Protein Kinase Antagonists in Therapy of Immunological and Inflammatory Diseases

CHAPTER88  ProteinKinaseAntagonistsinTherapyofImmunologicalandInflammatoryDiseases

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Protein Kinase Antagonists in Therapy of Immunological and Inflammatory Diseases Arian Laurence, Massimo Gadina, John J. O’Shea

Reversible phosphorylation is one of the major mechanisms controlling protein activity in all eukaryotic cells and, as such, is involved in all fundamental cellular processes, including cell cycle and cell growth, cell shape and movement, metabolism, differentiation, and apoptosis. This covalent modification is a major means for transmitting information from outside the cell and between the subcellular components within the cell. Phosphorylation is a major mechanism underlying normal signaling, as exemplified by insulin and other growth factors, but in addition, the importance of protein phosphorylation is supported by evidence that mutations and dysregulation of protein kinases play causal roles in human disease. This is especially true in cancer, in which mutant protein kinases or their upstream activators function as oncogenes. From the point of view of an immunologist, protein phosphorylation is also the major mechanism by which immune receptors, including the T-cell receptor (TCR), B-cell receptor, and natural killer (NK) cell receptor and Fc receptors, trigger signaling. As discussed in Chapter 4, the first step in signaling by multichain immune recognition receptors (e.g., the aforementioned receptors) is tyrosine phosphorylation of the receptor itself and adapter molecules, such as linker of activated T cells (LAT), mediated by Src family protein tyrosine kinases (PTKs). This leads to the recruitment of PTK members spleen tyrosine kinase (Syk) and Zap70 followed by phosphorylation of adapters, such as SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and the activation of Tec family PTKs. These initial steps trigger the activation of serine-threonine kinases, including the mitogen-activated protein kinases (MAPKs) and protein kinase C (PKC) family (Fig. 88.1). We now know a great deal of details about how the cascade of protein phosphorylation links events at the plasma membrane to calcium modulation, cytoskeletal rearrangement, gene transcription, and other canonical features of lymphocyte action (Chapter 12). Similarly, a critical first step in signaling by many cytokine receptors is the activation of phosphorylation. The receptors for classical growth factor cytokines, including stem cell factor and platelet-derived growth factor (PDGF) are receptor tyrosine kinases (RTKs), whereas the receptors for transforming growth factor family cytokines are receptor serine–threonine kinases. Type I and II cytokine receptors signal via the activation of receptor-associated Janus kinases (JAKs; see below, Table 88.1 and Table 88.2) (Chapter 9). Other cytokines like interleukin (IL)-1 and tumor necrosis factor (TNF) initiate signaling in a

kinase-independent manner but, nonetheless, signal through kinase cascades to exert their effects. It is clear that many aspects of protein phosphorylation are of major importance in immune and inflammatory mechanisms. The nonredundant functions of various kinases in different immune cells are exemplified by both studies in knock-out mice and humans with mutations. On the basis of these findings, targeting protein kinases has been proposed to be a useful strategy in the development of novel immunosuppressant drugs and is one of the most active areas of pharmaceutical drug development (Table 88.3), with much of the impetus coming from oncology. The field is so vast that it is impractical to comprehensively review all this information in one chapter; therefore we will focus both on important historical precedents in the field and then discuss drugs and targets that are most immunologically relevant. We will start by briefly reviewing some of the basics of kinase biochemistry.

STRUCTURE AND FUNCTION OF PROTEIN KINASES KEY CONCEPTS Kinase Families • 518 kinases in the genome • 90 protein tyrosine kinases (e.g., Janus kinases [JAKs]) • 400 protein serine/ threonine kinases: • AGC kinase family (e.g., protein kinase B/AKT) • CAMK kinase family (e.g., calmodulin-dependent kinase) • CMGC kinase family (e.g., MAP kinases: ERK, JNK, p38) • STE kinase family (e.g., MAPK kinases, MAPKK kinases) • TKL kinase family (e.g., IRAK) • Others: casein kinase family, GYC kinase family, IKK family

Protein kinases or phosphotransferases catalyze the transfer of the γ-phosphate from a purine nucleotide triphosphate (i.e., adenosine triphosphate [ATP] and guanosine triphosphate [GTP]) to the hydroxyl groups of their protein substrates. They generate phosphate monoesters by using protein alcohol groups (on serine and threonine residues) and/or protein phenolic groups (on tyrosine residues) as phosphate acceptors. Thus protein kinases can be classified by the amino acid substrate preference: serine/ threonine kinases, tyrosine kinases, and dual kinases (i.e., both serine/threonine and tyrosine residues can be phosphorylated).

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Antigen-presenting cell CD80 MHC TCR

CD28

P P

SLP-76

P P

GADS P PLCγ1 P P

RasGAP

GTP

LAT

fyn

Grb2

Ras

PIP3

GTP

PDK1

Sos RasGAP

p110

p85

P P13’K

P DaG

ZAP-70

P P

Ras

ITK

PIP3

CD3 Ick

P

P’tn

IP3 PKC

T cell

Raf

PKB

Downstream serine/threonine kinases and their effectors

FIG 88.1  The Proximal Signaling Events in Response to T-Cell Receptor (TCR) Activation by an Antigen-Presenting Cell. Tyrosine and lipid kinases are indicated in red, and serine/ threonine kinases are indicated in blue.

TABLE 88.1  Functions and Signaling of Cytokines Sharing the Common γ Chain Cytokine Receptor Functions

IL-2R

IL-4R

IL-7R

IL-9R

IL-15R

IL-21R

Control of peripheral self-tolerance (mice)

Regulation of B-cell function (in concert with interleukin [IL]-21 and IL-25) Immunoglobulin class switching

Thymocyte survival and development factor

Goblet cell hyperplasia

CD8 memory T-cell survival and proliferation factor

T- and B-cell proliferation factor

Peripheral T-cell survival factor Mediates homeostatic reconstitution of lymphopenic animals B-cell progenitor survival factor (mice)

Mucus production

Peripheral T cell survival factor Mediates homeostatic reconstitution of lymphopenic animals Natural killer (NK) cell development, differentiation and survival factor

Regulates immunoglobulin production (with IL-4)

PI3K, STAT(1), (3), 5

PI3K, Ras MAPK, STAT1,3,(5)

PI3K, Ras MAPK, STAT1,3,5

PI3K, Ras MAPK, STAT1,3,(5)

Development and maintenance of regulatory T cells

Downstream signaling pathways

Differentiation of helper and cytotoxic T cells

Differentiation of T helper cells (Th2 lineage)

In vitro expansion and differentiation of antigen-selected T and NK cells

Costimulant for growth in T, B, and mast cells

PI3K, Ras MAPK, STAT1,3,5

Inhibition of Th1 differentiation and macrophage activation PI3K, Ras MAPK, STAT6,5

NK cell proliferation and activation factor

CHAPTER 88  Protein Kinase Antagonists TABLE 88.2  The JAK Family of

Tyrosine Kinases

Gene Jak1 Jak2 Jak3 Tyk2

Murine Phenotype Associated With Gene Deletion Perinatal lethality, block in thymocyte development Embryonic lethality due to anemia T-, B-, and NK-cell lymphopenia, SCID phenotype Failure to clear Toxoplasma, reduced arthritis

Associated Receptor Many including IL-7R and IFNRs Erythropoietin receptor Common gamma chain receptor IL-12/23 receptor

JAK, Janus kinase; IL, interleukin; IFNR, interferon receptor; NK, natural killer; SCID, severe combined immune deficiency.

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Almost all protein kinases have catalytic domains that belong to a single eukaryotic protein kinase (ePK) superfamily. The common evolutionary ancestry of the kinase domain (also known as the catalytic domain), which consists of 250–300 amino acid residues, manifests as a highly conserved three-dimensional structure. In the human genome, there are 518 kinases, which are divided into eight major groups, in totality, representing 1.7% of the human genome. The PTK family has 90 members, one-third of which are RTKs, and the remainder are cytoplasmic proteins that typically function in close proximity to, and downstream of, receptor–ligand complexes. In terms of its catalytic role, the kinase domain has three functions: the binding of the ATP (or GTP) phosphate donor as a complex with a divalent cation (usually Mg2+ or Mn2+), the

TABLE 88.3  Selected Kinase Inhibitors and Related Drugs Mechanism

Compound

Kinase Inhibited

Comments

Direct binding to the kinase

Imatinib

Kit, platelet-derived growth factor (PDGFR), and Abl

Approved for treatment of chronic myeloid leukemia (CML), eosinophilic leukemia, and gastric stromal tumors Used in the treatment of chronic sclerodermatous graft-versus-host disease (GvHD) Under evaluation in multiple types of cancer and in combination with other cancer drugs US Food and Drug Administration (FDA) approved for the treatment of CML and PH + acute lymphoblastic leukemia (ALL) Phase II trial for acute myeloid leukemia (AML) and a number of solid cancers Approved for Philadelphia chromosome-positive CML; in Phase II trials for AML and ALL FDA approved for the treatment of CML Approved for treatment of adult CML and Philadelphia chromosome positive ALL Phase III trial for prostate cancer Phase II trial for CLL, Sarcomas Approved for the treatment of imatinib-resistant CML Phase II trial for glioblastoma FDA approved for the treatment of CLL, mantle-cell lymphoma (MCL), lymphoplasmacytic lymphoma (LPL) Phase III trial for the treatment of diffuse large B-cell lymphoma (DLBCL), posttransplantation lymphoproliferative disease (PTLD), pancreatic cancer Phase II trial for GvHD Phase II trial for RA, Phase I trial for CLL, non-Hodgkin lymphoma (NHL) Phase III trial for CLL, phase II trial for rheumatoid arthritis (RA) FDA approved for the treatment of mantle cell lymphoma (MCL) Approved for treatment of breast cancer Minor survival improvement in head and neck cancer Phase III trials for brain metastasis FDA approved for treatment of non–small cell lung carcinoma (NSCLC) Phase III trial for breast cancer and head and neck cancer Phase III trial for treatment of breast cancer Phase II trial for NSCLC Approved for use in NSCLC as a first-line therapy by FDA Successful phase III study in esophageal cancer Approved for treatment of pancreatic cancer and certain categories of NSCLC In phase II and III trials for various other cancers, including myeloproliferative disease Approved in 2011 for certain types of progressive medullary thyroid cancer Phase II trial for small cell lung cancer Phase I trial for solid tumors FDA approved for thyroid cancer and under phase III trials for hepatocellular cancer FDA approved for the treatment of Colorectal cancer and gastrointestinal stromal tumor (GIST) Phase III trials for hepatocellular cancer Orphan drug status for the treatment of hepatocellular cancer Approved for the treatment of idiopathic pulmonary fibrosis and NSLCLC adenocarcinoma In phase III trials for the treatment of ovarian and colorectal carcinoma

Ponatinib

Nilotinib Radotinib Dasatinib

PDGFR and Abl Multiple Src family tyrosine kinases, including Abl

Bosutinib Ibrutinib

Btk, Itk

Spebrutinib Acalabrutinib Lapatinib

ErbB-2 and epidermal growth factor receptor (EGFR) kinases

Afatinib Neratinib Gefitinib Erlotinib

HER1/EGFR, JAK2

Vandetanib Tivozanib Lenvatinib Regorafenib

VEGFR-2, EGFR, RET, and ErbB-1 tyrosine kinases Vascular endothelial growth factor receptor (VEGFR) kinases

Brivanib (N)intedanib

VEGFR-2, FGFR VEGFR, PDGFR, FGFR

Continued

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TABLE 88.3  Selected Kinase Inhibitors and Related Drugs—cont’d Mechanism

Compound

Kinase Inhibited

Comments

Cabozantinib

VEGFR, MET, RET

Axitinib

VEGFR1, 2 and 3, Abl (T315I), PDGFR-β and Kit

Sunitinib

Entrectinib

Inhibits multiple receptor tyrosine kinases (RTKs), including PDGFR3-alpha, PDGFR-β, VEGFR1, VEGFR2, VEGFR3, Kit, Flt3, CSF-1R, and RET Dual-specific inhibitor blocking both tyrosine and serine/threonine kinases, including RAF kinase, VEGFR-2, VEGFR-3, PDGFR-B, KIT, Flt3, and RET Inhibits multiple RTKs, including PDGFR3-α, PDGFR-β, VEGFR1 VEGFR2, VEGFR3, cKIT, Lck, c-FMS, FGFR-1, and FGFR-3 ALK, TrkA, TrkB, TrkC, ROS1

FDA approved for treatment of thyroid cancer Phase III registration for hepatocellular cancer Approved for treatment of renal cancer; has orphan drug status for follicular, medullary, and anaplastic thyroid carcinoma and metastatic or locally advanced papillary thyroid cancer Effective in imatinib resistant CML associated with the T315I mutation For treatment of gastrointestinal stromal tumor after disease progression on or intolerance of imatinib and for treatment of advanced renal cell carcinoma and pancreatic neuroendocrine tumors Completed phase II trials in NSCLC and breast cancer Potential benefit in Flt3 + AML

Crizotinib

ALK, c-Met

Filgotinib

JAK 1

ABT-494 Ruxolitinib

JAK1 and JAK 2

Sorafenib

Pazopanib

Baricitinib Gandotinib (LY2784544) Oclacitinib AC-410 Tofacitinib Peficitinib Decernotinib Momelotinib TG02/ SB-1317 Fedratinib AT-9283

Lestaurtinib

JAK3, JAK1, (JAK2)

JAK 3 JAK1, JAK2, IKKe, and TBK1 JAK2, Flt3, CDK1, -2, -7, -9 JAK2, Flt3, RET JAK2, JAK3, aurora A/B kinase, highly active against the Gleevecresistant T315l Abl mutation Flt3, TrkA, and JAK2

FDA approved for treatment of advanced renal cell carcinoma, thyroid cancer, and hepatocellular cancer In phase II trials for AML, ALL, CML, myelodysplastic syndrome (MDS), neurofibromatosis, portal hypertension

Approved for treatment of advanced renal cell cancer and soft tissue sarcoma Mixed results as a treatment for age related macular degeneration when used topically

Phase II trial for colorectal cancer, NSCLC Phase I trial for neuroblastoma Approved for treatment of ALK-positive NSCLC Phase II trial for anaplastic lymphoma kinase (ALK)–positive lymphoma Completed phase IIb studies in RA Completed phase II studies in Crohn disease In phase III trial for RA and phase II trial for Crohn disease FDA approved for myeloproliferative diseases In trials for treatment of GvHD Completed phase III trials for rheumatoid arthritis Completed phase IIb trials in psoriasis In phase II clinical trials for myeloproliferative disorders FDA approved for the treatment of canine allergic dermatitis In phase I clinical trials for autoimmune and myeloproliferative syndrome FDA approved for the treatment of RA Completed phase III trials for the treatment of ulcerative colitis (UC)and psoriasis Phase IIb for RA and UC Completed phase III studies in the treatment of RA In phase III trials for pancreatic adenocarcinoma In phase I clinical trials for ALL, AML, CLL In phase II trial for chronic beryllium disease Completed phase I trial for solid tumors in children

In phase III clinical trials for treatment of patients with AML who have an Flt3-activating mutation at first relapse from standard induction chemotherapy In phase II trials for psoriasis and pancreatic cancer

CHAPTER 88  Protein Kinase Antagonists

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TABLE 88.3  Selected Kinase Inhibitors and Related Drugs—cont’d Mechanism

Compound

Kinase Inhibited

Comments

Quizartinib

Class III receptor tyrosine kinases (Flt3, Kit, CSF1) Syk, Flt3

Completed phase II clinical trial for drug-resistant AML

Fostamatinib

PRT-062607/ BIIB057/ P505-15 R-348 Vemurafenib Dabrafenib Selumetinib

Syk

JAK/Syk BRAF

MKK family

Cobimetinib Trametinib Binimetinib PD-325901 CC 90003 XG102 RES-529 Uprosertib MK-2206 AZD-5363 Pexidartinib AZD 2014 CC 223 Sotrastaurin Ribociclib

ERK JNK AKT, MTOR, VEGFR AKT

CSF-1R, Kit MTORC1 and C2 PKC CDK4,6

Palbociclib

Indirect binding to kinase

Monoclonal antibodies binding to receptor tyrosine kinases Other mechanisms of action

Buparlisib Bardoxolone Fasudil KD-025

PI3K IKbK, COX2, NOSII, STAT3 ROCK1/ ROCK2 ROCK2

Sirolimus (Rapamycin) Everolimus

mTORC1

Temsirolimus Ridaforolimus Trastuzumab Cetuximab Bevacizumab

Barasertib

EGFR

VEGF—prevents binding to its receptor (Flt-1) Aurora kinase B (functions in the attachm ent of the mitotic spindle to the centromere)

Phase III for idiopathic thrombocytopenic purpura (ITP) Phase II for immunoglobulin A (IgA) nephropathy Failed phase III for RA, phase I for GvHD In phase I clinical trials for RA, CLL, NHL; preclinical for systemic lupus erythematosus (SLE) In phase II trials for ocular GvHD FDA approved for the treatment of BRAF mutated melanoma; phase II trial for colorectal cancer and myeloma FDA approved for the treatment of BRAF mutated melanoma Phase III trial for NSCLC and thyroid cancer Phase II for colorectal cancer and biliary cancer FDA approved for the treatment of melanoma Phase II trial for the treatment of breast cancer and Langerhans cell histiocytosis FDA approved for the treatment of BRAF mutated melanoma in combination with dabrafenib Phase III trial for the treatment of BRAF mutated melanoma Phase II trial for neurofibromatosis Phase I trial for solid tumors Phase III trial for ocular inflammation and sensorineural hearing loss Phase I trial for age-related macular degeneration Phase II trial for AML, solid tumors Phase II trial for solid and hematological malignancies Phase II trial for breast and gastric cancer Phase II trial for pigmented villonodular synovitis Phase II trial for DLBCL and solid tumors Phase II trial for NHL and solid tumors Phase II trial for DLBCL Phase III trial for breast cancer Phase II trial for gastrointestinal cancer FDA approved for the treatment of breast cancer Phase III trial for NSCLC Phase III trial for breast cancer Phase II trial for diabetic neuropathy and pulmonary hypertension Phase III for Raynaud phenomenon and scleroderma completed Phase II for psoriasis vulgaris Phase II for idiopathic pulmonary fibrosis FDA licensed for use in solid organ and bone marrow transplantation FDA licensed for use with cyclosporine in cardiac and renal transplantation FDA licensed for use in neuroendocrine tumors and renal cell carcinoma FDA licensed for use with MCL, renal cancer Phase II trial for coronary artery restenosis FDA licensed for use in HER2/neu positive breast carcinoma and metastatic gastric cancer FDA licensed for use in relapsed colorectal and head/neck cancers FDA licensed for use in metastatic breast and colorectal cancers, glioblastoma, and NSCLC (non-squamous cell histology) in combination with traditional chemotherapy Disrupts mitosis and cellular division in tumor cells; in phase III clinical trials for AML

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binding of the protein substrate, and the transfer of the γ-phosphate from ATP or GTP to the protein substrate. Despite the huge number of serine/threonine and tyrosine kinases, there is evidence of a common ancestor, and this is reflected in structural similarities, particularly in the active (ATP bound) confirmation. The major kinase domains of all typical protein kinases consists of two lobes (N-lobe and C-lobe) that surround the nucleotide binding site1 (Fig. 88.2). The smaller N-lobe consists of a cluster of β-pleated sheets with a single α helix. The larger C-lobe is made up of α helices. Within the C-lobe lies the substrate-binding site, typically a groove on the surface. A hinge region connects the two lobes. The hinge together with two loops emerging from each lobe form the ATP-binding pocket: the primary target for most kinase inhibitors. In many protein kinases, a loop emerging from the C-lobe must be phosphorylated for the kinase to be fully active (see Fig. 88.2). This is known as the activation loop. Substrates of PTKs often include the activation loop of downstream kinases, creating signaling cascades of proteins, which, in turn, phosphorylate each other; examples include the MAPKs (Fig. 88.3).

THE DISCOVERY OF KINASE INHIBITORS Given that protein kinases bind ATP, the notion that therapeutically useful kinase inhibitors could be generated was initially met with some skepticism. First, as there are more than 500 human kinases, many of which serve critical cellular functions, would it really be possible to attain the specificity needed? Second, protein kinases are not the only kinases—there are lipid kinases and nucleotide kinases, as well as many other ATP-binding

Phosphorylated active loop tyrosines

N-lobe ‘Gatekeeper’ active site residue Hinge region Inhibitor

C-lobe

FIG 88.2  Crystal Structure of the Janus Kinase 3 (JAK3) Domain Complexed With Staurosporine (pdb Accession Code 1YVJ). This structure captures the active conformation of JAK3 with both active loop tyrosine residues phosphorylated (green). The molecule can be described as halves, with the N-terminal lobe presented in blue and the C-terminal domain in red. These are linked by a hinge region that forms part of the active site. Highlighted in purple within the active site is the gatekeeper residue. Bound within this site is an analogue of the inhibitor staurosporine, and its proximity to the “gatekeeper” residue highlights why this residue and this region are critical for the specificity of inhibitors for individual protein kinases.

Activator

Ras-GTP

MAPKKK

c-Raf1

MEKK1

TAK1

MAPKK

MKK1

SEK1

MKK6

MAPK

ERK1

JNK1

p38 MAPK

TRAF6

Substrates

p90-RSK P P

P SRF SRF

EIK1

SRE

P

P

c-fos

p90-RSK

c-fos

P

P

c-Jun

AP-1

P

P

ATF2

P

P

ATF2 AP-1

FIG 88.3  A Summary of the Mitogen-Activated Protein Kinase (MAPK) Signal Transduction Pathways. Examples of receptors that activate Ras include the interleukin (IL)-2 and T-cell receptor (TCR). Examples of receptors that activate tumor necrosis factor receptor–associated factor 6 (TRAF6) include the IL-1 receptor.

CHAPTER 88  Protein Kinase Antagonists proteins, all of which share structural similarities with PTKs. Third, despite the many potential ways of designing a smallmolecule kinase inhibitor, in practice, the majority work by sitting within the ATP-binding pocket.2 A priori then, one might conclude that it would be impossible to generate an antagonist that did not target some other essential ATP-dependent process. Fortunately though, this skeptical view does not reflect reality.

THERAPEUTIC PRINCIPLES Protein Kinase Inhibitors As Drugs • Drugs can inhibit protein kinases with a high degree of specificity. • Multi-kinase inhibitors can be well tolerated and be more efficacious than single kinase inhibitors (e.g., second-generation Abl kinase inhibitors). • Conversely, unacceptable toxicity has limited the creation of clinical inhibitors to particular kinase members (e.g., p38 MAPK inhibitors).

Imatinib and Other First-Generation Protein Tyrosine Kinases Inhibitors The first protein kinase inhibitor approved by the US Food and Drug Administration (FDA) is imatinib (see Table 88.3). The mutated form of the Abl tyrosine kinase BCR-Abl represents a fusion protein that is the result of a chromosomal translocation (Philadelphia chromosome) observed in patients suffering from chronic myeloid leukemia (CML). The pathognomonic presence of BCR-Abl in CML has led to it becoming one of the most intensively studied PTKs. The fusion protein consists of an oligomerization domain, a PH domain, and a Dbl/cdc24 guanine nucleotide exchange factor homology domain that contains the N-terminal breakpoint cluster region (BCR) of the protein. The Abl half of the fusion protein contains a tyrosine kinase domain, a Src homology 2 (SH2) domain, and a DNA-binding domain, together with nuclear localization and nuclear export motifs. The Abl kinase is constitutively active within the fusion protein and has been implicated in initiating numerous signaling pathways that mediate cell survival and proliferation. In view of this, and the essential requirement for BCR-Abl kinase activity in CML, it was thought to be an ideal target despite the aforementioned caveats with targeting protein kinases. As predicted, imatinib has revolutionized the treatment of CML. This inhibitor has been remarkably successful in arresting the progression of the CML but is also well tolerated with acceptable side effects.3 Although conservation of the kinase ATP-binding pocket has posed a potential problem for designing kinase inhibitors, in practice, this has not happened for several reasons. Although different kinases may be structurally similar in an active ATPbound conformation, the inactive conformation is substantially less frequent and can be used to generate selective inhibitors.4 The ATP-binding region is made up of six polar amino acid residues that are invariant across whole families of kinases; similarly, there are numerous lipophilic residues that are highly conserved. In addition, this critical region contains an amino acid whose amide carbonyl binds to N-6 of adenine in the active conformation. The side chain of this amino acid sticks into the reaction pocket in the inactive state and, for this reason, is referred to as “the gatekeeper residue.”5 As the side chain is not involved in direct ATP binding, it varies across kinases, and variation of this gatekeeper residue is exploited by a number of inhibitors that are able to bind the inactive conformation of specific kinases. In the case of Abl kinase, the gatekeeper residue is threonine,

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which binds directly to a methyl group of the phenyl ring of the Abl kinase inhibitor imatinib.4 Across the collective kinase superfamily, almost any amino acid can appear as the gatekeeper, although in practice, it is typically a bulky nonpolar residue (methionine, tyrosine, phenylalanine, lysine).5 In principle, as more detailed structural information emerges from the many protein kinases, capitalizing on subtle differences in structure is expected to lead to the discovery of novel agents with improved potency and specificity. For instance, cyclin-dependent kinase 2 (CDK2) contains an additional pocket on its C-lobe next to the ATP-binding pocket.6 Several CDK2-specific inhibitors exploit this by binding to both pockets. Of further structural significance is the emergence of tumor drug resistance in response to the chronic use of protein kinase inhibitors. Mutant forms of BCR-Abl, Kit, and epidermal growth factor receptor (EGFR) have been associated with loss of drug activity and disease relapse. Interestingly, one of the most common sites of mutation is the otherwise conserved “gatekeeper residue.” The appearance of such mutations that cause resistance to imatinib have led to the need for other drugs with broader activity against a number of kinases. Thus in the setting of oncology, “multikinase” inhibitors, including dasatinib and sunitinib, have now entered clinical practice and have been approved by the FDA. Although a major problem in the treatment of malignancy, this is less likely to be an issue in the treatment of autoimmune disease; nonetheless, drugs used for oncological indications often end up being quite useful in the treatment of autoimmunity. Such precedents include cyclophosphamide, azathioprine, and methotrexate. Therefore it is not unreasonable to speculate that a number of the kinase inhibitors developed as anticancer agents may ultimately be used to treat inflammatory or immunological diseases. An unexpected finding for a clinically well-tolerated inhibitor, which was originally thought to be a highly specific inhibitor for Abl kinase, was the realization that imatinib has activity against several other PTKs.3 Consequently, it has been found to be useful in the treatment of a number of cancers that do not have abnormal Abl kinase activity. Imatinib has been used to treat gastrointestinal (GI) stromal tumor and hypereosinophilic syndrome through its effects on Kit and PDGFR-FIPIL1 kinases, respectively. In spite of efforts to develop highly specific kinase inhibitors, there is increasing evidence that a partial inhibition of multiple kinases is potentially less toxic than originally feared and may be important for the efficacy of many inhibitors. Second-generation Abl inhibitors, including dasatinib and bosutinib, are less selective than imatinib. This lack of specificity may contribute to their improved response rates in the treatment of CML.7 With respect to immune-mediated diseases, imatinib is used in the clinic for the treatment of fibrotic diseases, including skin fibrosis associated with chronic graft-versus-host disease (GvHD).8 At the time of writing, there are >20FDA-approved small molecule kinase inhibitors, most which are approved for oncological indications. In addition, there are numerous other kinase inhibitors in clinical trial or development (summarized in Table 88.3). Besides small-molecule direct inhibitors of kinases, which typically block the ATP-binding pocket, there are several alternative strategies. These include small inhibiting RNA to block protein expression of the kinase Syk (Excellair; ZaBeCor Pharmaceutical Co., Bala Cynwyd, PA), inhibition of associated proteins required to activate the target kinase as in the rapamycin derivatives and the use of monoclonal antibodies (mAbs) that inhibit ligand-dependent activation of transmembrane receptor

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kinases. These mAbs include and target the designated RTK: bevacizumab (vascular endothelial growth factor receptor [VEGFR]), ranibizumab (VEGFR), cetuximab (epidermal growth factor receptor [EGFR]), pertuzumab (human epidermal growth factor receptor [HER]), and trastuzumab (HER2/neu).

identifying mutations in patients with immunodeficiencies. Specifically, mutation of JAK3 results in a severe combined immune deficiency (SCID), characterized by an almost complete absence of T cells and NK cells with defective B cells. This phenocopies deficiency of the cognate receptor that associates with JAK3, the IL-2 receptor common γ chain, cγc (encoded by IL2RG), mutation of which underlies X-SCID (see Table 88.1; Fig. 88.4). The profound, but selective, phenotype associated with JAK3 deficiency led to the suggestion that targeting JAKs might be a strategy for the development of a new class of immunomodulatory drugs. There are now several FDA-licensed JAK inhibitors for the treatment of immune and neoplastic diseases, and more are in clinical trials. Tofacitinib, formerly designated CP-690,550, was one of the first JAK inhibitors to enter the clinic. It inhibits JAK3 and JAK1 and to a lesser extent JAK2, but has little effect on TYK2.9 Consequently, tofacitinib potently inhibits cγc cytokines but also blocks interferon (IFN)-γ, interleukin (IL)-6, and to a lesser extent

TARGETING CYTOKINE SIGNALING BY INHIBITING JANUS KINASES: TOFACITINIB, RUXOLITINIB, AND RELATED COMPOUNDS Cytokines regulate growth, survival, development, and differentiation of immune cells. Their importance in driving inflammatory and immunological responses has already made them attractive targets as antiinflammatory and immunosuppressive agents. As indicated above, a large subset of cytokines (roughly 60) signal through Janus kinases (JAKs) (see Table 88.2). The essential function of JAKs was documented by knock-out mice and by

Daclizumab IL-2R α β γ Tipifarnib

Sorafenib

GTP

Tofacitinib

IL-2

Ras Sos Grb2

Jak 1

Shc

Raf

Ick

MEK

Jak 3

PIP3 p85

P

PKB

P13’K

Other substrates leading to cell survival

P STAT

PDK1

p110

P

MAPK pathway P P

AMPK

STAT

STAT

TSC1 TSC2

Rheb Cell proliferation and survival

Sirolimus FKBP12

Raptor mTOR mLST8

4EBP1

S6K1

eIF4F

S6

Initiation of protein translation

Maintenance of protein translation

PDK1

Protein translation leading to cell growth

FIG 88.4  Signal Transduction Pathways Stemming From the Interleukin (IL)-2 Receptor in T Cells Culminating in the Activation of the Mammalian Target of Rapamycin (mTOR) Serine/Threonine Kinase. Tyrosine kinases are indicated in red, and serine/ threonine kinases are indicated in blue.

CHAPTER 88  Protein Kinase Antagonists IL-12 and IL-23. Functionally, tofacitinib inhibits T-helper 1 (Th1) cells and Th2 differentiation, as well as pathogenic Th17 cells.9 In addition to inhibiting adaptive immune responses, tofacitinib appears to inhibit innate immune responses as well.9 Tofacitinib passed phase III trials for the treatment of methotrexate-resistant rheumatoid arthritis (RA) and was FDA approved for this indication in 2012.10 It completed phase III trials in both ulcerative colitis and psoriasis with successful results.11 Other diseases in which tofacitinib is being studied include ankylosing spondylitis and juvenile idiopathic arthritis. Tofacitinib has been used in the treatment of alopecia areata, vitiligo, and atopic dermatitis.11 A variety of additional inhibitors that target JAK3, including decernotinib, peficitinib, and R-348 (Rigel), are under development or in clinical trials (see Table 88.3). As gene targeting of Jak2 in mice was embryonically lethal, it was initially thought that inhibition of JAK2 should be avoided. However, the discovery that gain-of-function mutations of JAK2 underlie primary polycythemia and myelofibrosis led to the idea that pharmacologically targeting JAK2 could be useful. The JAK1/ JAK2 inhibitor ruxolitinib was the first JAK inhibitor for the treatment of myelofibrosis to be approved by the FDA in 2011 and was subsequently approved for the treatment of primary polycythemia in 2014.11 As cγc cytokines employ both JAK1 and JAK3 for signaling, it might be expected that ruxolitinib and tofacitinib might block some of the same cytokines. It is therefore of interest to note that in a phase II study in RA, ruxolitinib was as efficacious as tofacitinib.12 A number of novel JAK1/JAK2 inhibitors under development both for the treatment of myeloproliferative and inflammatory diseases. The JAK1/JAK2 inhibitors baricitinib, filgotinib, and AC-410 (Daiichi Sankyo, Tokyo, Japan) all are in trials for the treatment of RA (see Table 88.3). The side effects of JAK inhibitors include infection, including serious infections, anemia, and leukopenia, presumably related to JAK2 inhibition and interference with cytokines, such erythropoietin, IL-11, and colony-stimulating factors.11 Little reduction in CD4 T cells has been seen, but significant reduction in NK cells and CD8 T cells does occur; just how significant this will be in terms of infection risk remains to be determined.11

TARGETING ANTIGEN RECEPTOR SIGNALING The first event in TCR signaling is the activation of the Src family kinase Lck (see Fig. 88.1), making it an attractive target as a therapy for autoimmune diseases and transplant rejection. Several Lck inhibitors have been developed and showed promise in preclinical models of allograft rejection and autoimmunity, although at the cost of inducing progressive lymphopenia. This is consistent with the finding that induced deficiency of Lck in mice leads to a progressive lymphopenia.13 Despite considerable effort, the discovery of a selective Lck inhibitor suitable for use as an immunosuppressive agent remains elusive. Lck activation leads to the recruitment of a second round of tyrosine kinases to the TCR complex; these include Zap70 or Syk. Deficiency of Zap70 causes SCID and preferential loss of CD8 T cells, but curiously, a successful Zap70 inhibitor has yet to be obtained. In contrast, Syk inhibitors have been generated; BIIB057 (Biogen, Cambridge, MA) is being investigated for the treatment of inflammatory diseases, and a second, fostamatinib, is being investigated for the treatment of immune thrombocytopenia and immunoglobulin A (IgA) nephropathy. The recruitment of Zap70/Syk results in activation of another class of PTKs, the Tec family kinases, Rlk and Itk in T cells and Btk in B cells. The importance of this class

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of kinases is exemplified by the fact that the mutations of BTK underlie Bruton agammaglobulinemia a condition characterized by the absence of all B cells (Chapter 34). Ibrutinib is the first BTK inhibitor approved by the FDA in 2014 for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia (CLL).14 Recent work has identified ibrutinib as an inhibitor of Itk, which suggests it may have value as therapy in autoimmune disease. Antigen receptor activation of phospholipase C (PLC)γ1 appears to require Lck, Zap70, and Tec kinases working in concert. The action of PLCγ1 leads to elevation in intracellular calcium, which in turn activates the phosphatase calcineurin. Calcineurin dephosphorylates and activates nuclear factor of activated T cells (NFAT), which translocates to the nucleus and in cooperation with activator protein-1 (AP-1) transcription factors induces transcription of IL-2 and other key lymphocyte activation genes. A number of potent clinically successful immunosuppressive drugs inhibit calcineurin, including cyclosporine and tacrolimus, drugs that have revolutionized organ transplantation.15 Despite their success, long-term use of these drugs is limited, as they may cause renal toxicity

CLINICAL PEARLS Mutations Reveal Key Functions of Kinases in Patients With Primary Immunodeficiencies • Common γ-chain deficiency, JAK3 deficiency: severe combined immunodeficiency • ZAP70 deficiency: severe combined immunodeficiency • TYK2 deficiency: rare cause of hyper-IgE syndrome

Protein Kinase C Family and NF-κB TCR signaling also leads to the activation of members of the PKC family, which, in turn, activate the transcription factor complex nuclear factor (NF)-κB and the Ras guanyl nucleotidereleasing proteins (RasGRPs). Genetic studies in mice have identified the importance of one member, PKC-θ in TCR signaling. Recent work has identified PKC-θ as an inhibitor of regulatory T cell function.16 Regulatory T cells (Tregs), unlike conventional effector T cells, act to constrain the immune system. Enhancing Treg numbers or function has been suggested as the treatment of a number of autoimmune diseases.17 For this reason, preserving or enhancing Treg function through the inhibition of PKC-θ is an attractive strategy. Along with many other receptors, TCR signaling leads to the action of NF-κB family transcription factors, which control the genes involved in cellular activation and resistance to apoptosis.18 In T cells, PKC-θ is the main isoform responsible for NF-κB activation. The novel PKC inhibitor Sotrastaurin, a potent inhibitor of the PKC-θ, β and α isoforms, is currently undergoing phase II trials for the treatment of diffuse large B-cell lymphoma. Early clinical testing on patients with psoriasis demonstrated that the drug is well tolerated and was able to inhibit T-cell proliferation, IL-2, and IFN-γ secretion. However, despite initial positive trial results, it was not effective in the treatment of psoriasis, ulcerative colitis, or liver transplantation. Downstream of several inflammatory signaling pathways, including PKC, lies the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).18 NF-κB has been implicated as a major mediator governing both cancer cell proliferation and survival and governing chronic inflammation. It is held in an inactive state by the binding of the inhibitor of κB (IκB). A cascade of protein kinases that include IκB kinases (IKK) and

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IKK kinases (IKKKs), of which PKC and PKB are members, regulate IκB.18 Several IKK inhibitors have been in development; however, all of them have failed clinical trials.

Lipid Kinases and Downstream Signaling Kinases are also important in phosphorylating lipids, and these modifications are very relevant in signal transduction through both the TCR and cytokine receptors. In addition to the production of inositol triphosphate by the action of PLCγ1, there is a second pathway of inositol lipid metabolism regulated by the TCR that is a response shared by costimulatory molecules, such as CD28, cytokines, and chemokines. This response is mediated by the class I group of phosphatidyl-inositol 3 kinases (PI3Ks), which is composed of four isoforms (PI3Kα to -δ), which phosphorylate the 3’-OH position of the inositol ring of phosphatidyl-inositol (4,5) bisphosphate (PI(4,5)P2) to produce PI(3,4,5)P3.19 This lipid and its metabolite PI(3,4)P2 bind to the pleckstrin homology (PH) domains of proteins and either induce localization of the protein to defined areas of the plasma membrane, where activation can occur, or induce conformational changes that allow for allosteric modifications of activity. Targets for D-3 phosphoinositides in T cells include a number of downstream protein serine/threonine kinases and the Rac-1 and RhoA guanine nucleotide exchange proteins. Small-molecule inhibitors of PI3’K, Wortmannin and LY294002, are both potent inhibitors of T-cell activation, although toxicity prevents either from being clinically useful. In contrast to the more widely expressed PI3’Kα and β, PI3’Kγ and -δ are only expressed in hematopoietic tissue and deletion of PI3’Kγ results in defective migration of neutrophils and macrophages to sites of inflammation without other pathology. This limited expression makes PI3’Kγ a potentially useful target, and selective PI3’Kγ inhibitors have shown to be effective in mouse models of collagen-induced arthritis.20 For similar reasons, PI3’Kδ is another attractive target for which the inhibitor idelalisib was FDA-approved for the treatment of lymphoma.21 Over 25 other inhibitors of PI3’K are currently being studied in preclinical and clinical trials and include buparlisib, which is in a phase III trial for the treatment of breast cancer. One PI(3,4,5)P3-regulated kinase activated by the TCR is protein kinase B (PKB/AKT).22 T cells stimulated in the presence of the PI3’K inhibitors Wortmannin or LY-294002 fail to activate and proliferate. The ability of cells to take up nutrients and switch on glycolysis is essential for lymphocyte activation; PKB is proposed to be the main effector that mediates the action of PI3’K on these fundamentally important metabolic pathways. There are three PKB isoforms (PKB-α, -β, -γ, or AKT1, -2, -3, respectively), and T cells lacking both Akt1 and Akt2 are greatly impaired.22 Specific inhibitors of PKB are in clinical trials with mixed results; MK-2206 (Merck, Kenilworth, NJ), AZD-5363 (Astra Zeneca, Cambridge, UK), and uprosertib are all in phase II trials for the treatment of several malignancies (see Table 88.3). In addition to glucose metabolism, the PI3’K-regulated serine/ threonine kinase mammalian target of rapamycin (mTOR) regulates protein synthesis in response to cellular nutrient and energy levels.23 It is activated not only by a number of growth factor receptors but also by cytokines, including IL-2. Many signaling pathways link growth factor receptors with activation of mTOR, including the adenosine monophosphate (AMP)– dependent kinase (AMPK) and phosphatidyl inositol 3’ kinase (PI3’K) (see Fig. 88.4). mTOR promotes cell growth by activation

of p70 S6K1 and inactivation of 4E-BP1,23 which are critical for translation of new proteins. As its name suggests, mTOR is inhibited by the macrolide rapamycin, now licensed as the drug sirolimus for the treatment of graft rejection. Sirolimus does not inhibit mTOR by directly binding to the ATP-binding pocket but acts indirectly, associating with FK506-binding protein 12 (FKBP12). This, in turn, inhibits the kinase complex made up of mTOR, mLST8, and raptor (mTORC1).23 It was hoped that sirolimus would be a potent anticancer drug, but it has met limited success in this regard. In contrast, it has been successfully used as an immunosuppressant, typically as part of a combination regimen for allograft rejection prophylaxis.24 In view of the ubiquitous expression of mTOR and its role in protein translation, it is not surprising that sirolimus would be associated with a number of side effects, including hyperlipidemia, hypertriglyceridemia, myelosuppression, and delayed wound healing. There is some evidence of renal toxicity, but this is minor compared with that caused by the calcineurin inhibitors cyclosporine and tacrolimus. Myelosuppression associated with sirolimus is typically dose related and rapidly reversible, even in patients receiving the drug for chronic GvHD. There are currently three rapamycin derivatives, namely, temsirolimus, everolimus, and AP23573, undergoing clinical trials. Everolimus is licensed for use in the management of heart, liver, and kidney transplantation in conjunction with cyclosporine. It is under investigation for the treatment of arthritis and a number of solid tumors. Temsirolimus is licensed for the treatment of renal cell carcinoma and mantle cell lymphoma. The introduction of novel rapamycin derivatives has been joined by the introduction of pan-mTOR direct inhibitors AZD2014 (AstraZeneca, Cambridge, UK) and CC-223 (Celgene, Summit, NJ), both of which are in phase II trials for the treatment of nonHodgkin lymphoma and solid tumors.

MAPK Pathways The MAPK family constitute a complex phospho-relay system of signal transduction, composed of three sequentially activated kinases that are themselves modulated by phosphorylation.25 Substrates of MAPK pathways include transcription factors, phospholipases, cytoskeletal proteins, and other protein kinases. Three main MAPK cascades have been identified in mammalian cells: the extracellular signal regulated kinase (ERK) cascade, the Jun kinase (JNK) cascade, and the p38 MAPK cascade. All start with a membrane-localized activator followed by three MAPKs that sequentially phosphorylate each other (see Fig. 88.3). The top level of kinases is termed MAPK kinase kinases (MAPKKKs, MKKKs, or Map3Ks). The middle level MAPK kinases (MAPKKs, MKKs, or Map2Ks) phosphorylate a common Thr-Xaa-Tyr motif, where Xaa is any amino acid. The lowest tier consists of the MAPKs that phosphorylate Ser/Thr-Pro motifs. Each pathway terminates in the phosphorylation of substrate proteins. The ERK Cascade ERK1 and 2 were identified as kinases that were activated in response to growth factor stimulation, which are mimicked by expression of constitutively active Ras. The link between active Ras and subsequent phosphorylation of the ERKs was made both by the discovery of the MAPK kinase MEK1 and its phosphorylation by the known Ras effector RAF1, now known as a MAPK kinase kinase (M3K). The ERK cascade is ubiquitous in mammalian cells and is generally considered one of the main effector pathways regulated by the GTPase p21ras. Activation of

CHAPTER 88  Protein Kinase Antagonists the TCR or the IL-2 receptor is able to trigger ERK signaling in T cells.25 Two small-molecule inhibitors of this serine/threonine kinase pathway are undergoing clinical trials: the farnesyl inhibitor of Ras tipifarnib, which is in phase III trials for the treatment of acute myeloid leukemia; and the multikinase inhibitor sorafenib, which is FDA approved for the treatment of renal cell carcinoma.26 Sorafenib is relatively well tolerated despite its ability to inhibit numerous kinases, including the M3K RAF, as well as receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR), VEGFR, Kit, and FLT-3. Its role as an immunosuppressant has yet to be explored. There are three RAF isoforms: A-RAF, B-RAF, and C-RAF. A number of cancers are associated with B-RAF mutations resulting in a constitutively active kinase, the best known of which is the V600E mutation. Two kinase inhibitors, vemurafenib and dabrafenib, which are highly active against the B-RAF V600E kinase, are FDA licensed for the treatment of melanoma. Their early use was associated with a partial and short-lived inhibition of the MAPK pathway as C-RAF signaling was preserved, which is often hyperactivated by upstream mutations in Ras signaling or by the presence of B-RAF V600E, which is able to dimerize and activate C-RAF even in the presence of inhibitors.27 Moreover, clinical trials are ongoing for the treatment of the histiocytosis known as ErdheimChester disease, for which B-RAF mutations have shown to be present in the vast majority of the case. The M2K MEK1 and the MAPK ERK1 lie downstream of RAF. Cobimetinib and trametinib are selective MEK inhibitors that are FDA licensed for the treatment of melanoma both as a single agent and in combination with B-RAF V600E inhibitors as they are able to block residual C-RAF signaling.28 Although all of these agents are seen as anticancer drugs rather than immunosuppressants, it is of note that cobimetinib is currently being assessed in the treatment of Langerhans cell histiocytosis (LCH). LCH is a clonal disorder characterized both by the B-RAF V600E mutation in histiocytes and by the presence of inflammatory lesions that often respond to simple corticosteroid therapy. Selective inhibitors of ERK have been developed: FR180204 has been shown to inhibit the development of collagen-induced arthritis in mice and is being considered as an agent in the treatment of RA. CC90003 (Celgene, Summit, NJ) is currently being assessed in a phase I trial in patients with solid tumors. The JNK Cascade Another limb of the MAPK pathway is the JNK pathway. Many inflammatory agents, including lipopolysaccharide (LPS), TNF-α, and IL-1 are able to activate the JNK pathway.25 In synoviocytes, this results in secretion of proteases implicated in joint destruction seen in RA.29 A number of small-molecule inhibitors of JNKs have been identified and are currently being investigated in the treatment of inflammation, cancer, and neurological diseases. XG102 (Xigen Ltd., Bedford, UK) is being assessed in phase III trials for the treatment of ocular inflammation and sensorineural hearing loss. The p38 MAPK Cascade This cascade was originally identified as part of a drug screen looking for inhibitors of TNF-α–mediated inflammatory responses.30 TLR-dependent production of IL-1 and TNF-α is p38 MAPK dependent. The success of TNF-α–blocking antibodies in the treatment of RA has led to much interest in the development of p38 MAPK inhibitors. However, although many p38 inhibitors have been reported, their development into therapeutic drugs

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has been frustrated by either unacceptable toxicity or poor efficacy. Currently, a new generation of p38 MAPK inhibitors are being assessed in phase II trials for the treatment of chronic obstructive pulmonary disease (COPD); losmapimod has recently failed phase II testing because of lack of efficacy, and AZD7624 (AstraZeneca, Cambridge, UK) and acumapimod are still being investigated.

CONCLUSIONS ON THE HORIZON • Many kinase inhibitors, initially designed as antitumor agents, have been repurposed as immunosuppressants (e.g., ruxolitinib). • Further understanding of the pathophysiology of autoimmune disease will lead to the increased use and creation of kinase inhibitors. • Increasing use of specific kinase inhibitors may improve our understanding of the pathophysiology of autoimmune disease.

Scientific advances in the 1990s have led to the discovery of many novel intracellular signaling pathways that link membranebound receptor and cytokine signaling with alteration of gene expression and cellular activation necessary to trigger an immune cell response. Many of these pathways are interlinked to make up a complex array of networks composed of enzymes, adaptor proteins, and transcription factors, all of which are potential targets for drug discovery in the quest to make a therapy that will be able to treat a specific autoimmune disease without an unacceptable degree of immunosuppression. Now more than a decade since the identification of many new targets, the first generation of drugs designed to interfere with specific immune cell signals are being brought to the clinic. The success of the anticancer BCR-Abl inhibitor imatinib and the immunosuppressive mTOR inhibitor sirolimus has placed the protein kinases center stage as targets of future drug discovery. As many of the key steps in the activation of an immune cell are often shared with those that allow a cancer cell to proliferate, many of these agents are being tested as anticancer drugs rather than as immunosuppressants. Despite this, some agents, such as the mTOR inhibitors, originally intended for the treatment of cancer have been far more successful in the field of immunology, and this may continue to be true for future modifiers of cell signaling. Conversely, JAK inhibitors could potentially be used in the treatment of lymphoma. Either way, we are likely to see a large number of novel immunosuppressants appear both serendipitously and intentionally as new protein kinase inhibitors are licensed for a wide range of debilitating illnesses. Please check your eBook at https://expertconsult.inkling.com/ for self-assessment questions. See inside cover for registration details.

REFERENCES 1. Hanks SK, Quinn AM, Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 1988;241:42–52. 2. Noble ME, Endicott JA, Johnson LN. Protein kinase inhibitors: insights into drug design from structure. Science 2004;303:1800–5. 3. Druker BJ, Lydon NB. Lessons learned from the development of an Abl tyrosine kinase inhibitor for chronic myelogenous leukemia. J Clin Invest 2000;105:3–7.

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4. Schindler T, Bornmann W, Pellicena P, et al. Structural mechanism for STI-571 inhibition of Abelson tyrosine kinase. Science 2000;289:1938–42. 5. Adams J, Huang P, Patrick D. A strategy for the design of multiplex inhibitors for kinase-mediated signalling in angiogenesis. Curr Opin Chem Biol 2002;6:486–92. 6. Davies TG, Pratt DJ, Endicott JA, et al. Structure-based design of cyclin-dependent kinase inhibitors. Pharmacol Ther 2002;93:125–33. 7. Kantarjian H, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med 2010;362:2260–70. 8. Arai S, et al. A Randomized Phase II Crossover Study of Imatinib or Rituximab for Cutaneous Sclerosis after Hematopoietic Cell Transplantation. Clin Cancer Res 2016;22:319–27. 9. Ghoreschi K, et al. Modulation of Innate and Adaptive Immune Responses by Tofacitinib (CP-690,550). J Immunol 2011;186:4234–43. 10. Fleischmann R, et al. Placebo-controlled trial of tofacitinib monotherapy in rheumatoid arthritis. N Engl J Med 2012;367:495–507. 11. Schwartz DM, Bonelli M, Gadina M, et al. Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases. Nat Rev Rheumatol 2016;12:25–36. 12. Mesa RA. Ruxolitinib, a selective JAK1 and JAK2 inhibitor for the treatment of myeloproliferative neoplasms and psoriasis. IDrugs 2010;13:394–403. 13. Zamoyska R, Basson A, Filby A, et al. The influence of the Src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation. Immunol Rev 2003;191:107–18. 14. Wang ML, Rule S, Martin P, et al. Targeting BTK with ibrutinib in relapsed or refractory mantle-cell lymphoma. N Engl J Med 2013;369:507–16. 15. Calne RY. Organ transplantation has come of age. Sci Prog 2010;93:141–50. 16. Zanin-Zhorov A, Ding Y, Kumari S, et al. Protein kinase C-theta mediates negative feedback on regulatory T cell function. Science 2010;328:372–6. 17. Riley JL, June CH, Blazar BR. Human T regulatory cell therapy: take a billion or so and call me in the morning. Immunity 2009;30:656–65.

18. DiDonato JA, Mercurio F, Karin M. NF-κB and the link between inflammation and cancer. Immunol Rev 2012;246:379–400. 19. Cantrell DA. Phosphoinositide 3-kinase signalling pathways. J Cell Sci 2001;114:1439–45. 20. Camps M, et al. Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis. Nat Med 2005;11:936–43. 21. Gopal AK, Kahl BS, de Vos S, et al. PI3Kdelta inhibition by idelalisib in patients with relapsed indolent lymphoma. N Engl J Med 2014;370:1008–18. 22. Juntilla MM, Wofford JA, Birnbaum MJ, et al. Akt1 and Akt2 are required for αβ thymocyte survival and differentiation. Proc Natl Acad Sci USA 2007;104:12105–10. 23. McManus EJ, Alessi DR. TSC1-TSC2: a complex tale of PKB-mediated S6K regulation. Nat Cell Biol 2002;4:E214–16. 24. Kreis H, Cisterne JM, Land W, et al. Sirolimus in association with mycophenolate mofetil induction for the prevention of acute graft rejection in renal allograft recipients. Transplantation 2000;69:1252–60. 25. Dong C, Davis RJ, Flavell RA. MAP kinases in the immune response. Annu Rev Immunol 2002;20:55–72. 26. Josephs DH, Ross PJ. Sorafenib in hepatocellular carcinoma. Br J Hosp Med (Lond) 2010;71:451–6. 27. Park EJ, Lee JH, Yu GY, et al. Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression. Cell 2010;140:197–208. 28. Larkin J, Ascierto PA, Dréno B, et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N Engl J Med 2014;371:1867–76. 29. Gum R, Wang H, Lengyel E, et al. Regulation of 92 kDa type IV collagenase expression by the Jun aminoterminal kinase—and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene 1997;14:1481–93. 30. Lee JC, Laydon JT, McDonnell PC, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 1994;372:739–46.

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MULTIPLE-CHOICE QUESTIONS 1. Specific inhibitors of individual protein kinases are possible because: A. Most kinase inhibitors are noncompetitive covalent inhibitors. B. Protein kinases can be divided into families of serine, threonine, and tyrosine kinases that are structurally very dissimilar. C. The adenosine triphosphate (ATP)–binding pocket of kinases are sufficiently distinct to allow generation of selective inhibitor. D. Most kinase inhibitors avoid the structurally similar ATPbinding pocket. E. Distinct kinases occupy different parts of the cell and do not interact with each other.

2. The first clinically successful direct tyrosine kinase inhibitor was: A. Ruxolitinib B. Infliximab C. Imatinib D. ZAP70 E. Methotrexate 3. Patients deficient in the following protein kinases suffer from a severe combined immunodeficiency (SCID): A. BCR-Abl B. JAK3 C. PTEN D. TYK2 E. BTK