabolic instability.104,106 Insufficient target inhibition has been noted for 38 in a T cell prolymphocytic leukemia study focusing on TCR signaling when compared to dual ITK/RLK inhibitor PRN694 (43, Figure 15).111 Imine 43 is interesting as it is both a dual Tec family kinase inhibitor and because it is a covalent inhibitor.111 Other covalent irreversible inhibitors that have been developed for ITK include acrylamides 44 and PF-06465469 (45).111−113 BTK inhibitor ibrutinub (27) also inhibits ITK through covalent modification of Cys442 and targeted interaction with this residue has been a central focus of many ITK inhibitor programs seeking potency and potential broader kinome selectivity.111−113 Thiazolopyridine 44 was optimized for inhalation and selected for development but was not progressed

Figure 15. Selected covalent inhibitors of ITK kinase activity.

into the clinic.112 Piperidine acrylamide 45, which bears some structural similarity to 27, was found to be a very potent irreversible covalent inhibitor of ITK.113 This compound also inhibited BTK with similar biochemical potency. Compound 43 is reported to have greater than 50× selectivity over BTK though it does have a narrow window of selectivity in biochemical assays when compared to TEC and RLK.111 Despite much excitement and effort to discover novel ITK inhibitors, so far there has been little progress in the clinic. There are several potential challenges to ITK inhibition that may account for the apparent discrepancy between preclinical and clinical progress. First, both RLK and TEC are expressed in T cells and there may be some redundancy in roles of TEC kinases in TCR signaling.114 Combining knockouts of both RLK and ITK leads to a more pronounced phenotype further relaying its dominance among the Tec family in T cells, though RLK can compensate for loss of ITK function revealing challenges with selective Tec family member inhibition.101 Also, while downstream consequences of TCR activation are reduced upon ITK knockout, they are not completely ablated leading to altered development and differentiation of distinct T cell subsets.100 A disconnect in translatability may also exist between kinase knockout and kinase dead mouse models and human disease intervention. Many studies providing support for the hypothesis of ITK inhibition in Th2-driven inflammatory diseases were carried out in mice lacking ITK. Because of this, the results of these experiments may be complicated by T cell signaling and developmental differences in these mice.115 A recent study of rechallenge in an ovalbumin-induced mouse model of asthma highlighted the additional complexities of ITK biology.101 Unexpectedly, compound 40 resulted in an increase in Th2 cytokines in a murine ovalbumin rechallenge asthma model. This is the opposite of the intended outcome and suggests that ITK may have different roles in initial versus repeated exposure to stimuli.101 There is currently only one ITK inhibitor under clinical evaluation, JTE-051, which is in phase II for RA (NCT02919475) and psoriasis (NCT03358290). While the chemical structure and discovery story have not been shared, the phase 2 study of JTE-051 in rheumatoid arthritis patients will be very informative. It remains to be determined what extent of kinase specificity would give optimal impact on an immune response. Redundancy among TEC kinases has led some to develop multi-Tec kinase inhibitors as mentioned for 43 and evaluation of a dual ITK/RLK inhibitor in patients will be informative if one enters clinical evaluation.116

4. INTERLEUKIN-1 RECEPTOR ACTIVATED KINASE The inhibitors outlined above largely seek to stop the response of the adaptive immune system in autoimmune diseases by targeting key signaling kinases in B and T cells. In these cases, the adaptive immune system has already been engaged by the M



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Dysregulation of TLR signaling has been implicated in several inflammatory diseases including RA, SLE, gout, and Lyme disease.121,122 Multiple TLR family members are found to be more highly expressed in several cell types including macrophages, monocytes, synoviocytes, and certain dendritic cells in RA patients compared to healthy individuals. TLR7, an endosomal TLR family member, and its eliciting of type 1 interferon production have been heavily implicated in SLE pathophysiology. Inhibition of IL-1 signaling is already an approved therapy with anakinra, a recombinant human interleukin-1−receptor antagonist that blocks the proinflammatory effects of interleukin-1β, approved for patients with RA. As such, novel strategies for inhibiting TLR and IL-1R signaling are of therapeutic interest. The IRAK family comprises four members, IRAK1 and IRAK4 which both have kinase activity and IRAK2 and IRAK3 (also called IRAKm) which are catalytically inactive.119,123 Both IRAK4 and IRAK1 are serine−threonine kinases that act as active kinases with additional scaffolding functions.124 IRAK1 catalytic activity is required for IFNβ production, but not all IRAK4 signaling is dependent on IRAK1 catalytic activity. This includes IL-1 or TLR-agonist stimulated signal propagation.6 Because of this, IRAK4 has been called the “master IRAK” as it is the only family indispensable in IL-1R and TLR signaling.120 There are several reports of small molecule kinase inhibitor programs targeting the IRAK4 kinase domain.123 The search for IRAK4-selective small molecule kinase inhibitors has endured since the early to mid-2000s and was recently comprehensively reviewed in this journal.123 As such, this section will focus on only those recent examples where IRAK4 inhibition has been evaluated in vivo. Of the multitude of IRAK1/4 or IRAK4-specific molecules reported, only three are known to have entered into the clinic: molecules from Bayer and BMS and PF-06650833 (Figure 17, 46). Neither

innate immune system to promote an inflammatory response to what is perceived as a nonself antigen.117,118 An alternative approach to blocking tissue damage is through targeting the innate immune system. The cells that comprise this system include natural killer cells, granulocytes such as neutrophils, basophils, eosinophils, and monocytes and macrophages which all play critical roles in this response. Macrophages and dendritic cells are particularly instrumental in both innate and adaptive immunity with dendritic cells comprising the most important antigen presenting cell, displaying antigens to T cells, forming a critical link between the innate and adaptive immune system.117,118 As the body’s first line of defense, the innate immune system must be sensitive and able to respond to immediate infections and injuries. Receptors that recognize pathogens are key regulators of innate immunity and either initiate signaling cascades themselves through kinase function or rely on other kinases to form critical nodes similar to examples above. One such example is the interleukin-1 receptor activated kinase (IRAK) family. The IRAK family is critical to the signaling transduction for the IL-1 receptor (IL-1R) family and for TLRs.6,119,120 TLRs are a family of transmembrane pattern recognition receptors (PPRs) that are critical in the detection of pathogen-associated molecular patterns (PAMPs) of foreign microbes such as lipopolysaccharide (LPS). Together with the IL-1R family, they enable cells to quickly respond to inflammatory cytokines by mounting the initial protective response through the activation and downstream function of IRAK. Recognition of PAMPs by the TLRs, either at the cell surface or in the endosomal compartment with the exception of TLR3, leads to recruitment of MyD88 to the Toll/IL-1R (TIR) domain followed by IRAK family members to form the myddosome complex, shown in Figure 16.121,122 The first IRAK family member involved is

Figure 17. Structures of IRAK4 kinase inhibitors PF-06650833 (46), pyridine 47, and BMS-986126 (48).

Bayer nor BMS has reported the chemical structures of the molecule(s) they advanced into human studies. However, they have reported on some structures including compounds (Figure 17, 47)125 and BMS-986126 (Figure 17, 48),121 respectively. The discovery story of the Pfizer clinical candidate (PF06650833, 46) was recently disclosed.126 Briefly, compound 46 was derived from a carboxamide discovered during a fragment screening approach where binding to hinge residues is through the carboxamide moiety off the isoquinoline (Figure 18). Advances in IRAK4 potency were then gained by growing into the space below the P-loop with the 5-hydroxymethyl pyrrolidinone. Displacement of a putative high-energy water molecule with the ethyl substituent off the pyrrolidinone then brings in further potency for the target. The IRAK family displays high structural similarity,123 and compound 46 inhibits IRAK1 at 91% at a single 200 nM screening concentration. The molecule also inhibits four other

Figure 16. TLR and IL-1R signaling through IRAK family members to upregulate type I IFN genes leading to proinflammatory cytokine production.

IRAK4. IRAK4 activation by autophosphorylation leads to IRAK1 phosphorylation which in turn leads to recruitment of TNF-receptor associated factor (TRAF), IRAK1:TRAF dissociation from the complex, and downstream signaling through multiple cascades leads to transcription and initiation of the inflammatory response. IL-1R also contains a TIR domain and is reliant on this pathway for signal transduction. N



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patients with IRAK4 or MyD88 deficiencies, which are indistinguishable in the clinic, is poor largely due to recurrent life-threatening bacterial diseases in infancy and early childhood though no death or invasive infectious diseases were reported after 8 and 14 years of age, respectively.128 As such, the impact of IRAK1/4 inhibition in certain disease populations will be interesting to understand and the industry looks forward to the trial outcomes of these first IRAK4 inhibitors to enter human studies.

5. NF-κB INDUCING KINASE (NIK) As mentioned above with the IRAK family, the NF-κB family of transcription factors regulate the expression of specific genes involved in the inflammation response as well as proliferation, differentiation, cell adhesion, and apoptosis.129 Transcription complexes comprise various homo- and heterodimeric subunits p50, p52, c-Rel, RelA (p65), and RelB. NF-κB signaling occurs through two distinct pathways: the canonical and noncanonical pathways. In the basal or resting state, the canonical pathway induces activation of an IκB-bound NF-κB species. Receptor activation leads to degradation of I-κB downstream of IKK α/ β/γ complexes, which results in translocation of canonical NFκB subunits, such as p65/p50, to the nucleus, where they trigger immune gene expression.130 The noncanonical NF-κB signaling requires NF-κB inducing kinase (NIK, also known as MAP3K14) as its central regulator (Figure 19).129−132 In the basal or resting state, NIK signaling is constitutively tempered by the continuous degradation of NIK protein through its association with TRAF2, TRAF3, and cellular inhibitors of apoptosis (cIAP) (Figure 19, left).132,133 This complex is responsible for mediating the ubiquitination and degradation of NIK. Once activated, though, the noncanonical pathway (Figure 19, right) depends on activation of IKKα dimers only, activates NF-κB transcription factors p52 and RelB, and requires NIK for these functions.129,131,132 Pathway activation occurs upon ligand binding to a subset of TNF superfamily receptors including CD40 (as illustrated in Figure 19), lymphotoxin β receptor, BAFF receptor, and receptor activator of NF-κB (RANK). Binding and receptor

Figure 18. Cocrystal structure of PF-06650833, 46, with IRAK4 (5UIU).

kinases at greater than 70% when screened against a panel of 278 kinases at this same concentration. However, the carboxamide displays subnanomolar potency for its target (IC50 = 0.2 nM) providing a potentially more than sufficient selectivity window.126 Kinase selectivity may be further enhanced in a cellular setting based on the high KM for ATP that IRAK4 displays.127 Compound 46 showed modest stability and exposure in vivo but elicited dose-dependent reduction in LPS-induced TNF-β in rodent serum. This is of interest as compound 48 elicited little activity downstream of TLR4 upon LPS stimulation, which can also activate an MyD88independent pathway.121 Compound 46 recently completed multiple phase I evaluations investigating modified release formulations and is currently recruiting RA patients with inadequate response to methotrexate in a phase II trial (NCT02996500). The question remains whether inhibition of such a critical component of innate immunity may cause too broad of an impact on sentinel innate immune function.120 At the same time, other IRAK1/4 inhibitors have been shown to only inhibit response in certain cell types such as plasmacytoid dendritic cells (pDCs), the major systemic source of IFNα, though dispensable in other cells.6,124 Clinical outcome of

Figure 19. Role of NIK in the noncanonical NF-κB pathway. O



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Figure 20. (A) Crystal structure of apo-murine NIK kinase domain where the pocket is filled by a sulfate (4G3C). (B) Overlay of inhibitors 51 and 53 in murine NIK kinase domain (4G3E, 5T8O).

Figure 21. NIK inhibitors described by Amgen and Genentech. Areas of the molecule that participate in binding the hinge region of the active site are shown in red. Areas occupying the back pocket are shown in blue.

autoreactive B cells and autoimmune disease pathology as BAFF has a large contribution to diseases such as lupus. Insufficient efficacy has been noted for inhibitors targeting IKK,15 and targeting the active NIK pathway may provide an alternative. Mice with a mutation in the nik gene (NIKaly/aly) that prevent NIK association with IKKα and NIK-knockout mice are unable to engage the noncanonical NF-κB pathway and show impaired B cell survival.4 NIK-knockout mice showed less bone erosion than wild-type mice in murine models of inflammatory arthritis.138 As NIK function is implicated in several autoimmune and inflammatory disorders and is positioned downstream of multiple receptors and upstream of proinflammatory gene expression, small molecule inhibition of NIK has been proposed as a therapeutic strategy. Despite recognition that NIK “may be an ideal target for clinical intervention”,139 there have only been a handful of reports describing discovery efforts and preclinical studies.140−144 This apparent discrepancy may have been in part due to an initial lack of protein structural information with early discovery efforts relying on virtual screens with homology models.142,143 Recently, however, both Amgen and Genentech identified stable protein constructs and reported crystal structures of the

activation triggers NIK dissociates from the complex. Without constitutive degradation, NIK accumulates in the cell and activates IKKα through phosphorylation. Once activated, IKKα phosphorylates p100, a precursor protein of NF-κB2 p52. Phosphorylation of p100 in turn leads to its ubiquitination and partial degradation to release mature p52. Transcription factor p52 heterodimerizes with RelB, and this complex translocates to the nucleus and initiates transcription of target genes leading to the release of chemokines and cytokines.132 The noncanonical NF-κB pathway is thought to regulate important functions including lymphoid organogenesis, B cell maturation and survival and bone metabolism.132 Dysregulation leading to pathway activation is linked to both autoimmune and hematological disorders.132−134 For example, while BAFF is necessary for activation and maturation of B cells,135 high levels are associated with autoimmune and inflammatory diseases such as RA, SLE, and MS.136 Similarly, while RANK signaling under normal circumstances regulates osteoclastogenesis, hyperactivity is associated in bone erosion. RA patients in particular show high BAFF serum levels as well as high expression levels of NIK in synovial endothelial cells.137 These observations suggest that increased BAFF signaling through the noncanonical pathway may result in the increased survival of P



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NIK kinase domain.141,145 The apo-NIK kinase domain was found to have an active “DFG-in” conformation without phosphorylation and a solvent-occupied back pocket behind the Met gatekeeper residue (Figure 20A). Both groups then used structure-based drug design methods to discover potent NIK inhibitors.140,142 Beginning with a high throughput screening campaign hit and a CHK1-based homology model, scientists at Amgen identified a series of inhibitors that interact with the hinge through an imidazopyridinyl pyrimidinamine (compound 49, Figure 21).142 After obtaining a cocrystal structure with an early lead, they found significant improvements in biochemical and cellular potency by appending a propargyl alcohol substituent to two indolederived scaffolds (50 and 51, Figure 21).141,142 The alkyne functional group passes through the narrow channel adjacent to the gatekeeper Met471 residue and forms hydrogen bonding interactions with amino acids lining the back pocket (Figure 20B, gold). Genentech also described their NIK inhibitor discovery efforts beginning with tricyclic HTS hit 52. This series of molecules takes advantage of the type I1/2 binding mode seen in the Amgen molecules. The carboxamide moiety forms two hydrogen bonds with the hinge region via Leu474 and Glu472, and the tricyclic core provides the vector required to access the back pocket (Figure 20B, salmon).140,141 Application of a propargyl alcohol moiety allows the molecule to extend past the gatekeeper into a deep back pocket and substitution with an isoxazole fills the space (Figure 21). Although imidazolocarboxamide 53 showed acceptable kinase selectivity, the authors realized the tricyclic core could bind other kinases through an alternative hinge-binding motif through the benzoxepin oxygen based on reported inhibitors to PI3K family members. To preclude this binding mode, the authors investigated two modifications: substitution of the imidazole ring to clash with the PI3K active site (amide 54) and removal of the oxygen atom (azabicyclo[4.1.1]octane 55) (Figure 21). Both compounds were found to be highly potent and selective NIK inhibitors, and in a cellular imaging study, amide 54 was shown to prevent nuclear translocation of p52 in HeLa cells. Although NIK inhibitors have yet to enter the clinic, recent preclinical studies in inflammation models show encouraging results. Small molecule NIK inhibitors have reduced angiogenesis in an RA synovial inflammation model and decreased both levels of p52 protein in the liver and the expression of proinflammatory genes in a mouse carbon tetrachlorideinduced acute liver inflammation model.144,146 Administration of 51 has been shown to protect mice from severe liver injury and death in a mouse model of liver inflammation. In a recent report, NIK inhibition by a small molecule kinase inhibitor mimicked the pharmacological effects of BAFF blockade both in vitro and in vivo in an experimental lupus model and demonstrated inhibition of BAFF-independent pathways and end points.130 With the recent advances in understanding NIK structure and function along with promising preclinical data, evaluation of NIK inhibitors in the clinical setting will be very interesting to see.

domains.147,148 RIPK1 is the only family member to contain a C-terminal death domain, through which RIPK1 can be recruited to signaling complexes that initiate different pathways (Figure 22). It also possesses an intermediate domain

Figure 22. RIPK1 regulation of multiple roles upon signaling by TNF. Abbreviations: TRADD, TNF-R1-associated death domain protein; TAK1, transforming growth factor β-activated kinase 1; IKK, IκB kinase; MLKL, mixed lineage kinase domain-like protein.

containing a RIP homotypic interaction motif (RHIM) that likely mediates protein−protein interactions. RIPK2 bears a caspase activation and recruitment domain. This protein is essential in regulating signaling downstream of the Crohn’s disease susceptibility protein, NOD2. In this role, RIPK2 is part of the protein complex that recognizes intracellular bacterial infection and helps tailor the cytokine response to eradicate an offending pathogen.149 RIPK3 has a unique C-terminus and a RHIM motif like that found in RIPK1. RIPK4 and RIPK5 are characterized by the ankyrin repeats in their C-terminus. While less well studied, RIPK4 (also known as protein kinase Cassociated kinase) is the causative gene in popliteal pterygium syndrome, a disease characterized by early lethality with multiple developmental abnormalities. RIPK6 and RIPK7 (also known as leucine-rich repeat kinases 1 and 2) contain potential recognition motifs for molecular patterns resulting from damage, stress, or pathogens as well as domains for stimulating kinase activity and have been associated with the pathogenesis of Parkinson’s disease.148,149 Molecular modeling and crystal structures have shown the kinase domains between at RIPK1, -2, and -3 are largely superimposable. However, there are subtle structural differences between these three kinases, which can help explain pharmacologic specificity as was elegantly reported by Abbott et al. in 2016.149 Furthermore, recent work has shown that, at least for RIPK2, Tyr474 can also be phosphorylated along with the previously known serine and threonine.149 RIPK1 and RIPK3 have well-established roles as central inducers of necrotic cell death (necroptosis) activation and apoptosis via caspase interaction.150 Emerging evidence now suggests additional roles for both RIPK1 and RIPK3 in the direct regulation of proinflammatory signaling as well with RIPK1 mediating the signaling switch between necroptosis and inflammatory gene expression.147,148,151 A range of triggers including extracellular factors linked to innate immune regulation, such as ligands for TNF receptor (TNF-R), TNF-α, interferon-α receptor (IFNαR), and TLR families, as well as viral infection, genotoxic stressors, and T cell activation, have been associated with RIP activation and induction.148,150 These hallmarks of the proinflammatory

6. RECEPTOR-INTERACTING PROTEIN KINASE (RIPK) The receptor interacting protein (RIP) kinase family has recently received attention as potential targets for disease intervention in the innate immune system. Seven members have been identified within the RIP family, and all share a homologous kinase domain but have distinct functional Q



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response then lead to necroptosis that damages tissues and liberates DAMPS, further stimulating the inflammatory response and/or upregulating inflammatory signals.147 RIPK1 acts as a molecular switch to determine which path the cell will trigger (Figure 22).147,148 However, the mechanisms by which these and other RIPK functions are exerted, be it scaffolding or through catalytic activity, are still under investigation.150−152 Given their key roles in inflammation, inhibitors of RIPK1, RIPK2, and RIPK3 have been discussed in the literature, though only molecules targeting RIPK1 and RIPK2 are currently known to be in the clinic. RIPK1 has effector functions downstream of multiple signaling events including TNFR-1 activation by TNF as shown in Figure 22.147 These different pathways result from modification of RIP by ubiquitination, deubiquitination, or phosphorylation along with protein−protein interactions.147,148,151 Upon TNF stimulation of TNFR-1, RIPK1 and TRADD are recruited to TNFR-1. Ubiquitination of RIP1 leads to recognition by ubiquitin-binding domain containing proteins transforming growth factor β-activated kinase 1 (TAK1) and IKK. TAK1/ IKK-mediated activation of NF-κB then leads to inflammation in one possible outcome.147 Deubiquitination results in the formation of the ripoptosome which leads to apoptosis via the caspase 8 cascade in another.147 Finally, autophosphorylation of RIPK1 and RIPK3 leads to formation of the necrosome. RIPK3 then interacts with mixed lineage kinase domain-like protein (MLKL). MLKL then can associate with other proteins leading to mitochondrial ROS production resulting in necroptosis or form oligomers leading to membrane rupture.147,151 With its role upstream of a number of inflammatory pathways, RIPK1 is positioned well within the innate immune response as a target for inhibition. Indeed, research by Najjar and others has shown RIPK1 activation by LPS in macrophages strongly increased expression of a broad range of inflammatory molecules while RIPK3’s functions were more contributory and context dependent.150,152 The only known RIPK1 inhibitor in the clinic is GSK2982772 (Figure 23, 57) which is undergoing evaluation

While initially not structurally enabled, a cocrystal structure of GSK2982772 gave insights into both components by suggesting it functions as a type III binding inhibitor. Compound 57 does not make interactions with the hinge but rather sits deep into the ATP binding pocket with the benzoxazepinone ring occupying the space normally occupied by the α phosphate of ATP. The binding mode leads to the single kinase specificity but also yields significant potency differences between primate and non-primate RIPK1 that was not seen in molecules using other binding modes. Specific mutations in mouse suggested that the loss in potency is a result of the inability of the activation loop in non-primate RIPK1 to adopt the required conformation to bind this template.154 While species differences contributed to poor potency in rodents, the molecule was able to prevent TNFinduced necrotic cell death and block spontaneous cytokine release in an UC explant assay.154 It will be very interesting to see how this first molecule targeting RIP signaling impacts disease progression in the clinic. Other RIPK inhibitors may not be far behind as selective inhibitors for RIPK1, RIPK2, and RIPK3 have been recently reported as well as RIPK1/RIPK3 inhibitors though their suitability as anti-inflammatory agents is unknown.155 Figure 24 highlights the broad range of chemotypes able to inhibit kinase function of RIP family members. These include additional putative type III binders such as compound 58,155 type II binders like the multikinase inhibitor ponatinib 59,156 and smaller chemotypes like necrostatin-1 analog nec-1a (60).157 Compound 58 is hypothesized to be a type III binder based on molecular docking studies.156 In simulation studies, 58 may form hydrophobic interactions with RIPK1 through both the phenyl group and the branched alkane. In addition, two hydrogen bonds, one between the carbonyl oxygen and a backbone NH and the other between the N-OH and a Val residue, are hypothesized.155 Of note is compound 58, a type III binder like 57, which does not show the difference in potency between primates and non-primates. As such, researchers were able to assess the molecule in various murine models and found compound 58 efficiently protected mice from TNF-α-induced multiorgan damage and death.155 Type II binder 59 establishes a single hydrogen bond to hinge residue Met98 as well hydrophobic interactions with nearby residues. The central linker forms two additional hydrogen bonds, one with a side chain of a Glu residue and the other with a backbone NH. Upon binding to the inactive “DLG-out” conformation, the trifluoromethyl group fills the hydrophobic pocket vacated by the DLG. Structural determination with necrostatin 60 shows the molecule binding largely outside the ATP binding site and locks RIPK1 in an inactive conformation.157 More recent reports show type I binders of RIPK2 GSK583 (61),158 imidazopyridine 62,159 and pyrazolopyrimidine WEHI345 (63).160 Quinoline 61 is an ATP competitive inhibitor that interacts with the hinge through a hydrogen bond between the quinolone N and the backbone of Met98. Other interactions include those by the 3-aminoindazole in a back pocket past the Thr gatekeeper and a hydrogen bond between the sulfone and the side chain of Ser25 side chain. The polar Ser25 residue is rare among protein kinases, with only three other kinases having a serine at this position, and is exploited in this case and by 62 for broader kinome selectivity. While 61 exhibits safety signals preventing advancement into the clinic, the molecule demonstrated inhibition of RIPK2

Figure 23. Development of RIPK1 clinical candidate GSK2982772 (compound 57) from DNA-encoded library hit 56 showing significant shift in kinase activity across species.153

in psoriasis (phase II NCT02776033), UC (NCT02903966), and RA (NCT02858492).153 The discovery of 57 was recently disclosed and presents a very interesting story of an atypical kinase inhibitor.153 The authors discussed in an earlier report the discovery of the highly novel and kinase selective benzoxazepinone pharmacophore 56 (Figure 23) using DNAencoded libraries.154 Of particular interest with both 56 and 57 is the excellent selectivity among the kinome along with unique species selectivity for primate versus non-primate RIPK1.153 R



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Figure 24. Examples of recently reported inhibitors of RIPK1, RIPK2, and RIPK3 showing various chemotypes employed.

lism, motility, and further amplification of signals in response to extracellular signals (Figure 25).166 These have been extensively

activity would modulate intestinal inflammation in both murine and human systems, supporting RIPK2 as an attractive therapeutic target for the treatment of IBD, and RIPK2 inhibitor GSK2983559 has just recently entered clinical evaluation (NCT03358407).158 Like quinoline 61, the pyridine of 62 forms a single hinge interaction with Met98 and there is a hydrogen bond interaction between the sulfone group and the side chain of Ser25 residue.159 In addition, compound 62 showed moderate but significant reduction in IL6 and TNF-α in PD studies designed to assess RIPK2 as a target for IBD treatment. Computational docking of compound 63 with a murine RIPK2 homology model hypothesizes interactions with hinge residues Glu96 and Met98 via pyrimidine N3 and the amine.160 The authors also provide evidence that in addition to blocking cytokine transcription and production, inhibition of RIPK2 is beneficial in a murine model for multiple sclerosis. Additional examples selected from the recent patent literature highlight the significant interest still in the RIP family. The discovery stories have yet to be shared and include pyridine 64, isoxazolidinone 65 and 1,5-dihydro-4H-pyrrolo[3,2-c]pyridin-4one 66, the lone RIPK3 inhibitor.161−163 There was initial hesitation in targeting RIPK1 as its expression is important to T cell survival and germline mice deficient in RIPK1 die within 3 days of birth due to multiorgan cell injury and inflammation.148,164 However, knock-in mice expressing kinase-inactive RIPK1 are viable and do not exhibit increased cell death and inflammation.164 Kaiser and others showed RIPK3-selective inhibition led to concentrationdependent induction of apoptosis, and there was growing concern that kinase intervention may lead to RHIM-driven apoptosis.165 How inhibition of mediators in the signaling between necroptosis and inflammatory gene expression affect both efficacy and safety will be extremely interesting to learn.

Figure 25. Core components and cross-talk between PI3K and MAPK signaling pathways.

reviewed and are being actively pursued in a variety of therapeutic areas, in particular oncology along with cardiovascular, inflammatory, and autoimmune diseases.13,167−169 The two pathways are intertwined, as shown in Figure 25, with both having mechanisms for activation and inhibition of the other.166 The MAPK signaling cascade will be highlighted first followed by the one PI3K family member of interest for immunological disease intervention, PI3Kδ. 7.A. MAPK Family. The keen focus on the MAPK pathway is due to its far reaching roles within immune cell signaling. Following immune cell stimulation and signaling cascade initiation, members of the MAPK kinase kinases (MAP3Ks) are activated and phosphorylate MAPK kinases (MAP2Ks). MAP2Ks, MAPK/ERK kinases (MEKs), and mitogen-activated

7. BROADLY EXPRESSED KINASE TARGETS: MAPK/PI3K FAMILIES The previous kinases all focus on key cell members of the immune response, be it innate or adaptive. However, some kinase families are widely distributed and have been pursued for intervention in immune response as well as in other disease settings such as cancer. Two prime examples of this are the MAPK family and the PI3K family. Several of the kinases mentioned above signal through highly conserved MAPK and PI3K-mediated cascades, the cell’s chief mechanisms for regulating cell survival, proliferation, differentiation, metaboS



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transcription factors, cytoskeletal proteins, and other signaling proteins.170 Inhibitors of several MAPK kinases have shown efficacy in animal models such as collagen-induced arthritis models, acute inflammatory response in rat and cynomolgus monkeys, and robust anti-inflammatory activity in chronic disease models.170−172 However, of the multiple MAPK small molecule inhibitors discussed in a review by Arthur and Ley,170 none are currently being evaluated in the clinic for immunological disorders. In addition, of the well over 100 studies currently enrolling or active in the clinic with MAPK family member inhibitors, only one is for immune modulation: inhaled AZD7624 in corticosteroid resistant asthmatics (phase II, NCT02753764). This is after showing positive responses in humans on attenuation of lung and systemic inflammation upon LPS challenge.173 The limited positive outcome for at least p38 MAPK inhibitors has been attributed to modest clinical efficacy and transient suppression of inflammation biomarkers observed in multiple studies.14,171 Hammaker et al. laid out a number of potential reasons behind this modest efficacy including isoform selection (p38α versus β), reliance on poorly translating animal models, and redundant signaling networks allowing for compensations.14 This may suggest that a more meaningful, sustained suppression of chronic inflammation is required. One potential mechanism for this is a new reported type I1/2 inhibitor of p38 kinase that significantly extends the target residence time compared to classical type 1 inhibitors.174 An example is shown in Figure 21, panels A and B, where skepinone-L analog 67 has a target half-life of >15 h.174 Other examples in that report extend this to over 42 h.174 Compound 67 binds to the hinge via residues Met109 and Gly110 while also forming a hydrogen bond with Asp168 and an edge-to-face interaction with Phe169 of the DFG-motif (Figure 27A).

protein kinase kinase (MKKs) then phosphorylate the MAPK subfamilies, ERK, p38, and Jun N-terminal kinase (JNK), leading to their activation (Figure 26).170 Different family

Figure 26. Signaling cascade for MAPKs including ERKs, JNKs, and p38. Activation of MAP3Ks following receptor binding or stress induction leads to phosphorylation and activation of MAP2Ks such as MEKs and MKKs which in turn phosphorylate the MAPK family members. Activated MAPKs then translocate to the nucleus, where they phosphorylate transcription factors and modulate gene expression.

members have distinct roles in translating the effects of stimulation into the correct physiological responses via the phosphorylation of a range of downstream substrates, including

Figure 27. Different DFG orientations of p38α with DFG-out molecule PF-03715455 (67) and type 11/2 skepinone-L analog 68: (A) X-ray cocrystal structure of 67 (5MTY); (B) chemical structures of 67 and 68 with reported binding kinetics;174,175 (C) X-ray cocrystal structure of 68 (2YIS); (D) overlay of p38α backbones showing different positions of D168 and F169 upon 67 and 68 binding. T



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Pfizer has also reported a type II or DFG-out inhaled p38α inhibitor, PF-03715455 (68, Figure 27). In comparing binding kinetics of the two, the type II inhibitor showed even greater improvement in residence time, 80 h, as is expected for the DFG-out binding mode.175 Figure 27 compares the conformation of the DFG residues with type I1/2 67 (panel A), type II inhibitor 68 (panel C), and an overlay of the backbone of both showing the different positions of the DFG (panel D). While the increased residence time could lead to more meaningful suppression, development of 68 was halted in COPD and asthma due to business reasons, not safety and/or efficacy concerns (NCT02366637). 7.B. Phosphoinositide 3-Kinase (PI3K). Another critically important kinase within the immune system is phosphoinositide 3-kinase δ (PI3Kδ). PI3Kδ is a member of the class I PI3K family of lipid signaling kinases that phosphorylate phosphoinositides and is activated by receptor tyrosine kinases.167,168 While other PI3K members are ubiquitously expressed, PI3Kδ is predominantly found in leukocytes. There, it functions downstream of a number of receptors important to immune cell function including TCRs and FcRs as well as chemokine receptors in B cells and neutrophils. The heterodimeric kinase complex forming PI3Kδ, namely, the p85α regulatory subunit and the p110δ catalytic subunit, is acutely activated in B cells and T cells after exposure to antigens. This complex controls many aspects of lymphocyte development and differentiation, in part via the AKT/protein kinase B, forkhead box O1, and mechanistic target of rapamycin pathways.176 As such, PI3Kδ acts as a key signal transduction node in cells of the immune system where it is involved in cell survival, differentiation, activation, cellular senescence, and corticosteroid resistance. PI3Kδ had been a promising target in the pharmaceutical industry for over a decade. Driving this tremendous interest in PI3Kδ as a potential target for therapeutic intervention are the outcomes of its activation, deletion, and upregulation. Both PI3Kδ knockout and kinase dead mice show reduction in progenitor B cells. Activation and upregulation of PI3Kδ have been found in the lungs of COPD patients, in asthma animal models, and in the newly described activated PI3Kδ syndrome (APDS or PASLI disease). In APDS, hyperactivation of PI3Kδ signaling leads to T cell senescence and/or death among other symptoms.167,168,170,176,177 On the basis of these and other studies, several classes of PI3Kδ-isoform selective inhibitors have been described in the literature.167 Examples are shown in Figure 28 including idelalisib (69) which is approved for chronic lymphocytic leukemia, 178 clinical candidates GSK2269557 (70),179 and seletalisib (UCB5857, 71)180 along with recently disclosed preclinical molecule pyrrolotriazine 72, which was found efficacious in a mouse arthritis model.181 Critical interactions with the ATP binding site include hydrogen bonding with the backbone amine of hinge residue Val and the carbonyl of hinge residue Glu for those with bidentate interactions (Figure 28: 69, 70, and 72). In addition, indole 70 interacts with the Asp residue of the DFG loop.179 Previously disclosed series have also been identified that interact with a Lys in the affinity pocket.167 Despite the tremendous effort placed on finding inhibitors, targeting PI3Kδ has not been without its challenges. Nonselective PI3K inhibitors have shown dose-limiting adverse events, some of which were hypothesized to be related to other family members.182 This led to the development of isoenzymeselective inhibitors.

Figure 28. PI3Kδ-selective inhibitors recently described in the literature along with their important interactions where disclosed. Fold isoform selectivity is shown in parentheses. Participants in hinge residue binding are shown in red at the top of the molecule. Additional specific interactions are also shown.

Oral PI3Kδ inhibitors developed for the treatment of B cell leukemia include 69, which exhibits >100× selectivity over other PI3K family isoforms. However, patients treated still suffer from considerable adverse effects. While 69 is efficacious in the lymphoma setting for which it is approved, unexpected autoimmune and infectious toxicities upon treatment demonstrate the need for careful development and monitoring, both alone and in combination.183 Clinical studies have shown high rates of autoimmune toxicities and increased rates of infection including opportunistic infections leading idelalisib to now carry a black-box warning. Phase I clinical evaluation of 71 in psoriasis patients and healthy volunteers (NCT02303509, NCT02207595) and the molecule was progressed to phase II in patients suffering from primary Sjö gren’s syndrome (NCT02610543). However, this trial was terminated early this year due to enrollment challenges (www.clinicaltrials.gov). Noted safety findings were reported in the recent disclosure of 72.181 Despite displaying >50% suppression of paw swelling in the mouse CIA efficacy model 2 and 5 mg/kg, QTc prolongation and hemodynamic effects were seen at doses down to 10 mg/kg to 20 mg/kg leaving a narrow safety margin for a chronic indication.181 Many of the autoimmune toxicities encountered are likely mediated through the on-target effects of inhibiting PI3Kδ in different lymphocyte subsets.167,168,176 These include Tregulatory lymphocytes which are critical in the maintenance of immune self-tolerance and downregulation and also appear to be instrumental in the mechanism of PI3K-associated autoimmune toxicities. A potential method for circumventing systemic exposure is being explored by indazole 70 (Figure 28) which is being dosed as an inhaled inhibitor.176 Evaluation in asthma patients has been completed (NCT01462617), though results have not yet been disclosed. These and results from current clinical trials for APDS (NCT02593539) and COPD U



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ORCID

(NCT03345407, NCT02522299) will be informative around whether a PI3Kδ inhibitor can be successful in a chronic inflammation and immune setting.

Marian C. Bryan: 0000-0002-3138-6888 Notes

The authors declare the following competing financial interest(s): Authors are Genentech employees and have stock/shares in Genentech, Inc.

8. FUTURE DIRECTIONS Despite a lack of progress in terms of numbers of clinical approvals for small molecule kinase inhibitors for inflammatory and autoimmune diseases, there is much promise for continued pursuit in the field. First, only a relatively small subset of the human kinome has been studied with most of the focus being on tyrosine kinases.12 With the rapid advancement of our understanding of inflammatory pathways (guided by advanced genetics and CRIPR technologies), additional targets for therapeutic intervention are expected. There has also been a dramatic increase in the ability to achieve high levels of selectivity, decreasing the likelihood of off-target effect and broadening the window for improved safety. With these improvements, there has been an expansion in novel strategies for targeting these kinases. These include inducing pockets in the ATP-binding site (BTK), selecting for type I1/2, type II, and type III inhibitors (RIP, p38, NIK), targeting pseudokinase domains (JAK), and targeting kinase-unique cysteine residues (JAK, BTK). When systemic exposure may not be tolerated for even single-kinase selective inhibitors due to on-target toxicities or when localized exposure is critical, alternative modalities may provide relief. Indeed, these are being explored for several of the kinases mentioned including JAK family inhibitors and PI3Kδ inhibitors where both inhaled and topical applications are being pursued. There is also a growing appreciation for the redundancy and cross-talk involved in the immune system, and inhibiting more than one kinase may be needed to increase efficacy through additive or synergistic effects.2 Toward this, specific combination therapies may prove highly beneficial due to the synergistic impact on multiple pathways and may allow for lower doses of different molecules giving better tolerability profiles and may avoid or delay the development of resistance or induction of broad immunosuppression.16 In cases where the level of inhibition for optimal impact is unknown, expected to be insufficient, or may come with tolerability challenges, either inhibiting multiple kinases with a single small molecule (e.g., dual ITK/RLK inhibitor 44, dual SYK/JAK inhibitor 26) or inhibiting multiple nodes via combination therapies (e.g., BTK/ JAK inhibitors with SYK inhibitor GS-9876) may be more suitable.116 In addition, they may prevent development of resistance as mentioned for JAK inhibitors. Certain diseases may be better suited for multikinase inhibitors including severe airway disease where treatment with inhaled, so-called narrow spectrum kinase inhibitors is more effective than p38α inhibitors alone at suppressing the release of inflammatory cytokines from macrophages in vitro and in rodent models.2 Finally, other targets, including family members of those described above like IRAK1,6,119 other RIP family members, and other MAP2Ks like the mixed lineage kinases (MLKs),184 are actively being pursued and may hold significant benefit. Despite the challenges that targeting kinases for inflammatory and autoimmune diseases have faced, there remains a great deal of optimism for the strategy and for those small molecule inhibitors under clinical evaluation.

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Biographies Marian C. Bryan studied chemistry and biochemistry at Clemson University prior to pursuing her Ph.D. in Chemistry under the direction of Professor Chi-Huey Wong at The Scripps Research Institute. Following an American Cancer Society postdoctoral fellowship with Professor Linda Hsieh-Wilson at The California Institute of Technology, she joined the Department of Medicinal Chemistry at Amgen, Inc., in 2006. In 2012, she joined Genentech, Inc., where she is a team leader and Senior Scientist in Discovery Small Molecule Research. Naomi S. Rajapaksa received her Bachelor’s degree in Chemistry from Stanford University. She obtained her Ph.D. from Harvard University where she studied asymmetric catalysis and total synthesis under the direction of Professor Eric N. Jacobsen. She then joined the infectious diseases medicinal chemistry group at Novartis. In 2016, she joined Genentech, Inc., where she is a scientist in the Small Molecule Discovery Chemistry department.

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ACKNOWLEDGMENTS The authors thank Dr. Brent McKenzie, Dr. Hans Brightbill, Swathi Sujatha-Bhaskar, and Dr. Tim Heffron for helpful discussions and feedback and Dr. James Kiefer for structural graphics support.

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ABBREVIATIONS USED APDS, activated PI3Kδ syndrome; BAFF, B cell activating factor; BCR, B cell receptor; BMX, bone marrow kinase; BTK, Bruton’s tyrosine kinase; CIA, collagen-induced arthritis; cIAP, cellular inhibitors of apoptosis; CLE, cutaneous lupus erythematosus; DAG, diacyl glycerol; DMARD, diseasemodifying antirheumatic drug treatment; FcR, Fc receptor; IFNαR, interferon-α receptor; IgE, immunoglobulin E; IKK, IκB kinase; IL1R, IL-1 receptor; IP3, inositol triphosphate; IRAK, interleukin-1 receptor activated kinase; ITAM, immunoreceptor tyrosine-based activation motif; ITK, interleukin-2 inducible T cell kinase; JAK, Janus kinase; JNK, Jun N-terminal kinase; mCAIA, mannan-induced collagen antibody induced arthritis; LAT, linker of activated T-cells; LPS, lipopolysaccharide; MAP2K, MAPK kinase; MAP3K, MAPK kinase kinase; MEK, MAPK/ERK kinase; MKK, mitogen-activated protein kinase kinase; MLKL, mixed lineage kinase domain-like protein; NFAT, nuclear factor of activated T cells; NIK, NFκB inducing kinase; NK, natural killer; PA, psoriatic arthritis; PAMP, pathogen-associated molecular pattern; pDC, plasmacytoid dendritic cell; PIP2, phosphoinositol diphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; PLCγ2, phospholipase C γ 2; PPR, pattern recognition receptor; p-STAT, phosphorylated STAT; RA, rheumatoid arthritis; RANK, receptor activator of NF-κB; RHIM, RIP homotypic interaction motif; RIPK, receptor-interacting protein kinase; RLK, resting lymphocyte kinase; SLE, systemic lupus erythematosus; STAT, signal transducers and activators of transcription; SYK, spleen tyrosine kinase; TAK1, transforming growth factor β-activated kinase 1; TCR, T cell receptor; TEC, tyrosine kinase expressed in hepatocellular carcinoma; Th, T helper; TIR, Toll/IL-1R;

AUTHOR INFORMATION

Corresponding Author

*Phone: +1 (650) 225-6532. E-mail: [email protected]. V



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(22) Wiegman, C.; Adcock, I.; Rothaul, A.; Main, M.; Morgan, F. Invitro evaluation of a new potent, selective pan-Janus kinase (JAK) inhibitor VR588. Eur. Respir. J. 2015, 46, PA2130. (23) Flanagan, M. E.; Blumenkopf, T. A.; Brissette, W. H.; Brown, M. F.; Casavant, J. M.; Shang-Poa, C.; Doty, J. L.; Elliott, E. A.; Fisher, M. B.; Hines, M.; Kent, C.; Kudlacz, E. M.; Lillie, B. M.; Magnuson, K. S.; McCurdy, S. P.; Munchhof, M. J.; Perry, B. D.; Sawyer, P. S.; Strelevitz, T. J.; Subramanyam, C.; Sun, J.; Whipple, D. A.; Changelian, P. S. Discovery of CP-690,550: a potent and selective Janus kinase (JAK) inhibitor for the treatment of autoimmune diseases and organ transplant rejection. J. Med. Chem. 2010, 53, 8468−8484. (24) Wollenhaupt, J.; Silverfield, J.; Lee, E. B.; Curtis, J. R.; Wood, S. P.; Soma, K.; Nduaka, C. I.; Benda, B.; Gruben, D.; Nakamura, H.; Komuro, Y.; Zwillich, S. H.; Wang, L.; Riese, R. J. Safety and efficacy of tofacitinib, an oral Janus kinase inhibitor, for the treatment of rheumatoid arthritis in open-label, longterm extension studies. J. Rheumatol. 2014, 41, 837−852. (25) Zerbini, C. A. F.; Lomonte, A. B. V. Tofacitinib for the treatment of rheumatoid arthritis. Expert Rev. Clin. Immunol. 2012, 8, 319−331. (26) Ito, M.; Yamazaki, S.; Yamagami, K.; Kuno, M.; Morita, Y.; Okuma, K.; Nakamura, K.; Chida, N.; Inami, M.; Inoue, T.; Shirakami, S.; Higashi, Y. A novel JAK inhibitor, peficitinib, demonstrates potent efficacy in a rat adjuvant-induced arthritis model. J. Pharmacol. Sci. 2017, 133, 25−33. (27) Wernig, G.; Kharas, M. G.; Okabe, R.; Moore, S. A.; Leeman, D. S.; Cullen, D. E.; Gozo, M.; McDowell, E. P.; Levine, R. L.; Doukas, J.; Mak, C. C.; Noronha, G.; Martin, M.; Ko, Y. D.; Lee, B. H.; Soll, R. M.; Tefferi, A.; Hood, J. D.; Gilliland, D. G. Efficacy of TG101348, a selective JAK2 inhibitor, in treatment of a murine model of JAK2V617F-induced polycythemia vera. Cancer Cell 2008, 13, 311− 320. (28) Wan, H.; Schroeder, G. M.; Hart, A. C.; Inghrim, J.; Grebinski, J.; Tokarski, J. S.; Lorenzi, M. V.; You, D.; McDevitt, T.; Penhallow, B.; Vuppugalla, R.; Zhang, Y.; Gu, X.; Iyer, R.; Lombardo, L. J.; Trainor, G. L.; Ruepp, S.; Lippy, J.; Blat, Y.; Sack, J. S.; Khan, J. A.; Stefanski, K.; Sleczka, B.; Mathur, A.; Sun, J.-H.; Wong, M. K.; Wu, D.-R.; Li, P.; Gupta, A.; Arunachalam, P. N.; Pragalathan, B.; Narayanan, S.; K.C., N.; Kuppusamy, P.; Purandare, A. V. Discovery of a highly selective JAK2 inhibitor, BMS-911543, for the treatment of myeloproliferative neoplasms. ACS Med. Chem. Lett. 2015, 6, 850−855. (29) Vazquez, M. L.; Kaila, N.; Strohbach, J. W.; Trzupek, J. D.; Brown, M. F.; Flanagan, M. E.; Mitton-Fry, M. J.; Johnson, T. A.; TenBrink, R. E.; Arnold, E. P.; Basak, A.; Heasley, S. E.; Kwon, S.; Langille, J.; Parikh, M. D.; Griffin, S. H.; Casavant, J. M.; Duclos, B. A.; Fenwick, A. E.; Harris, T. M.; Han, S.; Caspers, N.; Dowty, M. E.; Yang, X.; Banker, M. E.; Hegen, M.; Symanowicz, P. T.; Li, L.; Wang, L.; Lin, T. H.; Jussif, J.; Clark, J. D.; Telliez, J.-B.; Robinson, R. P.; Unwalla, R. Identification of N-{cis-3-[methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF04965842): a selective JAK1 clinical candidate for the treatment of autoimmune diseases. J. Med. Chem. 2018, 61, 1130−1152. (30) Voss, J. W.; Camp, H. S.; Padley, R. J. JAK1 Selective Inhibitors and Uses Thereof. WO2015061665, April 30, 2015. (31) Zak, M.; Hurley, C. A.; Ward, S. I.; Bergeron, P.; Barrett, K.; Balazs, M.; Blair, W. S.; Bull, R.; Chakravarty, P.; Chang, C.; Crackett, P.; Deshmukh, G.; DeVoss, J.; Dragovich, P. S.; Eigenbrot, C.; Ellwood, C.; Gaines, S.; Ghilardi, N.; Gibbons, P.; Gradl, S.; Gribling, P.; Hamman, C.; Harstad, E.; Hewitt, P.; Johnson, A.; Johnson, T.; Kenny, J. R.; Koehler, M. F. T.; Bir Kohli, P.; Labadie, S.; Lee, W. P.; Liao, J.; Liimatta, M.; Mendonca, R.; Narukulla, R.; Pulk, R.; Reeve, A.; Savage, S.; Shia, S.; Steffek, M.; Ubhayakar, S.; van Abbema, A.; Aliagas, I.; Avitabile-Woo, B.; Xiao, Y.; Yang, J.; Kulagowski, J. J. Identification of C-2 hydroxyethyl imidazopyrrolopyridines as potent JAK1 inhibitors with favorable physicochemical properties and high selectivity over JAK2. J. Med. Chem. 2013, 56, 4764−4785. (32) Liang, J.; Van Abbema, A.; Balazs, M.; Barrett, K.; Berezhkovsky, L.; Blair, W. S.; Chang, C.; Delarosa, D.; DeVoss, J.; Driscoll, J.; Eigenbrot, C.; Goodacre, S.; Ghilardi, N.; MacLeod, C.; Johnson, A.;

TNF-R, TNF receptor; TRAF, TNF-receptor associated factor; TYK2, tyrosine kinase 2

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