key: cord-0768131-fa0fvzei
authors: Zarrin, Ali A.; Bao, Katherine; Lupardus, Patrick; Vucic, Domagoj
title: Kinase inhibition in autoimmunity and inflammation
date: 2020-10-19
journal: Nat Rev Drug Discov
DOI: 10.1038/s41573-020-0082-8
sha: 3e62377cae27fa30cbdd5c9b5e0285c7b71b63e3
doc_id: 768131
cord_uid: fa0fvzei

Despite recent advances in the treatment of autoimmune and inflammatory diseases, unmet medical needs in some areas still exist. One of the main therapeutic approaches to alleviate dysregulated inflammation has been to target the activity of kinases that regulate production of inflammatory mediators. Small-molecule kinase inhibitors have the potential for broad efficacy, convenience and tissue penetrance, and thus often offer important advantages over biologics. However, designing kinase inhibitors with target selectivity and minimal off-target effects can be challenging. Nevertheless, immense progress has been made in advancing kinase inhibitors with desirable drug-like properties into the clinic, including inhibitors of JAKs, IRAK4, RIPKs, BTK, SYK and TPL2. This Review will address the latest discoveries around kinase inhibitors with an emphasis on clinically validated autoimmunity and inflammatory pathways.

Inflammation is a physiological response of the immune system to injury and infection. This process signals the immune system to heal and repair damaged tissue, as well as to defend itself against infective agents, such as viruses and bacteria. However, unresolved or inappro priately activated inflammation can become pathogenic 1 . Chronic inflammation is the primary cause of a broad spectrum of diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD; including gastrointesti nal conditions such as Crohn's disease and ulcerative colitis), chronic obstructive pulmonary disease (COPD), asthma, psoriasis and idiopathic pulmonary fibrosis (IPF), among others 1 . Viral and bacterial infections or other insults (such toxins, chemicals and so forth) can lead to uncon trolled acute inflammatory responses and injury often seen in patients with underlying pathogenic conditions (such as COPD or asthma). Acute lung injury (ALI) is a syndrome with diagnostic criteria based on hypoxaemia and a classical radiological appearance, with acute respi ratory distress syndrome (ARDS) at the severe end of the disease spectrum impairing gas exchange, leading to multiple organ failure widespread inflammation in the lungs and sepsis 2 . A recent example of virally induced ALI and ARDS includes SARS CoV2 infection, which is associated with a cytokine storm (characterized by high levels of IL6, IL12 and IL1β, and tumour necrosis fac tor (TNF)) and defective type I interferon activity 3 . This inflammatory response resembles the cytokine release syndrome observed in patients receiving chimeric antigen receptor (CAR) T cell therapy and bispecific T cell engaging antibodies, which can be treated with anti cytokine therapy targeting the IL6-IL6 receptor (IL6R) signalling pathway 3 . Although select biologics and kinase inhibitors (see below) are effective in treating various inflammatory diseases, a large proportion of patients are not responsive to current therapies, and effective treatment approaches for this subset of patients are needed 4 .

Autoimmune diseases refer to a spectrum of condi tions in which the immune system mistakenly attacks one's own body 5 . This autoimmune response often involves dysregulated adaptive immunity (mediated by B and T lymphocytes) towards anatomical self antigens (such as insulin) 5 . Certain human leukocyte antigen (HLA) genes have also been demonstrated to be predic tive of the development of autoimmune diseases. HLA molecules on antigen presenting cells present antigens to effector T cells in an interactive process required for antigen specific T cell activation. Effector T cells then generate local inflammation by producing inflamma tory cytokines or directly damaging the tissues, whereas CD4 + CD25 + regulatory T (T reg ) cells counteract the inflammatory response to maintain immune homeo stasis in tissues. Autoimmune diseases are on the rise and contribute to approximately 100 clinical indications affecting 3-5% of the population 5 . They are caused by the deregulation of such cellular dynamics resulting in organ damage, including systemic lupus erythema tosus (SLE; systemic disease with many organs targeted), type 1 diabetes (affecting the pancreas), multiple scle rosis (which affects the central nervous system), coeliac disease (which affects the small intestine), primary biliary cirrhosis (affecting the liver), chronic spontane ous urticaria (which affects the skin), immune throm bocytopenic purpura (ITP; platelets), autoimmune haemolytic anaemia (which affects red blood cells) and

Rheumatoid arthritis (RA) . A progressive autoimmune and inflammatory disease manifested by joint pain and swelling in the feet and hands that can cause permanent joint destruction and deformity.

Inflammatory bowel disease (IBD) . A group of disorders that involve chronic inflammation of the digestive tract, including ulcerative colitis and Crohn's disease.

JAK1 JAK2 IgA nephropathy (which affects kidney glomeruli), among others 5 . Autoinflammatory syndromes are a rare set of dis orders caused mostly by genetic mutations that affect innate immune cells (such as macrophages or neutro phils) and lead to uncontrolled activation of the immune system when there is no actual infection 6 . These patients often respond better to select anti inflammatory drugs (such as anti TNF or anti IL1β) but not broad immuno suppressives. Examples of autoinflammatory disorders include familial Mediterranean fever, TNF receptor associated periodic syndrome, cryopyrin associated periodic syndromes, deficiency of the IL1 receptor (IL1R) antagonist (IL1Rα), deficiency of the IL36Rα and interferonopathies 6 .

Although understanding signalling pathways in inflammatory and autoimmune diseases is challeng ing, preclinical and clinical research has been greatly instructive for therapeutic development. Biologics (such as antibody antagonists or fusion proteins) have validated several pathogenic pathways involved in these diseases (FIg. 1) . Examples include therapies that use inhibition of cytokine receptors and/or ligands (such as TNF and IL1), cellular depletion to reduce patho genic cellular response (such as anti CD20 antibodies for B cell depletion to limit autoantibody production or B cell mediated antigen presenting cell function) or inhibition of cellular differentiation (such as inhibi tion of macrophage colonystimulating factor 1 recep tor (CSF1R) to reduce macrophage differentiation). Recently, strategies to stimulate immune receptors to reset productive immunity are evolving as a powerful approach in drug development 7 .

However, there are still considerable unmet medical needs for the treatment of some inflammatory (COPD, IPF and IBD) and autoimmune (SLE, type 1 diabetes, primary biliary cirrhosis, Graves disease and multiple sclerosis) diseases, indicating the demand for effective therapeutics 5 . Even for diseases such as RA, for which several approved drugs exist, 74% of patients are not satisfied with their current treatment, according to surveys 8 . Thus, availability of effective treatments with disease modifying potential and minimal adverse effects to reset productive immunity is crucial.

Kinases (518 encoded in genome) are enzymes that phosphorylate up to one third of the proteome, and their utility as drugs is expanding in cancer, inflammatory and neurodegenerative diseases 9 . Owing to the distri bution of select kinases in multiple signalling cascades in immune cells, the use of small molecule kinase inhibi tors has the potential to disable inflammation in a tar geted fashion 10 . In addition, emerging data suggest that combination therapy with non overlapping therapeutics (such as combinations of biologics and kinase inhibitors) may be more effective than single agents 11, 12 . Therefore, a comprehensive understanding of signalling kinases combined with the ongoing clinical evaluations should lead to the discovery of effective therapies.

There has been tremendous progress in advancing several kinase inhibitors into preclinical and clini cal investigations. Janus associated kinase inhibitors (JAKis) have already been proven clinically beneficial for the treatment of RA and are being advanced in sev eral other indications (Crohn's disease, alopecia areata, psoriasis, Alzheimer disease); however, intense efforts are underway to optimize their selectivity and modes of delivery to reduce toxicity 13 . This Review captures the current efforts, progress and new discoveries around kinase inhibitors with an emphasis on key clinically vali dated pathways and targets with potential to mitigate human disease.

Tissue inflammation involves an influx of immune cells -including neutrophils, monocytes and macro phagesto various tissues, such as the skin, gut or lung 1 . This process is often regulated by a complex hierarchy of immune cells, cell surface receptors, signal transduction and the resultant gene transcription and translation of immunomodulating factors. Activation of receptors on immune cells drives signalling cascades that dic tate, maintain and amplify local or systemic immune responses. Accordingly, chronic or dysregulated signal ling can perpetuate inflammation and generate excessive levels of superoxide radicals, proteases, and cytokines and chemokines that can then cause tissue damage 14 . Importantly, the production of these pro inflammatory mediators is subject to multiple regulatory mechanisms at the transcriptional and post transcriptional levels. Early induction of the majority of inflammatory transcripts depends on transcription factor networks including NF κB (canonical and non canonical), signal transdu cers and activators of transcription (STATs), nuclear fac tor of activated T cells (NFATs) and interferon regulatory factors (IRFs). However, the net production of the corre sponding proteins depends, in part, on mitogen activated protein kinases (MAPK) and molecular programmes that regulate transcript stabi lity and translation 14 . The canonical NF κB pathway mediates the activation of transcription factors NF κB1 p50, transcription fac tor p65 (encoded by RELA) and protooncogene REL, whereas the noncanonical NF κB pathway selectively activates p100 sequestered NF κB members, predomi nantly NF κB2 p52 sub unit and RelB 15 . MAPK signal ling (such as that mediated by ERK1/2 and p38) regulates RNA stability and translation of cytokines, which enable Major inflammatory pathways and downstream kinases are depicted to show surface versus intracellular drug targets, highlighting drugs that are currently being clinically evaluated or already approved. All biologics that are included have mostly been evaluated in phase II trials and beyond. All small-molecule kinase inhibitors have been evaluated in phase I and beyond. BAFFR, B cell activating-factor receptor; BCMA, B cell maturation antigen; BCR, B cell receptor; BTK, Bruton's tyrosine kinase; CD40L, CD40 ligand; CSF1R, colony-stimulating factor 1 receptor; CTLA4, cytotoxic T lymphocyte-associated protein 4; FcεR, Fcε receptor; IKKε, inhibitor of NF-κB subunitε; IL-1R, IL-1 receptor; IRAK, IL-1R-associated kinase; ITK, IL-2inducible T cell kinase; JAK, Janus-associated kinase; MD2, myeloid differentiation factor 2; NF-κB, nuclear factorκ-light-chain-enhancer of activated B cells; NIK, NF-κB-inducing kinase; RANKL, receptor activator of NF-κB ligand; RIP1, receptor-interacting protein 1; RLK, resting lymphocyte kinase; ST2, IL-1R-like 1; SYK, spleen tyrosine kinase; TACI, transmembrane activator and CAML interactor; TBK1, TANK-binding kinase 1; TCR, T cell receptor; TEC, Tec protein tyrosine kinase; TLR, Toll-like receptor; TNF, tumour necrosis factor; TNFR, TNF receptor; TPL2, tumour progression locus 2; TSLP, thymic stromal lymphopoietin; TYK2, tyrosine kinase 2.

An inflammatory disease that causes inflammation and sores (ulcers) in the innermost lining of the colon and rectum.

Chronic obstructive pulmonary disease (COPD). A chronic inflammatory lung disease that causes obstructed airflow from the lungs accompanied by breathing difficulty, cough, mucus production and wheezing.

(IPF). A chronic and progressive fibrotic lung disease with unknown aetiology accompanied by scarring, resulting in persistent dry, hacking cough.

An abnormally low concentration of oxygen in the blood. ◀ Nature reviews | Drug Discovery immune cells to respond promptly 16 . The STAT family of transcription factors integrates the signalling cascade of several cytokine receptors and ligands 13 . Activated STATs bind to GAS (IFNγ activated sequence) DNA ele ments, and initiate transcription of target genes. Diverse outcomes of STAT signalling are not only determined by the expression of specific receptors but also by the interaction of STATs (such as STAT5) with cofactors, and by the cell specific activity of members of the suppressor of cytokine signalling (SOCS) family, which negatively regulate STAT function 13 . Therefore, complex positive and negative regulatory networks orchestrate immune responses.

The physiological or pathogenic immune response involves multiple receptors on different immune cells and their cognate ligands. Host immunity is divided into innate and adaptive immune responses 17 . The former reacts rapidly and non specifically to pathogens, whereas the latter responds in a slower but specific man ner, with the generation of long lived immunological memory 17 . Strict regulation of immune response is partly regulated by CD4 + T helper (T H ) cells because they regu late the function of other immune or even non immune cells 18 . Naive CD4 + T cells can differentiate into multi ple distinct T cell subsets, such as T H 1, T H 2, T H 17 and T reg cells, depending on the cytokine milieu 18 . T reg cells are essential in preventing autoimmune diseases and avoiding prolonged immunopathological processes and allergies acting via classic suppressive mechanisms on other immune cells as well as reparative functions 19 . B cells, in addition to their function in antibody pro duction, also express a high level of MHC class II and can present antigens to T H cells to mount an immune response 20 . Self reactive B cells and T cells can turn the immune system against its own body to cause various autoimmune disorders 5 .

The innate immune response is carried out by neu trophils and plasmacytoid dendritic cells (pDCs), baso phils, natural killer cells, innate lymphoid cells and granulocytes 21 . These cells express various cytokines and selected receptors and ligands to mount an immune response 21 . Cytokines and other inflammatory medi ators function as messengers that bind to specific receptors to regulate immune response 1 . The TNF superfamily contains 19 members that bind 29 recep tors that are expressed predominantly by immune cells and function as cytokines regulating diverse cellular functions, including immune response and inflam mation 22 . The IL1R family comprises ten members 23 , and includes several ligand binding receptors (IL1R1, IL1R2, IL1R4, IL1R5, IL1R6), two types of accessory chain (IL1R3, IL1R7), molecules that act as inhibitors of IL1 and IL18 cytokines (IL1R2, IL1R8, IL18BP) and two orphan receptors (IL1R9, IL1R10) with no known ligand 23 . The majority of the receptors from the IL1R family promote activation, proliferation, differen tiation and production of pro inflammatory cytokines from various cell types 23 . IL6 is a pleiotropic cytokine implicated in several diseases, including arthritis, sepsis, anaemia of chronic diseases, angiogenesis acute phase response, bone and cartilage metabolism disorders and cancer 24 . IL6 binds IL6R, which has two subunits, IL6Rα and IL6Rβ (also known as gp130). Cells only express gp130 and are not responsive to IL6 alone, but they can respond to a complex formed by IL6 bound to a naturally occurring soluble form of the IL6R, a process known as trans signalling and that controls the pro inflammatory responses of IL6 (ReF. 24 ). The dis covery of the IL6-IL6R axis provided a foundation to understand the biology of a group of related cytokines, including the IL12 family of cytokines (IL12, IL23, IL27, IL35), which use shared receptors and cytokine subunits 25 . IL12 is produced by innate cells, such as macrophages and dendritic cells, and binds to a hetero dimeric receptor formed by IL12Rβ1 and IL12Rβ2, which promotes development of IFNγ producing T H 1 cells from naive T cells 26 . IL23 is also produced by innate cells and signals through the IL23R and the shared subunit IL12Rβ1 (ReF. 27 ). IL23 is a hetero dimeric cytokine formed by the p19 and p40 subunits that binds the IL12Rβ1 and IL23R receptor complex expressed by several cells (natural killer cells, macro phages, dendritic cells, memory T cells and keratino cytes). Comparing the phenotypes of mice deficient in IL23 or IL12 receptor and ligand subunits established that IL23 is a main culprit in autoimmune disease models 27, 28 . IL23 facilitates the production of IL17 in T H 17 cells and acts on cellular targets -including keratinocytes, neutrophils, endothelial cells and fibro blasts -to stimulate production of various chemokines and cytokines, which, in turn, promote tissue inflam mation 28 . Correspondingly, the blockade of IL17 or IL23 is effective in managing the symptoms of certain diseases, such as psoriasis 28 .

IgG Fc receptors (FcRs) bind to antibodies to clear infected cells or invading pathogens 29 . This complex medi ates inflammatory signalling via the immuno receptor tyrosine based activation motif (ITAM) in phagocytic or cytotoxic cells to destroy microbes, or in infected cells by antibody mediated phagocytosis or antibody dependent cell mediated cytotoxicity 29 . In similar fash ion, autoantibodies and autoimmune complexes (auto antibody bound to self antigen) may serve as pathogenic factors in autoimmune or inflammatory injury, as they are responsible for the initiation of the inflammatory cascade and its resulting tissue damage 29 .

Toll like receptors (TLRs) are sensors of microbial antigens that recognize pathogen associated molec ular patterns (PAMPs), which are conserved structures found on microbial cell walls that activate the host innate immune response 30 . TLRs can also recognize damage associated molecular patterns (DAMPs) that are generated in the host following tissue injury or cellular activation 30 . There are ten TLRs identified in humans (TLR1-TLR10). Most TLRs are expressed on the cell surface and recognize antigens present on bacterial outer membranes. TLR3, TLR7, TLR8 and TLR9, however, are expressed intracellularly in endosomes and recog nize nucleic acid ligands from various sources, includ ing viruses or DAMPs 30 . Excessive TLR activation by DAMPs or PAMPs disrupts the immune homeostasis by sustained pro inflammatory cytokine production, and consequently contributes to the development of several inflammatory diseases 30 .

Plasmacytoid dendritic cells (pDCs). Specialized, immunomodulatory dendritic cells that produce large quantities of type I interferons in response to viral antigens, with limited capacity to present such antigens to T cells.

Antibodies reactive against an individual's own tissues or organs.

Targeting kinases in immunity Signalling from multiple cytokine receptors converges on a few kinases -such as JAK1 -which has made kinases potential targets to disable inflammation in a targeted fashion 13 . In cases such as JAK1, even partial target inhibition is sufficient to reduce several patho genic pathways simultaneously in the clinic 13 . Other kinases with restricted or preferred immune cell func tion are emerging as promising drug targets to alleviate dysregulated inflammation with reduced side effects. For example, the non canonical NF κB pathway is less universal and integrates signalling cascades down stream of selected immune receptors that are validated as attractive drug targets, such as CD40 and BAFFR (also known as tumour necrosis factor receptor super family member 13C (TNFRSF13)) in immunological disorders 15 or NF κB inducing kinase (NIK; also known as MAP3K14). In addition, select kinases (such as MAP3K8, also known as tumour progression locus 2 (TPL2)) participate in positive feedback loops in which kinase dependent production of pathogenic mediators re engage the original signalling cascade to activate the same kinase. TPL2 is transcriptionally induced and acti vated by the inflammatory receptors, including multi ple TLRs, TNF receptor 1 (TNFR1), IL1R1 and IL1R2 (ReF. 31 ). TPL2 also amplifies local inflammation by pro moting the production of TNF and IL1, which bind and activate their corresponding receptors 31 . Therefore, inhibition of TPL2 may disrupt the feedback loop and dampen such pathogenesis in diseases such as RA, IBD and psoriasis 31 .

It should be emphasized that challenges remain for targeting kinases and the success rate for the genera tion of selective small molecule inhibitors is low. This is because most kinase inhibitors are aimed to bind to the kinase pocket to compete with the ATP binding site, which is highly conserved across the kinome. In addi tion, signal transduction for some kinases may extend beyond its activity, with additional roles in creating structural docking hubs (such as IRAK4 as discussed below) 32, 33 . Another obstacle is the complexity of each signalling pathway in different cell types, which is not well understood in humans.

IRAK4 functions downstream of several innate immune cell receptors, such as TLRs and IL1Rs. Most recently, for the first time, positive clinical data have been reported with IRAK4 inhibitors in patients with RA, opening an opportunity for probing this tar get across several other inflammatory indications 34 . Receptor interacting serine/threonine protein (RIP) kinase inhibitors are under investigation in patients with RA, ulcerative colitis and neurodegenerative dis eases; multiple Bruton's tyrosine kinase (BTK) inhibi tors (BTKis) are being evaluated in lupus and RA 35 ; and a TPL2 inhibitor is advancing in clinical studies with promising potential for multiple inflammatory diseases 36 . Several inhibitors of tyrosine protein kinase SYK are currently being evaluated for the treatment of autoimmune haemolytic anaemia, IgA nephropathy and chronic spontaneous urticaria 37 . Beyond these advanced targets, the emerging genetic and functional data plus the availability of experimental tool kinase inhibitors support the utility of other kinases, including p38δ, p38γ, IL2 inducible T cell kinase (ITK), NIK, TANK binding kinase 1 (TBK1), inhibitor of NF κB subunit ε (IKKε), cyclin dependent kinase 8 (CDK8) and CDK19.

In the following sections, we provide an overview of several kinases that are positioned in key inflamma tory cascades and their utility as drug targets in various inflammatory diseases.

The JAK family of kinases includes JAK1, JAK2, JAK3 and non receptor tyrosine protein kinase TYK2 (ReF. 13 ). JAKs transduce signals from many cytokine receptors of the interleukin and interferon families as well as from growth hormone and erythropoietin (EPO) 13 (FIg. 2) . JAKs transduce signalling of IL2R, IL4R, IL5R, IL6R, IL13R and type I interferons, which all have been vali dated as pathogenic pathways in different diseases such as RA and asthma. TYK2 is activated downstream after receptor binding by IL23, IL12 and type I interferons, each of which are implicated in the pathogenesis of multiple inflammatory diseases 38 . Dedicated combina tions of STAT family members (STAT1-STAT6) unique to each receptor and the associated docking sites are recruited and phosphorylated by JAKs, leading to STAT dimerization and subsequent nuclear translocation for gene regulation 13 (FIg. 2) .

JAKis, both reversible and irreversible, have been advanced to clinical evaluation with varying degrees of selectivity 13 (FIg. 2) . Covalent inhibitors bind irreversibly to kinase pockets or to the adjacent chemically reactive amino acid (usually cysteine, lysine or aspartic acid) to form a bond and block activity (TABle 1) . Tofacitinib was the first JAKi approved for the treatment of RA and inhibits JAK3, JAK1 and, to a lesser degree, JAK2 (ReF. 13 ). Baricitinib is also approved for RA and inhibits JAK1 and JAK2 (ReF. 39 ). Peficitinib is a pan JAKi awaiting more clinical data 13 . PF06651600 is the only irreversible cova lent JAK3i tested for the treatment of RA, alopecia areata and ulcerative colitis with breakthrough designation 13, 40 . PF06651600 is unique among JAKis in that it inhibits only cytokine receptors that use the common γ chain. PF06651600 has selectivity for JAK3 owing to its cova lent interaction with a cysteine residue (Cys909) in the catalytic domain of JAK3, which, in the other JAK isoforms, is replaced by a serine residue 13 . Figlotininb (Gilead) is a selective JAK1i that is effective in modula ting the immune response (measured by cellular and serum biomarkers) and achieving clinical response in patients with moderate to severe RA 41 . JAK3 may be a potential target in asthma and organ transplants based on preclinical model studies that have described its selective activation downstream of IL4, IL15 and IL21 recep tors, all of which use the common γ chain [42] [43] [44] (FIg. 1) . PF06651600 can also inhibit the tyrosine protein kinase TEC family including BTK, bone marrow kinase (BMX), ITK, resting lymphocyte kinase (RLK) and Tec protein tyrosine kinase (TEC) by binding similarly to the Cys909 shared in the binding pocket of the kinase domain 40 . The inhibition of TEC kinases might expand the mechanism of action of JAK3 to other cell types, such as lymphocytes (see below). JAK2 is activated downstream of receptors

A process designed to expedite the development and review of drugs that are intended to treat a serious condition and for which preliminary clinical evidence indicates that the drug may demonstrate substantial improvement over available therapy on a clinically significant end point(s).

for the cytokines thymic stromal lymphopoietin (TSLP), IL13, IL23 and IFNγ (FIg. 1 ). Ruxolitinib inhibits JAK2 and, to a lesser extent, JAK1 and JAK3, and is approved for myelofibrosis and polycythemia vera 45 . Fedratinib targets JAK2 and was recently approved for myelofibro sis, showing significant improvements in symptoms and a reduction in spleen size 46 .

Cytokine antagonists have provided a precedent for the utility of selective JAKis in the clinic. The use of tezepelumab, an antibody against TSLP in adults with uncontrolled asthma, suggest that selective JAK2 inhibi tion might be beneficial 47 . In addition, given the poten tial side effects of systemic JAKis, other localized routes of administration of JAKis with unique biophysical properties (such as those restricted to the gut, or topi cal or inhaled routes) may be beneficial in intestinal 48 , dermatological 49, 50 or respiratory 33 diseases, although determining the dosing regimens may be challenging and it is not clear whether partial inhibition of systemic JAKi is sufficient in all of these indications 49, 50 .

A coding variant of TYK2 that protects from multiple autoimmune diseases 38 leads to the substitution of a pro line residue with alanine at position 1104 (P1104A) in the catalytic domain, preventing receptor mediated acti vation. This finding has enabled rationale designs to tar get TYK2 (ReFS 51,52 ), such as BMS986165, which blocks the receptor mediated activation of TYK2 allosteri cally, in a mechanism similar to that of the deactivating TYK2 P1104A coding variant. BMS986165 targets the pseudokinase domain of TYK2, which is promising in the discovery of a selective inhibitor of TYK2 that lim its off target effects in other kinases, particularly of the related JAK kinases. BMS986165 blocks IL12 and IL23 signalling in human cells and also prevents type I inter feron signalling, which showed protection from disease in mouse models of colitis or SLE 38 . BMS986165 was well tolerated in healthy volunteers during a phase I trial and dampened responses to an in vivo interferon challenge 38 . It was also beneficial in psoriasis in a phase II study, with a large phase III programme currently ongoing 53 . Another selective TYK2 inhibitor, PF06826647, is also being tested in moderate to severe psoriasis in an ongo ing phase II clinical trial (NCT03895372). Furthermore, another inhibitor that targets both TYK2 and JAK2, PF06826647, is also being tested in moderate to severe psoriasis in a phase II clinical trial (NCT03895372). IL-13   IL-2, IL-7,  IL-9, IL-15   JAK1 JAK3   EPO, TPO,  GH   JAK2 JAK2   IL-3, IL-5,  GM-CSF  IL-6   JAK1 JAK2   TSLP, LIF,  IL-11, OSM  IL-12, IL-23   Type I  interferons  (α/β)   IFNγ,  IL-10, IL-19, IL-20,  IL-22, IL-24, IL-26,  IL-28, IL-29   JAK2  JAK1  JAK2   TYK2  JAK2 JAK2  JAK1  JAK2   TYK2  JAK1  JAK2   TYK2 

A kinase-like domain that lacks at least one of the conserved catalytic residues.

www.nature.com/nrd JAK inhibition may benefit the management of COVID19 patients by reducing JAKdependent cytokine storms (such as that mediated by IL6 or IL12) 3 . Baricitinib could also impair SARSCoV2 endocytosis by inhibiting AP2 associated kinase 1 (AAK1) and the cyclin G associated kinase (GAK) kinases 54 . Several clini cal trials assessing the efficacy of JAKis in COVID19 are ongoing, including baricitinib, ruxolitinib, tofac itinib and the nebulized TD0903 molecule from Theravance Biopharma 3 . In an initial open label, small trial (NCT04358614, 12 patients), baricitinib treated patients achieved significantly greater improvements in clinical symptoms, lung function and hospitalization 55 . If successful, JAKis might be consi dered for patients with non COVID19induced ALI and ARDS. It is crucial to identify companion predictive and diagnostic biomark ers to improve the diagnosis and treatment of patients with ALI and/or ARDS 2 .

IL1Rs and TLRs share a conserved Toll/IL1R recep tor (TIR) domain on the cytoplasmic tails of each receptor, and therefore categorically use similar signal ling pathways 30 . The TIR domain on all IL1R family members (except for TLR3) recruits the TIR domain found on the carboxy terminus of myeloid differenti ation primary response 88 (MyD88) 30 . On its amino terminus, MyD88 contains a death domain that recruits respective death domains found on IL1R associated kinases (IRAKs), and together they form a signalling complex called the Myddosome 56,57 (FIg. 3) .

As with most kinases, IRAK activity is modulated, in part, by conformational changes and post translational modifications 30, 58 . At the Myddosome, IRAK4 is acti vated via trans autophosphorylation to then activate IRAK1 by phosphorylation 58 (FIg. 3) . Activated IRAK1 and TNFR associated factor 6 (TRAF6) dissociate from the Myddosome and activate TGFβ activated kinase 1 binding protein 1 (TAK1), a member of the MAPK kinase family [59] [60] [61] . TAK1 activates IKKβ in the IKK com plex, which phosphorylates NF κB inhibitor α (IκBα), resulting in MAPK activation and NF κB regulated transcription 61, 62 (FIg. 3) . IRAK2 may not be required for receptor mediated NF κB activation, but it has been observed that IRAK2 mediated post translational modi fications are important for mRNA stability and trans lation by facilitating nuclear export of NF κB regulated transcripts [63] [64] [65] . IRAK3 lacks kinase activity owing to the absence of an aspartate residue at the active site and, instead, inhibits IRAK signalling by binding to Nature reviews | Drug Discovery and arresting IRAK4, IRAK1 and IRAK2 in inactive states [66] [67] [68] [69] . TLR7, TLR8 and TLR9 induce the produc tion of both NF κB dependent cytokines as well as type I interferons 70 . In pDCs, MyD88 forms a complex with IRAK1, TRAF6, TRAF3, IKKα and IRF7. In this complex, IRAK1 may directly activate IRF7 to drive the expression of type I interferons. In conventional dendritic cells, activation of TLR7 and TLR9 results in IRF1 mediated IFNβ gene expression 71, 72 . Mutations in MYD88, IRAK4 or IRAK1 found in patients have revealed essential roles of these proteins in host defence. Patients with an autosomal recessive disorder who are deficient in IRAK4 or MYD88 are equally susceptible to a subset of pyogenic bacterial infections, but are resistant to other infections, including other bacteria, most viruses, fungi and parasites 73 . As the first kinase in the receptor signalling cascade, IRAK4 kinase activity is most critical in activating pathways downstream of IL1R family members (FIg. 3) and, therefore, is a prime target candidate for the treatment of several inflamma tory diseases [59] [60] [61] 74 . Mutations in the kinase domain in IRAK4 that abrogate its activity protect mice in sev eral inflammatory disease models, including septic shock 63,75-77 , SLE 78-80 , acute liver injury 81 , cardiovascular disease 82 and the APPPS1 Alzheimer disease model 83 .

The endosomal receptors TLR3, TLR7, TLR8 and TLR9 cannot discriminate between self and foreign nucleic acids, and therefore can pose serious threats to the development of autoimmunity. SLE development is attributed to the activation of endosomal TLRs. IRAK4 inhibition using BMS986126 in preclinical models of lupus (MRL/lpr and NZB/NZW) demonstrated strong attenuation of disease symptoms and minimal off target effects 78 . Similarly, IRAK4 inhibition using PF06650833 in patients with RA showed significant improvements in disease severity 34 . Interestingly, deletion of IRAK1 resulted in only a partial loss of signalling in immune cells in vitro 76, 84 . Only one IRAK1 deficient immuno compromised patient with deletions of several nearby genes has been reported 85 . In contrast to IRAK4, IRAK1 seems to be redundant downstream of the IL1R in human fibroblasts in vitro. However, IRAK1 is essential for signalling downstream of TLR2, TLR6, TLR4, TLR7 and TLR8 in fibroblasts as well as in mature B cells. Therefore, the functions of IRAK4 and IRAK1 may be cell type and receptor specific 86, 87 . Interestingly, com pared with IRAK4 deficiency in humans, which confers susceptibility to a few bacterial infection and decreases with age, Irak4 deficient mice are susceptible to multi ple bacterial, viral, fungal and parasitic infections at all ages 88 .

IRAK2 may partially compensate for a loss in acti vity of other IRAKs 63, 76, 89 . IRAK2 is required for the production of pro inflammatory cytokines 89, 90 where IRAK2-TRAF6 interaction becomes rate limiting after IRAK1 is degraded from the cells after prolonged TLR stimulation 89 . Allosteric inhibitors that disrupt the inter action between IRAK2 and TRAF6 may hold potential for therapeutic development in inflammatory diseases such as RA and SLE.

The precise mechanisms of IRAK4 signal transduc tion are still being studied. Leukocytes and fibroblasts from several IRAK4 deficient patients showed that IRAK4 kinase activity was more essential for TLRs and IL1R mediated signalling in innate immune cells than in fibroblasts 91 Fig. 3 | irAK4 is the upstream kinase that transduces TLrs and iL-1r signals. IL-1 receptor (IL-1R)-associated kinase 1 (IRAK1) and IRAK4 function is regulated by proteinprotein interactions and by post-translational modifications that may be uniquely regulated in different cell types to fine-tune an immune response. IL-1R or Toll-like receptor (TLR; except for TLR3) engagement causes the recruitment of adaptor protein myeloid differentiation primary response 88 (MyD88) to the intracellular Toll/IL-1R receptor (TIR) domains to initiate the Myddosome assembly. MyD88 recruits IRAK4 via death domain interactions. IRAK4 is activated via autophosphorylation and is also K63-ubiquitylated (grey circles). Phosphorylated IRAK4 can activate IRAK1, which facilitates IRAK1-tumour necrosis factor receptor-associated factor 6 (TRAF6) complex formation. K48 ubiquitylation (blue circles) of IRAK1 is required for the activation of TGFβ-activated kinase 1-binding protein 1 (TAK1), and its binding to TAB1 and TAB2 to drive the formation of the inhibitor of NF-κB (IKK) complex and subsequent NF-κB inhibitorα (IκBα) activation, which then leads to NF-κB, mitogen-activated protein kinase (MAPK) and interferon-regulatory factor (IRF) activation to induce the transcription of pro-inflammatory cytokines and cellular processes, such as proliferation and activation 61, 62 .

The mechanism of IRF activation by MyD88 is less understood. Downstream of IRAK4 activation, TAK1 and IKKβ complex can mediate IRF5 phosphorylation, nuclear translocation and transcription in monocytes 74 , but IRAK1 can also directly bind and phosphorylate IRF7 in plasmacytoid dendritic cells 263 . The mechanism of IRAK4 kinase activity-independent action is less understood but may involve K63-ubiquitylated signalling hubs and other novel molecular scaffolds 92 . AP-1, activator protein 1; CREB, cAMP response element-binding protein; P, phosphorus; TAB, TGFβ-activated kinase 1 binding protein; Ub, ubiquitin.

www.nature.com/nrd that facilitates IRAK1 and IRAK4 oligomerization is K63 ubiquitylation 92 (FIg. 3 ). During ubiquitylation, the C terminus of ubiquitin (Ub) is covalently attached to the lysine residue of substrate proteins. First, the Ub moiety is activated by E1 enzymes (also known as Ub activating enzymes). Following activation, an E2 Ub conjugating enzyme (UBC) transfers Ub from E1 to an E3 enzyme (also known as Ub ligases) to which the substrate protein is specifically bound. The Ub moiety contains several lysine (K) residues (such as K48 and K63) and a methionine at the N terminus (M1), which can attach another Ub to form a polyUb chain. K48 linked polyUb chains control targeting of a substrate to 26S pro teasomes for degradation. K63 linked polyUb chains provide additional structural scaffolding for proteinprotein interactions, which can facilitate signalling. IRAK1 has 31% sequence identity to IRAK4, and con tains two important lysine residues in the linker domain that are required for K63 linked polyUb chains, which is essential for activation of NF κB 92 . Receptor engagement has been shown to induce K63 linked polyUb chains on several subunits of the Myddosome, suggesting that this type of modification may be of particular importance in this pathway 92 . Because IRAK4 provides structural integ rity to the Myddosome, it is possible that IRAK4 kinase activity dependent and activity independent mecha nisms work in parallel to facilitate cytokine production downstream of the Myddosome 76,93,94 . Therefore, further characterization of post translational modifications of IRAK4 may yield kinase independent and cellspecific mechanisms of signalling. It is unclear why TLRs and IL1R have evolved to be uniquely complex across cell types and species. It is pos sible that different mechanisms of activation can facili tate the fine tuning of a complex immune response. Immune cells may be more sensitive to catalytic kinase activity in order to trigger, as well as terminate, the immune response, and other cell types such as epithelial cells or fibroblasts might rely less on kinase activation to tran siently control inflammation and avoid collateral tissue damage. Several IRAK4 inhibitors are currently being evaluated in the clinic, including PF06650833, BAY 1834845, CA4948 and R835 (FIg. 1) . PF06650833 was shown to improve clinical scores in a phase II trial in patients with active arthritis that is not responding to methotrexate 34 . There are no reports on IRAK1 or IRAK4 ubiquitylation specific modules that alter TLR or IL1R signalling in the clinical stage.

Receptor interacting protein kinases (RIPKs) are critical regulators of cell death and inflammation with important roles in the maintenance of tissue homeostasis 95 (FIg. 4) . RIPKs exert multiple signalling functions through their kinase activity, protein binding and post translational modifications 96 . Dysregulation of RIPK functions can lead to unbalance in multiple signalling pathways and cause severe inflammatory conditions, suggesting that these kinases are important sentinels of human health 95 .

RIP1 (also known as RIPK1) is a seminal component of TNF signalling that mediates proliferative NF κB and MAPK activation as well as apoptotic and necroptotic cell death pathways 97 (FIg. 4) . Necroptosis is referred to as a regulated form of necrosis or inflammatory cell death. Typically, necrosis or cellular injury is associated with unprogrammed cell death that results from cellular damage or invasion by pathogens, in contrast to orderly, programmed cell death via apoptosis 95 . Binding of TNF to the TNFR1 triggers the recruitment of adaptor proteins TNFR associated death domain (TRADD), TRAF2 and RIP1, and the Ub ligases inhibi tors of apoptosis 1 and 2 (IAP1/2) 98 . Ubiquitination of RIP1 mediated by IAP1/2 by addition of K63 linked and K11 linked polyUb chains leads to recruitment of additional signalling complexes, including the IKK complex (which includes NF κB essen tial modulator (NEMO)), TAK1 associated with TAB2 and TAB3, and the linear ubiquitin chain assembly complex (LUBAC; which consists of E3 ubiquitin protein ligase HOIP and cofactors HOIL1 and Sharpin) 96 . Linear ubiq uitylation of RIP1 and several other TNFR1 associated proteins further enhance TNF stimulated NF κB and MAPK signalling. Conversely, deubiquitinases A20 and CYLD function to disassemble such polyUb chains to limit NF κB and MAPK activation in order to promote cell death [99] [100] [101] .

TNF induces cell death via apoptosis and necroptosis, which largely depends on RIP1 and involves one trans location to the cytosol, where it binds FAS associated death domain (FADD) and caspase 8 (and caspase 10 in some cases) 98 (FIg. 4) . Caspases are effectors of apoptotic cell death; however, in cases in which caspase 8 is inhibi ted or insufficiently activated, RIP1 can engage RIP3 in a kinase dependent and RIP homology interaction motif (RHIM) dependent manner to form a necrosome 98 . Autophosphorylation of RIP1 and then RIP3 results in their full activation and leads to RIP3 mediated phos phorylation of the pseudokinase mixed lineage kinase domain like protein (MLKL), triggering its oligomeri zation and translocation to the plasma membrane to induce necroptotic cell death 102 .

RIP1, and to a lesser extent RIP3, are also polyubiq uitylated within the necrosome and dynamic changes in their ubiquitination status influence cell death and inflammatory responses 103, 104 . Overall, differential phos phorylation and ubiquitylation of RIP1 by numer ous kinases (TAK1, IKK2 and MK2; also known as MAPKAPK2) and E3 ligases (IAP1/2 and LUBAC) allows for spatial and temporal regulation of the transi tion from TNFR1 associated complexes, in which RIP1 is inactive, to the multiple cytoplasmic complexes where RIP1 kinase activity is crucial for activating programmes of cell death 96 .

Both the discovery of RIP1 specific kinase inhibi tors (necrostatins, GNE684 and other classes) and the lack of detrimental phenotypes observed in RIP1 kinase dead mice have suggested that RIP1 is a promising clinical target [105] [106] [107] . Such is not the case with RIP3, as RIP3 inhibitors can activate apoptotic cell death and genetic RIP3 kinase inactivation is lethal 106, 107 . Although targeting RIP1 activity represents an appealing strat egy for the treatment of inflammatory pathologies and tissue damage, RIP1 relevance for various diseases must first be better characterized, as tissue specific and/or cellular specific processes that lead to RIP1

A condition characteristic of primary immunodeficiency due to myeloid differentiation primary response 88 deficiency in which patients have increased susceptibility to infections owing to their inability to signal through Toll-like receptors to activate inflammation.

Nature reviews | Drug Discovery kinase activation do not always behave similarly 108 . For example, ischaemia-reperfusion kidney injury or myocardial infarction can be ameliorated by RIP1 kinase inhibition or inactivation in rodents 108 . Similarly, in Rip3 knockout mice, and to a lesser extent in Mlkl knockout mice, the absence of RIP3 and MLKL promotes survival and reduces nephropathy in kidney injury models, indicating a critical role for necroptosis in kidney tissue damage 108, 109 . Similarly, in mice, RIP1 inhibition ameliorates collagen antibody induced arthri tis, skin inflammation caused by mutant Sharpin or colitis caused by intestinal deletion of Nemo 105 . In skin inflammation models, necroptosis also plays an impor tant part in RIP1 inhibition as RIP1 inhibition reduces skin inflammation and phosphorylation of necrop tosis markers RIP3 and MLKL 110, 111 . Conversely, RIP1 inhibition does not affect tumour growth or survival in pancreatic tumour models driven by mutant KRAS, or lung metastasis in a B16 melanoma model 112 .

Another interesting feature of RIP1 kinase activity is its involvement in the interplay of multiple cell death and survival pathways. Although kinase activity of RIP1 is clearly instrumental for necroptotic cell death, it has also been implicated in TNF induced apoptosis when key NF κB signalling kinases (such as TAK1 or IKK) are inhibited or deleted 113, 114 . Caspase 8 negatively impacts RIP1 activation [115] [116] [117] but it is still unclear how RIP1 kinase activity regulates caspase activation. It is possible that a threshold of caspase activation determines which regu lated cell death pathway is activated, but RIP1 mediated apoptosis and necroptosis are unlikely to be mutually exclusive. Additionally, defective autophagy caused by and mixed-lineage kinase domain-like protein (MLKL)) to cause tissue damage and an inflammatory milieu. RIP1 inhibition can block RIP1-mediated apoptosis and necroptosis, and reduce inflammation by inhibiting inflammatory cell death. The kinase domain of RIP2 allows the binding of E3 ligase X-linked inhibitor of apoptosis protein (XIAP) and subsequent RIP2 ubiquitylation, which is a critical mediator of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) inflammatory signalling. Consequently, RIP2 kinase inhibitors prevent XIAP binding and RIP2 ubiquitylation to inhibit NOD2 pathway-activated NF-κB and mitogen-activated protein kinase (MAPK) signalling, and consequent production and release of pro-inflammatory cytokines, thus blocking inflammation. FADD, FAS-associated death domain; IAP1/2, inhibitor of apoptosis 1 and 2; LUBAC, linear ubiquitin chain assembly complex; MDP, muramyl dipeptide; P, phosphorus; TNFR1, TNF receptor 1; TRADD, TNFR-associated death domain; TRAF2, TNFR-associated factor 2; Ub, ubiquitin.

www.nature.com/nrd deletion of the gene encoding for autophagy related protein 161 (ATG16L1) in intestinal organoids or the intestinal epithelium of mice results in TNF driven necroptotic death, which can be rescued by RIP1 kinase inhibitors 105, 118 . Therefore, RIP1 kinase activity can aggre gate multiple signalling inputs that induce regulated cell death to mediate tissue damage and inflammation. This is evident in both acute and chronic animal disease models. In the acute kidney injury model or the TNF induced shock model (systemic inflammatory response syn drome), genetic or chemical inhibition of RIP1 kinase efficiently blocks tissue damage 105 RIP2 does not participate in cell death signalling but, instead, is a mediator of NOD1 and NOD2 inflamma tory signalling 122 (FIg. 4) . NOD2 is mutated in numerous inflammatory diseases such as Crohn's disease, Blau syndrome and very earlyonset sarcoidosis 123 . NOD2 recognizes bacterial peptidoglycans, such as muramyl dipeptide (MDP) that results in NOD2 activa tion and subsequent recruitment of RIP2 and its E3 ligase, XIAP 122, 124 . XIAP (and potentially other E3 ligases) promotes K63 linked ubiquitylation of RIP2, which enables LUBAC mediated linear ubiquitylation of RIP2 and subsequent activation of NF κB and MAPK to promote cytokine and chemokine production 96 . The kinase domain of RIP2 serves as a docking module that enables XIAP to bind 125 . Mutational inactivation of RIP2 kinase activity had no effect on RIP2mediated NOD2 signalling as it did not prevent RIP2 binding to XIAP 125 . However, RIP2 kinase inhibitors that disrupted RIP2-XIAP interactions were successful in blocking NOD2 signalling 125, 126 . This is a rare example of a kinase whose enzymatic activity is not needed for its biolo gical role. Instead, RIP2 ubiquitylation by XIAP enables the assembly of signalling complexes and stimulation of inflammatory responses 124, 125 . Therefore, targeting RIP2 kinase to disrupt the interaction between RIP2 and XIAP may be effective in NOD2 mediated diseases.

Tec family kinases are primarily expressed in haemato poietic cells and have important roles in the develop ment and function of leukocytes downstream of SRC and SYK 127 . Among Tec kinases, BTK and ITK are attrac tive drug targets given their established roles in B cell and T cell activation, respectively 128 . In addition, BTK also regulates Fcε receptor (FcεR) signalling in mast cells, positing it as an attractive drug in IgE mediated diseases such as allergy, asthma and atopic dermatitis 128 .

In T cells, ITK positively regulates TCR signalling to induce the production of IL2, IL4 and IL13 (ReFS 127,129 ) (FIg. 5a) . When peptide MHC binds to the cognate TCR, ITK is directly phosphorylated by the tyrosine protein kinase LCK, and subsequently undergoes autophos phorylation. ITK associates with the LAT-SLP76 com plex through its two SRC homology domains SH2 and SH3, creating a signalling complex that is dependent on upstream LCK and Zap70 signalling 127 . ITK is then able to phosphorylate phospholipase Cγ (PLCγ), which cleaves phosphatidylinositol 4,5 bisphosphate (PIP 2 ) at the plasma membrane, generating the secondary messen gers inositol trisphosphate (IP3) and diacyl glycerol (DAG). IP3 and DAG primarily activate NFAT and calcium signalling, which targets gene promoter activation including IL2, IL4 and IL13 (FIg. 5a) .

Comparing Itk knockout versus ITK inhibitor stud ies has revealed novel insights into ITK function 130 . ITK plays a critical part in priming T cells; however, in rechallenged antigen experienced T cells, ITK regu lates activation induced cell death 130 , highlighting dif ferences between Itk knockout and kinase inhibitor studies 130 . Activation induced cell death is a mecha nism of programmed cell death evolved to dampen an ongoing immune response and involves interactions of TNFRSF6 (also known as FAS and CD95) and its ligand TNFL6 (also known as FASL or CD95L) on neighbour ing cells 131 

This surprising finding has drawn a new paradigm for the utility of this target 130 . Therefore, although the inhibition of ITK may not be beneficial in the treat ment of asthma, this mechanism of ITK activity may be bene ficial in cancer immunotherapy to promote T cell survival 132 . JTE051 is the only ITK inhibitor under clinical evaluation in the treatments of RA and psoriasis (NCT03358290).

BTK integrates BCR signalling to regulate B cell development (FIg. 3b) . Mutations that inactivate BTK block B cell development causing X-linked agammaglobulinaemia 133 . Types I and III interferon production are impaired in BTK deficient patients during viral infections such as polio, but not during influenza 134 . Different isotypes of immunoglobulins exert their effector functions, in part, by binding to the respective FcRs 135 . IgE antibodies bind FcεR on mast cells and basophils to trigger degranulation and acute inflammation 135 , whereas IgG binds FcγR on macro phages, pDCs and natural killer cells to promote cellu lar activation or phagocytosis 135 . In certain autoimmune diseases, self reactive IgG binds to self antigens, such as nucleic acids in SLE, and forms immune complexes 135 . BTK positively regulates FcεR signalling in mast cells and basophils [136] [137] [138] and FcγR signalling in macrophages or pDCs to internalize and deliver immune complexes 139 (FIg. 3b) . TLR induced B cell differentiation and prolif eration is dependent on BTK, whereas the function of BTK in TLR signalling in myeloid cells is not well understood 140 (FIg. 3b) . In B cells, BCR activation exposes the ITAM to LYN/SYK/BTK, which activates PLCγ2 and phosphatidylinositol 3 kinase (PI3K). Active PLCγ2 and PI3K allow for calcium signalling, which is required for the activation of transcription factors, such as NFAT and NF κB, that regu late proliferation,

A genetic immunosuppressive condition that results in a severe reduction in the number of B cells and, thereby, in the production of protective antibodies. Patients with this condition are predominantly males and are at greater risk of recurrent opportunistic infections.

Nature reviews | Drug Discovery survival and cytokine expression 141, 142 (FIg. 5b) . Similar to BTK deficient mice, PLCγ2 deficient mice have defects in B cell development 143 . These mice also have defective FCεR and FCγRII/III signalling in mast cells and natu ral killer cells, respectively, but macrophage function and numbers are not altered 143 . The function of BTK in pDCs is less understood; however, one report suggests that BTK regulates TLR9, but not TLR7, signalling in human pDCs 144 . These overlapping phenotypes support the idea that BTKis could effectively target B cell dif ferentiation, mast cell and basophil associated immune pathologies but no other myeloid cells.

Cell specific BTK activity may be determined by post translational modifications. Phosphorylation at Y551 is important in FCεR and FCγR signalling whereas Y223 activation is essential for BCR signalling 145 (FIg. 5b) . In preclinical rodent models, BTK inhibition is pro tective against the development of arthritis or SLE by reducing autoantibody production and inflammatory cytokines 139, 140, [146] [147] [148] ; encouragingly, BTK inhibition was more efficient than BAFF blockade or SYK inhibition 140 . However, animal models are, unfortunately, often not representative of human disease and are likely to empha size limited pathways in disease progression. For exam ple, NZW/NZB F1 mice are an SLE like model that is highly B cell dependent 11 . Not surprisingly, treatment with either anti CD20 B cell depletion or BTK is pro tective in this model 140 . However, further investigation into the function of BTK in macrophages and pDCs (additional pathogenic cell types in autoimmune 149 . In this study, the authors show that in pDCs IRAK4 inhibition is more effective at blocking immune complex mediated inflammation downstream of endosomal TLRs. Several BTKis are being investigated in the clinic. In phase II trials for the treatment of relapsing multiple sclerosis, evobrutinib (a covalent BTKi) reduces enhanc ing brain lesion plaques after 3 months 150 . Branebrutinib (BMS986195, a covalent BTKi) has advanced into clini cal studies for the treatment of RA, Sjögren's syn drome and SLE 151 . Fenebrutinib (GDC0853, a rever sible BTKi) reduces disease activity in combination with methotrexate in patients with RA with an inade quate response to TNF based therapy 152 . PRN1008 is a novel reversible covalent BTKi that has shown pro mising results in phase II in pemphigus (NCT02704429) and is being further investigated in a phase III global trial (NCT03762265). Covalent BTKis often block the activities of other kinases, namely RLK, ITK and TEC 132 . Although off target effects are generally considered to increase the risk of adverse effects, select cases have demonstrated that they can be beneficial. For example, it was recently shown that specific inhibition of ITK by ibrutinib was beneficial in cancer by promoting the expansion of T cells via reducing activation induced cell death and FASL expression 130, 132 . Although the toler ance of less selective but efficacious molecules such as ibruti nib is higher in oncology, the bar for safety on daily medicines for chronic inflammatory indications such as RA and IBD is much higher, and for kinase drugs this safety is linked to selectivity. Additional clinical data that evaluate the generation of covalent or reversible BTKis should help us to better understand the function of BTK in different cell types to determine which drugs pro vide the optimal therapeutic index with minimal safety liability. Given the uncertainties around BTK activity in inflammation, combination therapy with BTK or other anti inflammatory molecules might be desired to explore in RA or SLE. BTKis are also being tested for their ability to interfere with the cytokine storm in severe COVID19 patients; preclinical studies and case series have sug gested that the BTKi ibrutinib may provide protection against severe lung injury 153 .

SYK is an Src family member essential in FcR and BCR signalling, and functions in parallel to its homologue, tyrosine protein kinase Zap70, in TCR signalling 154 , which makes it an attractive drug target in the treatments of chronic inflammation and autoimmunity (FIg. 3b) . SYK contains two SH2 domains and a Cterminal kinase domain connected by linkers A and B, respec tively. These linkers are bound together, rendering the SH2 domains inactive at steady state 154 . Receptor activation causes the release and autophosphorylation of the SH2 domains that enable docking at receptor ITAMs 155, 156 . Further phosphorylation of SYK causes it to dissociate from ITAMs and activate context specific signalling cascades (including its own degradation) via Tec family tyrosine kinases, lipid kinases, phospholi pases and guanine mediated exchange factors 154, [157] [158] [159] . SYK plays an important part in various immunologically relevant pathways, including the PI3K-AKT, Ras-ERK, PLCγ-NFAT, Vav1-Rac and IKK-NF κB pathways. Accordingly, targeting SYK has implications in several of the cellular processes that these pathways regulate, such as phagocytosis, cytokine production, degranula tion, proliferation, B cell maturation, osteoclastogenesis and platelet activation 160 (FIg. 5b) .

FcγRs expressed on myeloid cells internalize IgG opsonized particles through SYKdependent phagocy tosis and have a critical role in protective inflammatory responses 161 (FIg. 5b) . However, activation of FcγR by IgG immune complexes against autoantigens represents a key hallmark of RA, chronic spontaneous urticaria, ITP and SLE. Syk deficient mice are perinatally lethal and lack mature B cells in utero, which suggests that SYK has significant roles in general development and in the immune system 162, 163 . Syk deficient bone mar row chimaera mouse models, however, were viable and resistant to the arthritogenic serum induced model of arthritis, likely caused by both a lack of mature B cells and impaired FcγR internalization 164 .

Numerous smallmolecule inhibitors generated by Rigel Pharmaceuticals have entered clinical investiga tion (FIg. 1) . R406 (and its prodrug R788 or fostamati nib) showed efficacy in the prevention of arthritis in mice 165, 166 . In clinical trials, R406 and R788 have shown moderate efficacy in achieving the American College of Rheumatology 20% criterion, although they are less robust compared with anti inflammatory drugs such as TNF antagonists 167, 168 . Fostamatinib resulted in improved symptoms of RA likely owing to both on target and off target effects 169 ; it also resulted in several adverse side effects, and thus the development of this drug in RA was discontinued 167, 170, 171 . Fostamatinib is currently approved for the treatment of ITP, an autoimmune disease in which autoreactive IgG antibodies target and destroy platelets via macro phages through SYK dependent, FcγR mediated phagocytosis 172, 173 . Fostamatinib did not meet its primary end point in IgA nephropathy (NCT02112838) and is being further developed to treat autoimmune haemolytic anaemia (NCT04138927) and renal transplantation (NCT03991780).

Developing highly selective inhibitors is critical but is the most challenging facet of small molecule discovery. R406 is a good example of how off target activities that were not appreciated early on complicated its clinical development. In an initial in vitro selectivity assessment, fostamatinib bound to other targets (such as FLT3, LYN and LCK), but showed 5 fold to 100 fold greater inhibi tion of SYK than other tyrosine kinases when tested in a phosphorylation assay 169 . A decade later, the compre hensive pharmacological profile of fostamatinib, using a broad range of in vitro assays followed by functional and cellular assays, challenged key targets at therapeu tically relevant concentrations 174 . Using a larger kinase selectivity panel, fostamatinib inhibited 117 kinases, 100 of which had half maximal inhibitory concentra tion (IC50) values within 3 fold of the IC50 value for SYK, including FLT1, KDR (also known as vascular Pemphigus Skin disorders that cause blisters or pus-filled bumps. lesions can also form in the mucus membranes (soft linings of the eyes, nose, mouth, throat and genitals).

A standard criterion to measure the effectiveness of various arthritis medications or treatments. It means a 20% improvement in tender or swollen joint counts and other parameters. Nature reviews | Drug Discovery endothelial growth factor receptor 2 (VEGFR2)), SRC and KIT, which are associated with increased blood pressure, based on the analysis of published literature 174 . Among non kinases, antagonist activity was found against adeno sine A3 receptor 174 . Therefore, investment in gene rating a selective molecule using robust pharma cological profiling facilitates assessment of each target in the clinic with confidence. By contrast, polypharma cological effects complicate the interpretations of the clinical data to evaluate the desired target and, often, it is too costly to repeat such trials with new molecules.

IgE is commonly induced during allergic reac tions and causes the crosslinking of the highaffinity FCεRI expressed on mast cells. Following stimulation of FCεRI, SYK is immediately recruited and activated. SYK dependent activation of PI3K and AKT was shown to cause mast cell degranulation of histamine and the production of leukotrienes, prostaglandins and cytokines 175 . A study of B cell lymphomas demonstrated that a subset of malignant B cells with receptor hyper stimulation have linked SYK activity to cell survival and proliferation 176 . Gilead has developed GS9973 (ento spletinib), which has greater selectivity for SYK over R406 and is currently in clinical trials for the treatment of chronic lymphocytic lymphoma [177] [178] [179] (FIg. 1) .

MAPK: TPL2, p38γ, p38δ and ERK5 MAPKs are a highly conserved family of serine/threonine protein kinases that are induced in response to stress and inflammation and regulate proliferation, dif ferentiation, survival, apoptosis and other cellular processes 16 . MAPKs are downstream of several immune and cytokine receptors, such as TLRs, IL1R, TNFR, CSF1R and IL17R, as well as growth receptors, such as EGF, FGF and VEGF. Several inhibitors for the major MAPKs such as p38α/β, MEK1/2 and ERK1/2 have been advanced into the clinic and reviewed extensively 10, 16 . Of particular interest are MAPK kinases that have tissue or cell specific expression and prominent function in inflammation, including TPL2, p38γ and p38δ.

TPL2 is activated by several receptors, including TNFR, IL1R, TLR, CD40R, IL17R and Gαi2 transduced GPCR signals 180 (FIg. 6) . At steady state, TPL2 forms a complex with A20 binding inhibitor of NF κB (ABIN2) and only a small fraction (5% in the case of macrophages) of cellular NF κB1 (p105 subunit) 181, 182 . The kinase domain of TPL2 interacts with the death domain of p105 and this interaction blocks substrate access to the TPL2 active site 183 . Full length p105 is an REL protein specific transcription inhibitor, whereas its processed form is a 50 kDa protein (p50), DNA binding subunit of the NF κB complex 16 . Upon receptor engagement, IKKβ phosphorylates p105 at Ser927 and Ser932, leading to its degradation 16 . As a result, TPL2 is released from the complex, then autophosphorylated and/or trans phosphorylated 180 and activates targeting of its substrates MEK1 and MEK2 (ReF. 184 ) as well as MEK3 and MEK6 (ReF. 31 ) to regulate ERK1 and ERK2 or p38α and p38δ in inflammation. The TPL2 action on downstream ERK and p38 has a profound effect on the net outcome of an inflammatory response via several transcriptional and post transcriptional mechanisms 180 .

First, it can regulate gene transcription via CREB/AP1 activation 185 . The TPL2/ERK/p38 axis can determine the stability and abundance of the AU rich element (ARE) mRNAs, which is a feature of many cytokines and chemokines (such as TNF or IL6) 184 . Second, nucleo cytoplasmic localization of select genes (such as TNF) can be modulated 184 . In addition, other regulatory processes, such as CAP dependent RNA translation and protein export and processing via disintegrin and metallo proteinase domain containing protein 17 (ADAM17; also known as TACE), are regulated by TPL2 (ReF. 186 ). Thus, the net effect of TPL2 inhibition has a profound impact on inflammatory outputs without compromising the NF κB pathway (FIg. 6) . Each subunit of the TPL2 complex is essential for its stability. This was demon strated by p105 deficiency, which resulted in reduced protein levels but not transcript levels of both TPL2 and ABIN2 (ReF. 187 ), whereas TPL2 protein levels were greatly reduced in ABIN2 deficient mouse cells 16 . TPL2 defi ciency also reduces ABIN2 protein levels 16 . Accordingly, it has been challenging to determine the exact func tions of TPL2, ABIN2 or p105 in disease models until recently. TPL2 inhibition or mice expressing kinase dead mutant TPL2 have since been developed in order to probe the function of TPL2 catalytic activity in preclini cal models 31, 188 . Recent studies have shown that inhibi ting TPL2 catalytic activity is protective in preclinical models of multiple sclerosis 188 , as well as arthritis 31 and psoriasis 31 .

Tpl2 deficient mice are protected from numerous inflammatory and autoimmune diseases 180 . Analysis of mice expressing kinase dead TPL2 (D270A), in which ABIN2 expression is unaltered, showed that TPL2 kinase activity is a critical mediator in TNFR, TLR and IL1R signalling by positively regulating MAPK 31 . When challenged with a TLR agonist (such as lipopolysaccha ride (LPS)), these mice produced significantly fewer inflammatory cytokines and showed fewer immune cell infiltrates 31 . At the molecular level, TPL2 not only activates MEK1 and MEK2 but also activates isoforms p38γ and p38δ, which are key regulators of mRNA stabil ity and translation machinery for inflammation related proteins 31, 180 . This role of TPL2 is most profound in neutro phils, monocytes and macrophages 31 . This selec tive action of TPL2 on specific MAPKs is intriguing and should expand the utility of this target for several indications (see below). Recent studies with catalytically inactive ABIN2 (D310N) in gut inflammation showed that ABIN2 regulates IL1β dependent induction of cyclooxygenase 2 (COX2) and PGE2 secretion 189 , func tions previously thought to be regulated by TPL2 (ReF. 190 ). Pan kinase inhibitors, such as p38 or MEK inhibitors, suggest that the complex and often overlapping nature of different kinases in signalling would likely lead to pro blems with toxicity or inhibitor specificity 16 . Targeting an immune specific MAP3K such as TPL2 will likely maximize safety margins over broad MAPK inhibitors (such as p38i) in the clinic. Gilead has advanced its TPL2 inhibitor (GS4875) to the clinic for the treatment of ulcerative colitis 36 (FIg. 1) .

p38α, p38β, p38γ and p38δ isoforms are uniquely expressed throughout mammalian tissues 191 . Activation www.nature.com/nrd of p38 is cell type specific, receptor specific and signal strength specific 192, 193 . All p38 family members share the Thr Gly Tyr (TGY) activation motif, which is dually phosphorylated by MKK3 and MKK6 (and, in some cases, MKK4) 192 . p38α and p38β have been extensively characterized, but much less is known about p38γ and p38δ. p38γ (also known as ERK6, SAPK3 or MAPK12) and p38δ (also known as SAPK4 or MAPK13) are similar to each other (70% identity), and are both not as similar to p38α (~60% identity) 191 . Intriguingly, p38γ and p38δ regu late protein stability of TPL2 in both macrophages and dendritic cells 194 . As TPL2-ABIN2 signalling is required for LPS induced TNF and IL1β production, mice lacking p38γ or p38δ were less sensitive to septic shock and hepatitis after LPS treatment 194, 195 . Furthermore, TPL2 positively regulates p38δ, suggesting a feedback loop between inflammation and homeostatic conditions 31, 196 . In mouse models of collagen induced arthritis, defi ciency of p38γ or p38δ caused decreased serum IL17, IFNγ and autoantibody production 197 . p38δ was found to be highly expressed in neutrophils, and its deficiency in myeloid cells caused impaired neutrophil recruitment in a murine model of peritonitis 198, 199 . p38 MAPKs may also create structural scaffolds independent of kinase activity. p38γ was required for p38γ-ERK complex formation and Ras mediated oncogenic transformation 200 . 264 . Subsequently, IKKβ phosphorylates IKKα, targeting IKKα for proteasomal degradation. Then, released RelA/p50 dimers are translocated to the nucleus and modulate target gene expression. IKKβ also phosphorylates the target residues S927 and S932 in p105, leading to its proteasomal degradation that results in TPL2 liberation. IKKβ phosphorylates TPL2 at residue S400 to enhance its kinase activity. Free TPL2 then activates MEK1 and MEK2 as well as MEK3 and MEK6 to positively regulate ERK1/2 or p38α/δ to regulate gene transcription via cAMP response element-binding protein (CREB)/activator protein 1 (AP-1) as well as mRNA stability and protein production. The function of A20-binding inhibitor of NF-κB (ABIN2) is not completely understood and involves regulation of protaglandin E2 (PGE2) and cyclooxygenase 2 (COX2) in fibroblasts. TPL2 binds to a small fraction of p105 but the p50 domain of processed p105 can directly impact gene transcription 16 . Post-transcriptional regulation of cytokines and chemokines by MAPKs involves AU-rich elements (AREs) on messenger RNAs to dictate their stability (in the case of tumour necrosis factor (TNF) or IL-6, for instance) or cellular localization (TNF). In addition, other regulatory processes such as CAP-dependent RNA translation and protein export via TNFα-converting enzyme (TACE) (in the case of TNF) are regulated by the TPL2/MAPK axis. Therefore, the net effect of TPL2 inhibition has a profound effect on inflammatory outputs without compromising the NF-κB pathway. IL-1R, IL-1 receptor; P, phosphorus; TLR, Toll-like receptor; TNFR, TNF receptor; Ub, ubiquitin.

Several inhibitors against p38 have been investi gated in the clinic 10 . However, most p38 inhibitors to date (such as SB203580 and SB202190, among others) target isoforms p38α and p38β and lack inhibitory activity for p38γ and p38δ [201] [202] [203] . Targeting of p38α/β has not been very successful owing to toxicity, pleio tropic effects on various cell types, poor predictability of animal models or lack of efficacy in humans 204, 205 . A unique inhibitor of p38α, CDD450, was recently reported to selectively block p38α activation of the pro inflammatory kinase MK2 while sparing p38α activation of MAPK activated protein kinase 5 (also known as PRAK) and cAMP dependent transcription factor ATF2 (ReF. 206 ). CDD450 promotes IL1B, TNF and IL6 mRNA decay attenuating arthritis in rats 206 . CDD450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non MK2 substrates involved in anti inflammatory and housekeeping responses 206 . Thus far, only three inhibitors that effectively inhibit p38γ and p38δ have been considered for clinical eval uation. BIRB796 (which inhibits all p38 isoforms) was evaluated for RA, psoriasis and Crohn's disease, but generated hepatotoxicity and, therefore, clinical evaluation was discontinued 207 . RV568 -a p38α and p38γ inhibitor -was more effective than BIRB796 at suppressing inflammation in cell based assays, in vivo with rodent smoke emphysema models of COPD 112, 208 . Pirfenidone may target p38γ and has been approved for the treatment of IPF but is continuing to be evaluated in scleroderma associated interstitial lung disease, kidney disease, wound healing, fibrosis, cardiomyopathy and fibroids [209] [210] [211] [212] [213] [214] . SU002 and SU005 are newly identified molecules with better specificity for p38γ and p38δ but not p38α and p38β (ReF. 215 ). Although degrees of compen sation exist, understanding the regulation and functions unique to each isoform will reveal cellspecific and tissue specific mechanisms in inflammation and malignancy.

ERK5 (also known as MAPK7) is a ubiquitously expressed MAPK that is activated by MEKK2, MEKK3 and MEK5 (ReF. 216 ). ERK5 functions downstream of cel lular stress, several immune receptors (such as TLRs, IL1R, TNFR or IL17R), CSF1R and growth recep tors (EGF, FGF or VEGF) 216 . Among these receptors, CSF1R signalling has gained attention given its role in macrophage differentiation and the important func tion of macrophages in cancer or inflammation 217 . Several CSF1R inhibitors (PLX3397, JNJ40346527, ARRY382, ABT869, BLZ945) have moved to clini cal trials 218 (FIg. 1 ). ERK5 has a kinase domain located at the N terminal half of the protein that is homolo gous to the ERK2 kinase domain 219 . In contrast to other MAPKs, ERK5 has a unique C terminal transcriptional activation domain 220, 221 . Therefore, ERK5 is able to acti vate trans cription not only by phosphorylating trans cription factors but also by acting as a transcriptional coactivator itself. The role of ERK5 kinase activity in regulating inflammation is controversial. One study suggests that ERK5 kinase activity regulates cytokine production and the recruitment of immune cells 222 , but this finding has been challenged and this effect was attributed to off target BET bromodomain activity using biochemical and cellular assays 223 . Intriguingly, both studies show that ERK5 knockdown in epithelial cells and primary human vascular endothelial cells reduced inflammatory cytokine production 222, 223 . These data suggest that the noncatalytic activity of ERK5 posi tively regulates the inflammatory response. Consistent with this, in mice with ERK5 deficient keratinocytes, ERK5 was shown to be an essential component of skin inflammation 224 . Germline Mapk7 deficient mice are embryonic lethal at embryonic day 10.5 owing to loss of vasculature integrity 225 . Similar phenotypes were seen in mice lacking MEK5 and MEKK3, which suggests that this pathway is linear and important in both vasculo genesis and angiogenesis 216 . Initial developmental defects may primarily be due to the function of ERK5 in endo thelial cells. Induced genetic ablation of Mapk7 is lethal in mice up to 3 weeks of age, which suggests that full ablation of this pathway may not be safe. However, given the complex functional domains of ERK5, including its action as a kinase or transcription factor, knockout studies could be misleading to determine the outcome of its kinase inhibition. Correspondingly, mice treated with ERK5 inhibitors are viable at least for the duration of published studies, but the selectivity and potency of this ERK5 inhibitor requires further investigation 222 . Generation of selective ERK5 (allosteric or degraders) or MEK5 inhibitors for chronic dosing should help to better evaluate this target in cancer and acute or chronic inflammation.

TBK1 and its homologue IKKε are two serine/threonine protein kinases that activate IRFs to induce type I inter feron genes and interferon stimulated genes (ISGs) 226 , which are associated with several autoinflammatory or autoimmune diseases such as interferonopathies 227 and SLE 228 . In contrast to SLE, interferonopathies comprise monogenic Mendelian diseases characterized by distur bance of the homeostatic control of interferonmediated immune responses 227 with various genetic and molecular features. These pathologies include Aicardi-Goutières syndrome (an encephalopathy that affects newborn brains), familial chilblain lupus (childhood lupus), spondyloenchondrodysplasia (skeletal anomalies), interferon stimulated gene 15 (ISG15) deficiency and stimulator of interferon genes (STING)associated vascu lopathy with disease onset during infancy 229 . The disturbance of both the innate and the adaptive immune system may potentiate autoimmunity in some cases, for example SLE, in which a systemic immune response is triggered against selfantigens (such as nuclear anti gens) in multiple organs such as the kidney and skin 227 . TBK1 and IKKε are also induced in the liver and adipose tissue by high fat diet mediated NF κB activation 230 .

TBK1 and IKKε regulate many innate immune receptors, including TLRs, RIGIlike receptors (RLRs; which sense cytosolic nucleic acids) and STING (which is important for establishing an immediate antiviral state during acute infection) 30 (FIg. 7) . AAK1 and GAK are host kinases that regulate clathrin adaptor pro tein (AP) mediated trafficking in the endocytic and can then bind to create a mitochondrial antiviral-signalling protein (MAVS), mitochondrial-associated membranes and peroxisomes, which in turn activate TANK-binding kinase 1 (TBK1) and inhibitor of NF-κB subunitε (IKKε) to activate interferon-regulatory factor 3 (IRF3) and IRF7. Double-stranded DNA (dsDNA; such as self-DNA) can induce an allosteric structural change in cGAS that, in turn, activates second messengers to promote stimulator of interferon genes (STING) to undergo dimerization to form a complex with TBK1 and IKKε that phosphorylates IRF3 to activate gene transcription. DNA-dependent activator of interferon-regulatory factors (DAI; also known as Z-DNA-binding protein 1 (ZBP1)) can also act as a sensor of Z-dsDNA (left-handed double-helical structure), often acquired during viral infection in a cell type-specific manner. DAI recruits TBK1 and IRF3 upon DNA binding and may get further phosphorylated to amplify the DAI/TBK1/IRF3 circuit 265 . Numb-associated kinases (adaptor protein 2 (AP2)-associated protein kinase 1 (AAK1) and cyclin G-associated kinase (GAK)) regulate intracellular viral trafficking during entry, assembly and release of unrelated viruses. Disruption of endocytosis by inhibiting AAK1/GAK can prevent the virus passage into cells. Endosomal TLRs (TLR7/8/9) recruit myeloid differentiation primary response 88 (MyD88) and signal through IL-1 receptor-associated kinase 4 (IRAK4) to activate IRFs similar to cell surface TLRs (see FIg. 3 ). TIR-domain-containing adapter-inducing IFNβ (TRIF)-dependent and MyD88-independent signalling could also activate TBK1/IKKε to activate IRF-mediated gene transcription. P, phosphorus; ssDNA, single-strand DNA.

secretory pathways 231 . This pathway is important in the assembly and entry of certain viruses that hijack clathrin mediated pathways, and is being investigated for antiviral therapeutics 232 . Given the central role of TBK1 and IKKε in the production of type I interfer ons, both TBK1 and IKKε are attractive drug targets especially in interferonopathies. Mutations in TBK1 result in neuroinflammatory and neurodegenera tive disorders of the central nervous system, such as amyotrophic lateral sclerosis, which in part might be a consequence of dysregulated interferon signalling 233 . Whether TBK1 kinase inhibition results in amyotrophic lateral sclerosis pathology is unclear. TBK1 is constitutively and broadly expressed, but IKKε expression is inducible and limited to specific cell types, including lymphocytes 234 . Both phospho rylation and ubiquitylation regulate TBK1 signalling. Deubiquitination of K63 linked polyubiquitin chains from TBK1 terminates TBK1 activation and neg atively regulates the antiviral immune response 235 . Mutation studies of TBK1 in cells suggest that TBK1 has a dominant role in interferon production and may be an essential component of antiviral immunity 236 . IKKε deficient mice are susceptible to few viral infec tions, including γ herpesvirus (γHV68) DNA virus 235 . Intriguingly, IKKε may positively regulate IL17R sig nalling via its interaction with NF κB activator 1 (ACT1) to induce expression of pro inflammatory cytokines 237 . IKKε deficient mice are viable whereas TBK1 defi ciency is embryonic lethal, which raises concerns about systemic inhibition of TBK1 (ReFS 238, 239 ). Multiple TBK inhibitors are currently under development mainly at the preclinical stage, including the original BX795 mol ecule (developed by Amgen with suboptimal potency of 1 μM), MRT67307 (developed by the University of Dundee, similar to BX795 with much improved potency of 19 nM), AZ13102909 (developed by Astrazeneca with an IC50 of 5 nM), Domainex TBK1i (IC50 of 2 nM), and Myrexis MPI0485520 (potency of 0.5 nM) and compound II (developed by University of Texas Southwestern Medical Center with potency of 13 nM) 240, 241 . Finally, amlexanox is an antiinflammatory drug approved by the FDA to treat recurrent aphthous ulcers of the mouth and asthma that was later shown to inhibit TBK1 and IKKε, although at much lower potency of 1000 nM.

The generation of selective IKKε or TBK1 inhibitors has been extremely challenging, owing to the high degree of homology within the kinase domains of the two proteins 240 . Studies with current IKKε or TBK1 inhibi tors in preclinical models of interferonopathies (such as compound II in three prime repair exonuclease 1 (Trex1) knockout mice) 241 as well as neuroinflammatory mouse models (such as MRT67307 inhibitor in experi mental autoimmune encephalomyelitis) 242 suggest that targeting IKKε and TBK1 might be safe and beneficial in certain autoimmune or autoinflammatory disorders.

NIK is an integral component of noncanonical NFκB signalling, and is found downstream of a sub set of TNFR superfamily members including BAFF, CD40, TWEAK, RANK, TNFR2, FN14, CD27, CD30 and OX40 (ReF. 243 ) (FIg. 8) . NIK deficiency results in combined immunodeficiency syndrome accompanied by B cell lymphopenia, impaired differentiation of mem ory B cells, abnormal natural killer cell development and function as well as aberrant T cell responses 244 . NIK mutant mice have disorganized lymph nodes, Peyer's patches and splenic and thymic structures that lead to reduced B cell numbers and immunoglobulins 15 . Conditional NIK mutant mice and bone marrow chi meric mice have revealed the cell specific functions of NIK [245] [246] [247] . Broad deletion of Map3k14 in adult mice recapitulates the B cell effects observed with germline deletion 247 . B cells from mice in which Map3k14 is deleted fail to respond to BAFF stimulation, which supports the essential role of NIK in BAFF receptor signalling 248 . Intriguingly, T cell specific deletion of Map3k14 revealed non redundant functions of NIK in T cells 245 . Furthermore, deletion of Map3k14 in myeloid cells compromises CD40 signalling and cross priming in dendritic cells 246 .

NIK activation is partly regulated by intricate mecha nisms that regulate its protein stability (FIg. 8) . At steady state, NIK protein levels remain low as it is actively degraded by the proteasome via a Ub ligase complex comprising TRAF3, TRAF2 and IAP1/2 (ReF. 15 ) (FIg. 8) . When the non canonical NF κB pathway is activated, NIK activates IKKα, which in turn regulates the pro cessing of NF κB p100 to p52. p52 dimerizes with RelB, and the complex translocates to the nucleus to trigger transcription of target genes 15 (FIg. 8) .

Systemic canonical NF κB inhibition (and proteo somal inhibitors) may not offer a sufficient therapeutic index in chronic indications given the broad require ments for NF κB signalling in biological processes. However, inhibiting immune regulatory nodes such as NIK offer plausible therapeutic approaches for the treatment of diseases in which non canonical NF κB signalling is aberrantly activated, including RA, IBD, SLE, IgA nephropathy, metabolic syndrome and multiple sclerosis. Inhibition of NIK with the inhibitor NIK SMI1 provided evidence that NIK kinase activity positively regulates several important pathways, such as BAFF, OX40, CD40, inducible T cell costimulator (ICOS), IL21 and TNFRSF12A (also known as TWEAK recep tor) signalling, and its inhibition was protective in pre clinical models of lupus 247 . This finding corroborates the findings from the NIK mutant mice, in which its action downstream of the same immune receptors had been established 244, 247 . In a 4 week NZB/W mouse model, NIK inhibition decreased the frequency and numbers of splenic B cells, germinal centre B cells and plasma cells 247 . NIK inhibition was superior to BAFF blockade in the suppression of autoantibody titre 247 . In addition, NIK SMI1 significantly reduced the frequency and numbers of splenic T effector memory and T follicular helper as well as the production of cytokines: IL21, P40 subunit of IL12 and IL23. In a 9 week NZB/W efficacy study, NIK SMI1 improved survival and renal function 247 . NIK SMI1 is fairly selective, inhibiting only 3 out of 222 off target kinases (KHS1, LRRK2 and PKD1 (PKCμ)) to an extent >75% at a concentration of 1 μM 247 . In rodent models,

A disease of the central nervous system that affects nerves in the brain and spinal cord, causing the progressive loss of muscle control.

Rare disorders caused by mutations in different genes involved in the development and function of B cells and T cells.

Peyer's patches lymphoid nodules located on the outer lining of the small intestine, serving as monitors of the intestinal contents, often bacteria of the microbiome, to prevent the outgrowth of pathogenic bacteria in the gut.

www.nature.com/nrd the chronic dosing of NIK SMI1 is safe, but additional toxicity studies with higher doses of NIK inhibitors with improved potency and pharmacokinetic properties are needed to ensure that these molecules are safe before they are moved into the clinic. NIK SMI1 should inhibit several pathogenic pathways that are each individually validated in the clinic, including BAFFR and CD40/ CD40L. Belimumab is an approved drug in SLE. Several modulatory antibodies or fusion proteins -anti CD40 (CFZ533 (NCT02291029), leselumab (NCT01585233), I655064 (NCT03385564) and FFP104 (NCT02465944)) and anti CD40L (dapirolizumab (NCT02804763) and letolizumab (NCT02273960)) -are being investigated in the clinic for various indications such as psoriasis, RA, Crohn's disease, SLE, ITP, primary biliary cirrhosis and transplantation 249 .

CDK8 and its paralog CDK19 (which share 97% protein homology with each other) regulate RNA polymerase II (RNAP II) activity 250 . Certain subsets of CDKs (CDK1, CDK2, CDK4 and CDK6) and the corresponding cyc lins are directly involved in cell cycle regulation 251 . Overexpression of CDK8 or CDK19 was found in sev eral cancers and led to the discovery of multiple CDK8 inhibitors 250 NF-κB-inducing kinase (NIK) itself is regulated at the basal level by a destruction complex, and signal-induced non-canonical NF-κB signalling involves NIK stabilization. The canonical NF-κB pathway involves activation of inhibitor of NF-κB (IKK) complex by TGFβ-activated kinase 1-binding protein 1 (TAK1), IKK-mediated NF-κB inhibitorα (IκBα) phosphorylation and subsequent degradation, resulting in rapid and transient nuclear translocation of the NF-κB heterodimer RelA/p50. NIK protein levels remain low via active degradation using ubiquitin ligase complex, comprising tumour necrosis factor receptor-associated factor 3 (TRAF3), TRAF2 and inhibitor of apoptosis 1 and 2 (IAP1/2). Receptor activation by agonists recruits this complex to the receptor where activated IAP mediates K48 ubiquitylation and proteasomal degradation of TRAF3, resulting in stabilization and accumulation of NIK. Subsequently, NIK activates IKKα to trigger p100 phosphorylation and processing to enforce persistent activation of RelB/p52 complex to activate gene transcription. BAFF, B cell-activating factor; BCR, B cell receptor; LTβ, lymphotoxinβ; P, phosphorus; TCR, T cell receptor; TLR, Toll-like receptor; TWEAK, tumour necrosis factor-related weak inducer of apoptosis; Ub, ubiquitin.

inhibitor of both CDK8 and CDK19, upregulates IL10 production by enhancing AP1 activity 256 , which indi cates that CDK8 and CDK19 have roles in innate immu nity. Therefore, the function of the CDK module in transcription seems to be contextdependent, such that its biological function may vary among different cell types or in response to distinct stimuli. Two independent studies have demonstrated that inhibitors against both CDK8 and CDK19 promoted T reg cell differentiation 257, 258 . Akamatsu et al. elegantly carried out a functional screen using a compound library of close to 5,000 inhibitors with different mole cular scaffolds to assess their effects on the differen tiation of naive CD4 + T cells into FOXP3 + T reg cells 258 . The authors showed that inhibition of CDK8 and CDK19 can activate STAT5, which positively regulates FOXP3 expression, in a TGFβ independent manner 258 . Inhibition of CDK8 and CDK19 in vivo enhanced the development of antigen specific T reg cells, which dam pened autoimmunity in preclinical models of experi mental autoimmune encephalomyelitis and non obese diabetes 258 . In a separate study, Guo et al. used CDK8 and CDK19 inhibitors (CCT251921 or Senexin A), which enhanced TGFβ signalling and drove T reg cell differenti ation. This pathway depends partially on the attenu ation of IFNγ-STAT1 signalling and on elevated SMAD2 phosphorylation 257 . Although the mechanisms of CDK8 and CDK19 inhibition elucidated in these studies dif fer, the net effects seem to promote T reg cell differen tiation from effector T cells. More studies are needed to understand how inhibition of CDK8 and CDK9 can reprogramme conventional T cells to T reg cells. Pharmacological inhibition of CDK8 and CDK19 may have potential in T cell conversion of differentiated, antigen specific effector memory T cells into FOXP3 + T reg cells for the treatment of autoimmune or inflamma tory diseases. Currently, a CDK8 inhibitor, BCD115, has been used in clinical trials to treat advancedstage and metastatic breast cancer via directly blocking cancer cell differentiation (NCT03065010). In tumours, pathogenic tissue resident T reg cells counteract productive immunity by inhibitory mechanisms, such as the downregulation of HLA and CD80/86 on dendritic cells and the modu lation of the cytokine milieu such as TGFβ and IL2 production 259 . Therefore, CDK8 and CDK19 inhibitors may be a powerful tool to induce T reg cell differentiation in vitro applicable to T reg cell based cellular therapies. However, given the likelihood of the broad action of these kinases in other cell types, these inhibitors may not be safe for chronic dosing.

Tremendous progress has been made to advance vari ous drugs, including inhibitors of JAKs, TYK2, IRAK4, BTK, SYK, RIPs and TPL2, into the clinic. The diversity of immunological pathways targeted by these molecules provides a golden opportunity to understand human immunology and to better design targeted therapeutics for multiple debilitating inflammatory and autoimmune diseases.

Potent kinase inhibitors are designed to be selective with minimal off target effects, often by targeting the ATPbinding site of the kinase domain. However, as the ATPbinding site is relatively conserved among kinases, the design and discovery of selective kinase inhibitors still remain challenging. Furthermore, because many kinases induced during inflammation also regu late non inflammatory pathways, kinase inhibition may also result in unknown ontarget effects. It will be critical to identify several independent lead chemical scaffolds. The continued availability of extensive and diverse smallmolecule libraries increases the likelihood of finding multiple candidates. The use of bioinformatics combined with machine learning can further mine the information provided by such libra ries to expedite align ments and capture molecules with pharmaco logical and selectivity potential. Artificial intelligence with a large repository of structured medi cal information -including numerous connections extracted from scien tific litera ture by machine learning -holds great potential to rapidly nominate rational targets 260 . One example is the identification of JAKis as a possible treatment for COVID19 patients, after the finding that bariti cinib inhibits clathrinmediated endocytosis 54 of the virus. In addition, advances in structural methodo logies (such as cryoelectron microscopy) will broaden structurebased design for molecule deve lopment. Adaptation of inno vative strategies, including platforms that determine biochemical and cellular targets as well as profiling selectivity against proteome, should help to prioritize better molecules. Novel deve lopments in kinase inhibitor design to target allosteric pseudokinase domains (such as TYK2) may broaden possibilities for target selectivity. TYK2 pseudokinase inhibitors have shown that using human genetics with the ana lysis of rare coding variants enables both the identification and the design of novel drug targets 51 . Improving organspecific (such as gut, lung or skin) 48 and/or cellspecific 261 delivery of kinase inhibitors should also improve therapeutic efficacy with reduced side effects.

Emerging evidence suggests that kinase function is not limited to catalytic activity and may also serve as structural scaffolds. Thus, the function of some kinases (such as IRAK4) may be only partially susceptible to activitybased small molecule inhibition, suggesting that full suppression can only be achieved by additive modali ties such as targeting protein-protein interac tions, conformational antagonism or protein degra dation. Depending on the pathway, partial or full suppression of kinase function may be advantageous to calibrate desired safety and efficacy outcomes for the disease indication. However, this also creates uncertain ties around the efficacy of the molecule in the clinic, as, traditionally, full suppression of the pathways is the most desired outcome in order to tune optimal dose adjustments. Recent positive clinical data with IRAK4 inhibitors argue that, ultimately, clinical assessment of the target is needed to determine its net effect on disease outcomes 34 .

Identifying the appropriate patient populations within a given disease indication is an important consideration as many inflammatory diseases are heterogeneous in nature, and, therefore, require differential treatments for effective symptom amelioration. For example, BTK www.nature.com/nrd inhibition may only be a suitable treatment for a fraction of patients as RA is a heterogeneous disease and different subsets of patients are responsive to different treatments, including B cell depletion, TNF blockade or JAKi thera pies 262 . Defining such subpopulations requires predictive disease biomarkers and a deeper understanding of the molecular mechanisms of kinase activity and the path ways they participate in. Identifying biomarker profiles predictive of efficacy of inhibitor specific treatments remains a significant challenge.

As we gain confidence in the safety and efficacy of novel small molecules, an additional therapeutic strategy will be to use them in combination therapy. There is a strong scientific rationale to consider therapeutics with non overlapping mechanisms of action. Oral treatment with kinase inhibitors may be advantageous given that these drugs can be dosed to partially inhibit a pathway and often are taken on a daily basis owing to their short half life. By contrast, biologics often ablate downstream signalling and persist for a few days to weeks. The use of multiple inhibitors, or both inhibitors and biologics, holds promise in treating chronic inflammation, in part, to address molecules that did not meet expectations in the clinic when used as single agents. Although this is a promising route to explore, combination therapy will require completion of safety studies with single agents as well as careful trial design in order to monitor undesired safety effects.

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Resolution of inflammation: a new therapeutic frontier

Acute respiratory distress syndrome

HiJAKing SARS-CoV-2? The potential role of JAK inhibitors in the management of COVID-19

Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases

Human autoimmune diseases: a comprehensive update

Systemic autoinflammatory diseases

Immune checkpoints as therapeutic targets in autoimmunity

Patient perceptions of unmet medical need in rheumatoid arthritis: a cross-sectional survey in the USA

This comprehensive review on various kinases and their utility also describes the technologies that are enabling efficient generation of highly optimized kinase inhibitors

Targeting protein kinases for the development of anti-inflammatory drugs

Dual B cell immunotherapy is superior to individual anti-CD20 depletion or BAFF blockade in murine models of spontaneous or accelerated lupus

Safety of combination therapy with two bDMARDs in patients with rheumatoid arthritis: a systematic review and meta-analysis

JAK inhibition as a therapeutic strategy for immune and inflammatory diseases

Transcriptional control of the inflammatory response

The non-canonical NF-κB pathway in immunity and inflammation

Mitogen-activated protein kinases in innate immunity

Immunological memory: lessons from the past and a look to the future

The functional plasticity of T cell subsets

Regulatory T cells in skin injury: at the crossroads of tolerance and tissue repair

The other function: class II-restricted antigen presentation by B cells

Innate and adaptive immune memory: an evolutionary continuum in the host's response to pathogens

Structural principles of tumor necrosis factor superfamily signaling

The family of the interleukin-1 receptors

The pro-and anti-inflammatory properties of the cytokine interleukin-6

New directions in the basic and translational biology of interleukin-27

Development of TH1 CD4 + T cells through IL-12 produced by Listeria-induced macrophages

Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain

IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases

Modulation of immune complexinduced inflammation in vivo by the coordinate expression of activation and inhibitory Fc receptors

Pattern recognition receptors and inflammation

This report shows that TPL2 activates both ERK and p38 signalling to impact neutrophilic inflammation

Structural basis for the noncatalytic functions of protein kinases

The secret life of kinases: functions beyond catalysis

Efficacy and safety of the selective interleukin-1 receptor associated kinase 4 inhibitor, PF-06650833, in patients with active rheumatoid arthritis and inadequate response to methotrexate

Review of Bruton tyrosine kinase inhibitors for the treatment of relapsed or refractory mantle cell lymphoma

GS-4875, a first-in-class TPL2 inhibitor suppresses MEK-ERK inflammatory signaling and proinflammatory cytokine production in primary human monocytes

FDA approves first-in-class SYK inhibitor

This remarkable study shows that inhibiting the pseudo-kinase activity of TYK2 with the novel molecule BMS-986165 can reduce kinase activity with much greater selectivity for TYK2

Baricitinib: a 2018 novel FDA-approved small molecule inhibiting janus kinases

PF-06651600, a dual JAK3/TEC family kinase inhibitor

Filgotinib, a JAK1 inhibitor, modulates disease-related biomarkers in rheumatoid arthritis: results from two randomized, controlled phase 2b trials

Prevention of organ allograft rejection by a specific Janus kinase 3 inhibitor

The JAK-3 inhibitor CP-690550 is a potent anti-inflammatory agent in a murine model of pulmonary eosinophilia

The novel JAK-3 inhibitor CP-690550 is a potent immunosuppressive agent in various murine models

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders

Beyond ruxolitinib: fedratinib and other emergent treatment options for myelofibrosis

Tezepelumab in adults with uncontrolled asthma

Development of gut-selective pan-Janus kinase inhibitor TD-1473 for ulcerative colitis: a translational medicine program

Design and synthesis of a pan-Janus kinase inhibitor clinical candidate (PF-06263276) suitable for inhaled and topical delivery for the treatment of inflammatory diseases of the lungs and skin

Lung-restricted inhibition of Janus kinase 1 is effective in rodent models of asthma

Resolving TYK2 locus genotypeto-phenotype differences in autoimmunity

Tyrosine kinase 2 variant influences T lymphocyte polarization and multiple sclerosis susceptibility

Phase 2 trial of selective tyrosine kinase 2 inhibition in psoriasis

Baricitinib as potential treatment for 2019-nCoV acute respiratory disease

Baricitinib therapy in COVID-19: a pilot study on safety and clinical impact

An oligomeric signaling platform formed by the Toll-like receptor signal transducers MyD88 and IRAK-4

Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling

IRAK4 dimerization and transautophosphorylation are induced by Myddosome assembly

Sequential control of Toll-like receptor-dependent responses by IRAK1 and IRAK2

IRAK-4: a novel member of the IRAK family with the properties of an IRAK-kinase

TRAF6 is a signal transducer for interleukin-1

Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol

Interleukin-1 receptor-associated kinase 2 is critical for lipopolysaccharide-mediated post-transcriptional control

Distinct molecular mechanism for initiating TRAF6 signalling

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs

IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family

IRAK-M is a negative regulator of Toll-like receptor signaling

IRAK-M mediates Toll-like receptor/ IL-1R-induced NFκB activation and cytokine production

The interleukin-1 receptor-associated kinase M selectively inhibits the alternative, instead of the classical NFκB pathway

Cofactors required for TLR7-and TLR9-dependent innate immune responses

Evidence for licensing of IFN-γ-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program

Interferon-regulatory-factor 1 controls Toll-like receptor 9-mediated IFN-β production in myeloid dendritic cells

IRAK4 and NEMO mutations in otherwise healthy children with recurrent invasive pneumococcal disease

IRAK4 kinase activity controls Toll-like receptor-induced inflammation through the transcription factor IRF5 in primary human monocytes

Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling

IL-1 receptor-associated kinase modulates host responsiveness to endotoxin

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor-and Toll-like receptor 7-mediated signaling and gene expression

This paper is the first demonstration that selective IRAK4 inhibitor can dampen disease in preclinical models of lupus

Deficiency in IRAK4 activity attenuates manifestations of murine lupus

Suppression of IRAK1 or IRAK4 catalytic activity, but not type 1 IFN signaling, prevents lupus nephritis in mice expressing a ubiquitin binding-defective mutant of ABIN1

The dietary flavonoid kaempferol mediates anti-inflammatory responses via the Src, Syk, IRAK1, and IRAK4 molecular targets

Genetic ablation of IRAK4 kinase activity inhibits vascular lesion formation

Loss of interleukin receptorassociated kinase 4 signaling suppresses amyloid pathology and alters microglial phenotype in a mouse model of Alzheimer's disease

Impaired cytokine signaling in mice lacking the IL-1 receptor-associated kinase

Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use

Inflammation: speciesspecific TLR signalling -insight into human disease

Experimental and natural infections in MyD88-and IRAK-4-deficient mice and humans

Two phases of inflammatory mediator production defined by the study of IRAK2 and IRAK1 knock-in mice

Human interleukin-1 receptor-associated kinase-2 is essential for Toll-like receptor-mediated transcriptional and post-transcriptional regulation of tumor necrosis factor α

A narrow repertoire of transcriptional modules responsive to pyogenic bacteria is impaired in patients carrying loss-of-function mutations in MYD88 or IRAK4

Activation of the canonical IKK complex by K63/M1-linked hybrid ubiquitin chains

Mechanism of dysfunction of human variants of the IRAK4 kinase and a role for its kinase activity in interleukin-1 receptor signaling

This study shows that IRAK4 has a scaffold function in Myddosome formation and that its kinase activity is dispensable for Myddosome assembly

The diverse role of RIP kinases in necroptosis and inflammation

Diverse ubiquitin linkages regulate RIP kinases-mediated inflammatory and cell death signaling

RIPK1 and RIPK3: critical regulators of inflammation and cell death

Intracellular regulation of TNF activity in health and disease

cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination

c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor α (TNFα)-induced NF-κB activation

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling

Multitasking kinase RIPK1 regulates cell death and inflammation

Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL

This study describes the instrumental and protective role for RIP1 kinase inhibition in inflammatory disease despite its lack of relevance in tumour progression and metastasis

Activity of protein kinase RIPK3 determines whether cells die by necroptosis or apoptosis

RIP3 induces apoptosis independent of pronecrotic kinase activity

RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury

Cytotoxicity of crystals involves RIPK3-MLKL-mediated necroptosis

RIP1 kinase activity is critical for skin inflammation but not for viral propagation

Inhibition of keratinocyte necroptosis mediated by RIPK1/RIPK3/MLKL provides a protective effect against psoriatic inflammation

IL-13-induced airway mucus production is attenuated by MAPK13 inhibition

NF-κB-independent role of IKKα/ IKKβ in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling

Regulation of RIPK1 activation by TAK1-mediated phosphorylation dictates apoptosis and necroptosis

A dominant autoinflammatory disease caused by non-cleavable variants of RIPK1

Cleavage of RIPK1 by caspase-8 is crucial for limiting apoptosis and necroptosis

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease

Autophagy protein ATG16L1 prevents necroptosis in the intestinal epithelium

Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury

Targeting RIPK1 for the treatment of human diseases

Discovery of a first-in-class receptor interacting protein 1 (RIP1) kinase specific clinical candidate (GSK2982772) for the treatment of inflammatory diseases

This paper describes the first RIP1 inhibitor that entered clinical trials

NOD1 and NOD2: signaling, host defense, and inflammatory disease

Autoinflammatory granulomatous diseases: from Blau syndrome and early-onset sarcoidosis to NOD2-mediated disease and Crohn's disease

The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity

This study describes a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signalling and provides a molecular basis for the design of novel therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors

Small molecule inhibitors reveal an indispensable scaffolding role of RIPK2 in NOD2 signaling

TEC-family kinases: regulators of T-helper-cell differentiation

Inhibitors of BTK and ITK: state of the new drugs for cancer, autoimmunity and inflammatory diseases

Tec family kinases modulate thresholds for thymocyte development and selection

Inhibition of the kinase ITK in a mouse model of asthma reduces cell death and fails to inhibit the inflammatory response

Apoptosis by death factor

Ibrutinib treatment improves T cell number and function in CLL patients

Defective B cell development and function in Btk-deficient mice

Type I and III interferon productions are impaired in X-linked agammaglobulinemia patients toward poliovirus but not influenza virus

Fcγ receptors as regulators of immune responses

Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fcε RI cross-linking

Involvement of Bruton's tyrosine kinase in FcεRI-dependent mast cell degranulation and cytokine production

BTK inhibition is a potent approach to block IgE-mediated histamine release in human basophils

Specific Btk inhibition suppresses B cell-and myeloid cell-mediated arthritis

Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis

Requirement of phospholipase C-γ 2 activation in surface immunoglobulin M-induced B cell apoptosis

Btk/Tec kinases regulate sustained increases in intracellular Ca 2+ following B-cell receptor activation

Phospholipase Cγ2 is essential in the functions of B cell and several Fc receptors

Bruton's tyrosine kinase regulates TLR9 but not TLR7 signaling in human plasmacytoid dendritic cells

Ability of Bruton's tyrosine kinase inhibitors to sequester Y551 and prevent phosphorylation determines potency for inhibition of fc receptor but not B-cell receptor signaling

Bruton's tyrosine kinase inhibitor BMS-986142 in experimental models of rheumatoid arthritis enhances efficacy of agents representing clinical standard-of-care

Therapeutic blockade of immune complex-mediated glomerulonephritis by highly selective inhibition of Bruton's tyrosine yinase

Btk inhibition treats TLR7/IFN driven murine lupus

This paper compares IRAK inhibition versus BTK inhibition and shows that IRAK4 has a major role in both TLR and immune-complex signalling

Placebo-controlled trial of an oral BTK inhibitor in multiple sclerosis

Discovery of branebrutinib (BMS-986195): a strategy for identifying a highly potent and selective covalent inhibitor providing rapid in vivo unactivation of Bruton's tyrosine kinase (BTK)

Efficacy and safety of fenebrutinib, a BTK inhibitor, compared to placebo in rheumatoid arthritis patients with active disease despite TNF inhibitor treatment: randomized, double blind, phase 2 study

The BTK-inhibitor ibrutinib may protect against pulmonary injury in COVID-19 infected patients

The SYK tyrosine kinase: a crucial player in diverse biological functions

Structural and biophysical characterization of the Syk activation switch

Structural basis for the inhibition of tyrosine kinase activity of ZAP-70

Syk activation and dissociation from the B-cell antigen receptor is mediated by phosphorylation of tyrosine 130

Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323

Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain

Getting Syk: spleen tyrosine kinase as a therapeutic target

Syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis

Syk tyrosine kinase required for mouse viability and B-cell development

Perinatal lethality and blocked B-cell development in mice lacking the tyrosine kinase Syk

Genetic deficiency of Syk protects mice from autoantibodyinduced arthritis

Inflammation and bone erosion are suppressed in models of rheumatoid arthritis following treatment with a novel Syk inhibitor

Specific inhibition of spleen tyrosine kinase suppresses leukocyte immune function and inflammation in animal models of rheumatoid arthritis

A phase III, multicenter, randomized, double-blind, placebo-controlled, parallel-group study of 2 dosing regimens of fostamatinib in patients with rheumatoid arthritis with an inadequate response to a tumor necrosis factor-alpha antagonist

Treatment of rheumatoid arthritis with a Syk kinase inhibitor: a twelve-week, randomized, placebo-controlled trial

R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation

Fostamatinib, an oral spleen tyrosine kinase inhibitor, in the treatment of rheumatoid arthritis: a meta-analysis of randomized controlled trials

An oral spleen tyrosine kinase (Syk) inhibitor for rheumatoid arthritis

Fostamatinib: first global approval

Fostamatinib for persistent/chronic adult immune thrombocytopenia

In vitro pharmacological profiling of R406 identifies molecular targets underlying the clinical effects of fostamatinib

This paper reports the utility of various biochemical and cellular assays to identify several off-target effects of R406/fostamatinib

Distinct roles for the linker region tyrosines of Syk in FcεRI signaling in primary mast cells

ZAP-70 directly enhances IgM signaling in chronic lymphocytic leukemia

This article describes a recently developed smallmolecule inhibitor, GS-9973, which is highly specific for SYK, and its clinical application in autoimmunity and oncology

An open-label phase 2 trial of entospletinib (GS-9973), a selective spleen tyrosine kinase inhibitor, in chronic lymphocytic leukemia

Phase 2 study of idelalisib and entospletinib: pneumonitis limits combination therapy in relapsed refractory CLL and NHL

TPL2 kinase action and control of inflammation

Lipopolysaccharide activation of the TPL-2/MEK/ extracellular signal-regulated kinase mitogenactivated protein kinase cascade is regulated by IκB kinase-induced proteolysis of NF-κB1 p105

ABIN-2 forms a ternary complex with TPL-2 and NF-κB1 p105 and is essential for TPL-2 protein stability

NF-κB1 p105 negatively regulates TPL-2 MEK kinase activity

TNF-α induction by LPS is regulated posttranscriptionally via a Tpl2/ERKdependent pathway

Tpl2/cot signals activate ERK, JNK, and NF-κB in a cell-type and stimulus-specific manner

TPL2-mediated activation of ERK1 and ERK2 regulates the processing of pre-TNFα in LPS-stimulated macrophages

NF-κB1/p105 regulates lipopolysaccharide-stimulated MAP kinase signaling by governing the stability and function of the Tpl2 kinase

Regulation of experimental autoimmune encephalomyelitis by TPL-2 kinase

ABIN2 function is required to suppress DSS-induced colitis by a Tpl2-independent mechanism

Intestinal myofibroblast-specific Tpl2-Cox-2-PGE2 pathway links innate sensing to epithelial homeostasis

p38 MAP-kinases pathway regulation, function and role in human diseases

Differential activation of p38MAPK isoforms by MKK6 and MKK3

Characterization of the structure and function of the fourth member of p38 group mitogenactivated protein kinases, p38δ

p38γ and p38δ kinases regulate the Tolllike receptor 4 (TLR4)-induced cytokine production by controlling ERK1/2 protein kinase pathway activation

Eukaryotic elongation factor 2 controls TNF-α translation in LPS-induced hepatitis

TPL2 meets p38MAPK: emergence of a novel positive feedback loop in inflammation

Alternative p38 MAPKs are essential for collagen-induced arthritis

Regulation of PTEN activity by p38δ-PKD1 signaling in neutrophils confers inflammatory responses in the lung

Regulation of PKD by the MAPK p38δ in insulin secretion and glucose homeostasis

Essential role of p38γ in K-Ras transformation independent of phosphorylation

Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution

Structural basis for p38α MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity

Acquisition of sensitivity of stressactivated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket

T. p38 mitogenactivated protein kinase inhibitors: a review on pharmacophore mapping and QSAR studies

p38 MAPK : stress responses from molecular mechanisms to therapeutics

Selective inhibition of the p38α MAPK-MK2 axis inhibits inflammatory cues including inflammasome priming signals

Oral p38 mitogen-activated protein kinase inhibition with BIRB 796 for active Crohn's disease: a randomized, double-blind, placebo-controlled trial

RV568, a narrow-spectrum kinase inhibitor with p38 MAPK-α and -γ selectivity, suppresses COPD inflammation

A controlled clinical trial with pirfenidone in the treatment of pathological skin scarring caused by burns in pediatric patients

Pirfenidone in the treatment of idiopathic pulmonary fibrosis: an evidence-based review of its place in therapy

An open-label, phase II study of the safety and tolerability of pirfenidone in patients with scleroderma-associated interstitial lung disease: the LOTUSS trial

Pirfenidone gel in patients with localized scleroderma: a phase II study

Pirfenidone for diabetic nephropathy

Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and enhances CB2 gene expression in patients with chronic hepatitis C

Comparative chemical array screening for p38γ/δ MAPK inhibitors using a single gatekeeper residue difference between p38α/β and p38γ/δ

ERK5: structure, regulation and function

Function of CSF1 and IL34 in macrophage homeostasis, inflammation, and cancer

Recent advances in colony stimulating factor-1 receptor/c-FMS as an emerging target for various therapeutic implications

Molecular cloning of mouse ERK5/BMK1 splice variants and characterization of ERK5 functional domains

The unique C-terminal tail of the mitogen-activated protein kinase ERK5 regulates its activation and nuclear shuttling

ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain

Extracellular signal-regulated kinase 5 promotes acute cellular and systemic inflammation

ERK5 kinase activity is dispensable for cellular immune response and proliferation

ERK5 is a critical mediator of inflammation-driven cancer

Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure

Mechanisms of type-I-and type-IIinterferon-mediated signalling

Genetic interferonopathies: an overview

Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus

Type I interferonopathies in pediatric rheumatology

This paper shows that amlexanox inhibits TBK1 and, in obese mice, can elevate energy expenditure through increased thermogenesis, producing weight loss

AP-2-associated protein kinase 1 and cyclin G-associated kinase regulate hepatitis C virus entry and are potential drug targets

This paper reveals that by regulating HCV entry and its life cycle, AAK1 and GAK represent potential targets for antiviral strategies

Repurposing of kinase inhibitors as broad-spectrum antiviral drugs

Human TBK1: a gatekeeper of neuroinflammation

A new family of IKK-related kinases may function as IκB kinase kinases

Ubiquitin-specific protease 2b negatively regulates IFN-β production and antiviral activity by targeting TANK-binding kinase 1

This report shows that USP2b deubiquitinates K63-linked polyubiquitin chains from TBK1 to terminate TBK1 activation

IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts

The inducible kinase IKKi is required for IL-17-dependent signaling associated with neutrophilia and pulmonary inflammation

The roles of two IκB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection

Multiple functions of the IKKrelated kinase IKKε in interferon-mediated antiviral immunity

Therapeutic potential of targeting TBK1 in autoimmune diseases and interferonopathies

This report demonstrates that TBK inhibitor is efficacious in interferonopathy mouse models

This paper shows that TBK1 regulates AKT1/ mTORC1 to control T cell activation and egress from draining lymph nodes using conditional T cell-specific knockout mice and TBK1 inhibitors

Noncanonical NF-κB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK

Biallelic loss-of-function mutation in NIK causes a primary immunodeficiency with multifaceted aberrant lymphoid immunity

Cell intrinsic role of NF-κB-inducing kinase in regulating T cell-mediated immune and autoimmune responses

Dendritic cells require NIK for CD40-dependent cross-priming of CD8 + T cells

This study shows that selective NIK inhibition can inhibit multiple immunological pathways, including BAFF, OX40, CD40, ICOS, IL-21 and TWEAK

Conditional deletion of NF-κBinducing kinase (NIK) in adult mice disrupts mature B cell survival and activation

Targeting the CD40-CD40L pathway in autoimmune diseases: humoral immunity and beyond

A conserved mediator-CDK8 kinase module association regulates mediator-RNA polymerase II interaction

CDKs and cancer: a changing paradigm

Cortistatin A is a high-affinity ligand of protein kinases ROCK, CDK8, and CDK11

Cyclin-dependent kinase 8 mediates chemotherapy-induced tumor-promoting paracrine activities

Discovery of potent, selective, and orally bioavailable small-molecule modulators of the mediator complex-associated kinases CDK8 and CDK19

Structure-based optimization of potent, selective, and orally bioavailable CDK8 inhibitors discovered by high-throughput screening

Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells

Inhibition of Cdk8/Cdk19 activity promotes T reg cell differentiation and suppresses autoimmune diseases

Conversion of antigen-specific effector/memory T cells into Foxp3-expressing T reg cells by inhibition of CDK8/19

Turning the tide against regulatory T cells

Artificial intelligence in drug discovery

Specific delivery of kinase inhibitors in nonmalignant and malignant diseases

Heterogeneity of autoimmune diseases: pathophysiologic insights from genetics and implications for new therapies

Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7-and TLR9-mediated interferon-α induction

Regulation and function of NF-κB transcription factors in the immune system

Innate immune recognition of DNA: a recent history

The authors thank Allison Bruce for superb graphic design and figure illustrations.

A.A.Z. conceived the review topic with major contributions to writing and revisions of the entire manuscript. K.B. contributed to IRAK4, Syk, p38 sections and revisions. D.V. contributed to RIPK section and revisions. P.L. contributed to Table 1 .

A.A.Z. is an employee of TRexBio and holds stock in TRexBio and the Roche Group. K.B. is an employee of Genentech. P.L. is an employee of Synthekine and holds stock in Synthekine and the Roche Group. D.V. is an employee of Genentech and holds stock and options in the Roche Group.

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