Essay: JAK1/2 Inhibitors in Clinical Study

INTRODUCTION
The importance of hormones and interferons (IFNs) such as prolactin, growth hormone and erythropoietin has been recognized for the last decades. The discovery of a plethora of other cytokines came with the molecular biology era. Cytokines, which we now know regulate all aspects of cell differentiation and development, represent a collection of structurally distinct ligands that bind to different classes of receptors. A major subgroup of cytokines, which can bind to receptors termed Type I/II cytokine receptors, comprises roughly 60 factors. From an immunology perspective, these cytokines, including IFN-γ, Type I IFNs, colony stimulating factors and many interleukins, are significant for constraining immune and inflammatory responses, initiating innate immunity and orchestrating adaptive immune mechanisms. 1[JAKs and STATs in Immunoregulation and Immune-Mediated Disease]
Cytokines’ biological functions mainly depend on cytokine-mediated gene repression or activation. Currently, main strategies for the treatment of immune response and related diseases against cytokine responses are inhibiting cytokine receptors (biological agents) and regulating related cytokine signaling pathways (small molecule inhibitors).
Efforts have been made for years to develop small molecule immune modulators, which come with advantages comparing to biologic agents of currently available biologic agents, including convenience and the expectation that they would be significantly less expensive. 2[Novel small-molecular therapeutics for rheumatoid arthritis]
Janus kinase (JAK) family is one of the most important and popular molecular targets for therapy. As shown in Fig.1, JAKs family includes JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2). JAK3 and TYK2 are mainly responsible for immune responses. JAK1 and JAK2, which have known for broad functions, have roles that range from host hematopoiesis and defense to growth and neural development. JAKs selectively associate with the cytoplasmic domains of various cytokine receptors and cytokine binding activates JAKs, which in turn phosphorylate cytokine receptors. Members of the signal transducers and activators of transcription (STAT) proteins family, including STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6, are then allowed to selectively bind to cytokine receptors. STATs, as DNA-binding proteins, subsequently become tyrosine-phosphorylated, which allows them to dimerize, translocate to the nucleus, and regulate gene expression. STAT1, STAT2, STAT4, and STAT6 have restricted functions, playing roles in immunoregulation and host defense. It has been reported that complete deficiency of STAT3 or STAT5A and STAT5B is lethal in mice. These proteins consistently have the broad, critical functions. 3[JAKs and STATs in Immunity, Immunodeficiency, and Cancer]
Fig. I: Signaling commences when the Ligand induces receptor dimerization; II: This brings the receptor associated Jak kinase into apposition, enabling them to reciprocally trans phosphorylate each other. The kinases, now activate, phosphorylate a distal tyrosine on the receptor; III: This receptor phosphoryl residue is recognized by the SH2 domain of a STAT protein, drawing them into the receptor complex, where they activated by tyrosine phosphorylation; IV: The activated STAT proteins are now rendered competent for heteo- or homodimerizations, ad nuclear translation (4), ad GAS binding. Jak-ax denotes a Jak kinase that can be either identical to a different form Jak-a. Stat-a denotes a STAT protein that can be either identical to or different from Stat-a. The a-chain of the receptor binds ligand. while the β-chain transduces signals. [transcriptional response to polypeptide ligands: the JAK-STAT pathway] [Janus kinases: components of multiple signaling pathways]
So far, mutation experiments of all protein subtypes have proved that JAK family plays a vital role in the occurrence and development of immune diseases. There is a huge number of cytokines that signal through JAKs, and mutations and polymorphisms of genes encoding JAK kinases have been linked to diseases of immunity. For example, JAK3 mediates all gamma-cytokine signaling. Thus, it renders mutations in JAK3, including the phenotype of gamma-deficiencies and leads to SCID.4,5 Loss of TYK2 function leads to more minor immunodeficiencies.6 A large amount of evidence from genome-wide association studies has confirmed that JAK is present in the pathogenesis of autoimmune diseases. For example, TYK2 is associated with SLE, and JAK2 polymorphism is associated with Crohn’s disease and Bechet disease.7-9 [Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases]. In addition, investigations on JAK-STAT pathway for possible other clinical indications are still in progress. Especially, the utilization of JAK-STAT to regulate tumor microenvironment has incurred great interest in medicinal chemists.10 [STATs in cancer inflammation and immunity: a leading role for STAT3]
Several reviews have been published recently on JAK-STAT pathway with emphasis on its inhibitors, biological functions, and different strategies of regulating JAK-STAT signaling in the immune system. 22,29,44–51 It is necessary to …!! We reviewed the latest small molecule modulators of JAK-STAT pathway which are currently in clinical research, based on ….!!!!/classified by…
THE BIOLOGICAL FUNCTION OF JAKs
JAK Enzyme Structure and Signaling
The JAKs, the relatively large proteins (120−130 kDa) of the tyrosine kinase family, possess two kinase domains including a true kinase domain and a likely inactive pseudo-kinase domain.5 There are 90 protein tyrosine kinases (PTKs), many of which serve crucial functions.11-13 PTKs can be further divided into the two main subgroups, receptor tyrosine kinases (RTKs) and non-receptor, or cytosolic, tyrosine kinases. RTKs contain an extracellular ligand binding domain, intracellular cytoplasmic kinase domain and transmembrane region. The cytoplasmic PTKs can be divided into nine subfamilies including the sarcoma (Src), C-terminal Src kinase (Csk), Activated CDC42 kinase (Ack), focal adhesion kinase (FAK), tyrosine kinase expressed in hepatocellular carcinoma (Tec), Fes , spleen tyrosine kinase (Syk), Abl , and Jak classes. [Selectivity and therapeutic inhibition of kinases: to be or not to be?]
The domain structure of JAKs. The domains JH1–JH7 are based on sequence similarity of four known JAKs. JH1 is the kinase domain, which contains two tyrosines that can be phosphorylated after ligand stimulation. JH2 is the pseudo-kinase domain. The JH6 and JH7 domains mediate the binding of JAKs to receptors.14
JAK family has a high degree of sequence homology, the highest homology observed in the catalytic domain. These enzymes contain seven different homologous regions (JH) and four structure domains (Figure). One of JAK’s features is the two structurally related domains JH1 and JH2.15 JH1 is the active kinase catalytic domain, whereas JH2 is known as a pseudo kinase domain that does not have a catalytic function but is thought to play a key role in concert with JH1.16 Interestingly, recent work has shown that the JH2 domain of JAK2 may negatively control the JAK2 function by phosphorylating the critical residues within the JH2 domain.17 The JH3 and JH4 domains are primarily play a structural role in stabilizing the confirmation of the JAKs, while the domains JH5-JH7 (FERM domain) have been believed to interact with the JH1 domain as well as interact directly with the intracellular domain of the cytokine receptor.18,19 Upon cytokine binding and conformational changes, the JAKs are activated and become phosphorylated especially in the activation loop of the kinase (JH1) domain on tyrosine residues. [Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases]
Currently, the precise functions of the pseudo kinase domain (JH2) as well as JH3-JH7 domains are still under investigation. The sequence of the JH3-JH7 domain is not similar to any of the characterized protein motifs. Considering the variety of interactions and functions performed by JAK kinase family members, it seems possible that these domains facilitate some key functions such as protein-protein interactions, recruitment of substrates, etc. [Janus kinases: components of multiple signaling pathways]
The specific tyrosine residues in the domain of the activated JAK phosphorylated receptor cells are capable of forming a recruitment site for STATs. The recruitment of STAT receptors also causes JAK to phosphorylate STAT, leading to its dimerization and subsequent migration to the nucleus, which binds to specific DNA binding sites that regulate gene transcription leading to changes in cell function. [Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases]
JAK
JAKs was found when various methods were attempted for identifying novel protein tyrosine kinases. The first JAK kinase, called TYK2, was obtained when the T cell library was screened using low stringency hybridization techniques.20,21 Only the other members of the JAK family are identified and characterized, and the unique structure and function of TYK2 becomes apparent. Polymerase chain reaction (PCR) of degenerate oligonucleotides across the severe conserved kinase domain of members of the protein tyrosine kinase Src family led to the identification of partial cDNA clones of JAK1 and JAK2.22 The full-length cDNA of JAK1 and JAK2 was then cloned using a partial cDNA fragment as a probe.23,24 Their findings and several other members of the tyrosine kinase family contributed to the use of the acronym JAK (Just Another Kinase). Subsequent sequencing studies have shown that the JAK family of PTK is significantly different from other classes of PTK by the presence of additional kinase domains. In order to express this unique structural feature, these kinases are called “Janus kinases”, which involve an ancient Roman door and a door on both sides. [Janus kinases: components of multiple signaling pathways]
The association of JAK with a given cytokine receptor is determined by association with a specific receptor chain. For example, JAK3 conjugated to gamma-co-stranding is always paired with JAK1 and this arrangement controls six known gamma-common cytokines IL-2, IL-4, IL-7, IL-9, IL- 15 and IL-21, which are mainly related to adaptive immune function.25 However, JAK1 also regulates signaling through a wide range of cytokine receptors and affects several proinflammatory cytokines, such as IL-6 and type I interferons associated with innate immune responses with JAK2 and TYK2不好理解. JAK2 is the only member of the JAKs that is paired with itself. In this combination, JAK2 controls signal transduction of various cytokines and growth factors such as IL-3, IL-5, granulocyte macrophage colony stimulating factor (GM-CSF), erythropoietin (EPO) And thrombopoietin (TPO). [Discovery and Development of Janus Kinase (JAK) Inhibitors for Inflammatory Diseases]
DISEASES AND CONDITIONS INVOLVING JAK
[Jak3 deficiency blocks innate lymphoid cell development]
Primary Immunodeficiency
From studying patients with primary immunodeficiency, a striking example of the importance of JAKs and STAT has been noticed.26 Mutations of JAK3 and TYK2 s are known cause of primary immunodeficiency including severe combined immunodeficiency (SCID) among the four JAK.
Impaired development or function of lymphocytes causes SCID. The receptor chain shared by interleukins 2, 4, 7, 9, 15, and 21 is the common interleukin-2Rγ chain, γc; the gene encoding this protein is located at Xq13. Mutations in the gene encoding interleukin-2Rγ (IL2RG) cause X-linked SCIDs characterized by a marked reduction in the number and function of T cells and natural killer (NK) cells but preserving the B cell number impaired function (T−B+NK−). When the JAK3 mutation was identified in most patients with no Tc mutation in T−B+NK− SCID, the Jc3-dependent dependence of γc signal was established.26 On the contrary, mutations in the alpha chain of the interleukin-7 receptor specifically damage T cells may lead to T−B+NK+ SCID because interleukin-15 signaling required for NK cell development is maintained.
Only two TYK2 autosomal recessive mutations have been reported in humans and their phenotypes are different. One child has atopic dermatitis, moderately elevated IgE levels, and severe viral, bacterial, and fungal infections.27 The other child had severe infection after vaccination with neurobrucellosis, bacille Calmette– Guérin (BCG), and infection with herpes simplex virus (HSV), but only very minor elevations in IgE levels and no atopy.28
Immunoregulation and Genetic Links to Immune-Mediated Disease
Animal models provide strong evidence that the JAK–STAT pathway and cytokines that use this pathway play key roles in the pathogenesis of allergic, asthma, autoimmune and other immune-mediated diseases. Recently, genome-wide association studies have supported the notion that cytokine and cytokine signaling are involved in human disease. Inherited variation in genes encoding cytokines and cytokine receptors and their respective JAKs and STATs are associated with a marked increase in the risk of immune-mediated diseases. Multiple genes in the IL-23 signaling pathway lead to a series of surprising autoimmune diseases including inflammatory bowel disease, psoriasis, ankylosing spondylitis and Behcet’s disease.29-31Polymorphisms of JAK2 and STAT3 are associated with these disorders.
Cancer
JAKs plays a key role in host defense and autoimmunity of hematologic cancers.32 The initial evidence for these effects stems from the identification of constitutive activation of JAK and STAT in cancer patients.33,34 JAK2 mutations are associated with myeloproliferative neoplasms, clonal cancers arising from hematopoietic progenitor cells, which include essential thrombocythemia, polycythemia vera, and primary myelofibrosis.35-38
The JAK2 V617F mutant is located within the JH2 “kinase-like” domains.39,40 It is catalytically active to phosphorylate and activate the kinase domain.41 Thus, this mutation results in a constitutively activated kinase that renders hematopoietic cells independent of exogenous growth factors, resulting in polycythemia vera and other myeloproliferative processes.42,43 These observations confirm the carcinogenic importance of the V617F mutation.
Somatically acquired mutations in JAK2 have been detected in high-risk patients with B-cell acute lymphoblastic leukemia (ALL) (9%) and in patients with B-cell ALL associated with Down’s syndrome (34%), most often affecting the R683 residue.44-46 Other JAKs can be activated by mutations in hematologic cancers, including JAK3 in patients with T-cell ALL, patients with NK-cell or T-cell lymphoma and patients with adult T-cell leukemia or lymphoma,47-49 and JAK1 in patients with T-cell ALL, patients with acute myeloid leukemia and patients with B-cell ALL who have a poor prognosis.46,50-52
The function of receptors associated with JAKs can also be altered by chromosomal rearrangements or cancer mutations resulting in constitutive JAK activity. Activating mutations that affect the thrombopoietin receptor MPL occur in about 9% of myelofibrosis patients and all patients lack the JAK2 V617F mutation, resulting in constitutive activation of JAK2 by the MPL receptor.53 In about 50% of B-cell ALL patients, the JAK2-binding cytokine receptor CRLF2 is over-expressed by chromosomal rearrangements, including 34% of Down’s syndrome patients and 9% of children with high-risk B-cell ALL.45,54-56 Many leukemias that have been altered in CRLF2 also have JAK2 mutations, suggesting that this receptor acts as a constitutive signaling platform for mutant JAK2. Approximately 10% of T-cell ALL patients have a mutant interleukin-7 receptor alpha subunit, resulting in constitutive JAK1 activation.57,58 Gain-of function mutations in granulocyte-colony stimulating factor receptors are associated with acute myeloid leukemia and severe congenital neutropenia.59 In addition to the hematopoietic lineage, an in-frame deletion affecting glycoprotein 130, the signal component of the interleukin-6 receptor, is present in 60% of patients with inflammatory hepatocellular adenoma resulting in JAK2 activation.60 JAKs can also be activated by the secretion of autocrine cytokines in several lymphoma subtypes. In primary mediastinal B cell lymphomas and Hodgkin’s lymphoma, autocrine interleukin-13 signaling activates JAK2 and its activity is further intensified by amplification of the JAK2 locus.61,62 Therefore, JAK2 inhibition of both types of lymphomas is lethal to the cell line.62
Autocrine secretion of interleukins 6 and 10 activates JAKs in the activated B-cell-like (ABC) subtype of diffuse large-B-cell lymphoma, promoting the survival of malignant cells.63-65 It is well-known that the autocrine cytokine ring is initiated by activating mutations that affect MYD88, an adapter protein in Toll-like receptor signaling.64 The most common MYD88 mutant L265P, occurs in 36% of patients with primary central nervous system lymphoma, and it is also present in 29% of patients with ABC diffuse large-B-cell lymphoma66 and 69% of patients with leg-type primary cutaneous lymphoma, both of which phenotypically resemble ABC diffuse large-B-cell lymphoma.MYD88 L265P is also common in Wald Enstrom’s macroglobulinemia67 (90% of cases) and occurs in chronic lymphocytic leukemias64,68 (10 to 11% of cases) and a subset of marginal-zone lymphomas69,70 (3 to 10% of cases). In ABC diffuse large B-cell lymphoma, the MYD88 mutant spontaneously coordinates the active signaling complex consisting of the interleukin-1-related kinase IRAK1 and IRAK4 and thus participates in the nuclear factor κB and p38 MAP kinase pathways and leads to interleukin-6 and interleukin-10 and autocrine JAK activation.
Downstream effectors of JAK signaling in hematopoietic cancers include the PI-3 kinase and Ras pathways and STAT transcription factors.32 The JAK2 V617F mutation does not cause myeloproliferative disease in mice lacking STAT5,71-73 and STAT3 is required for the survival of ABC diffuse large B-cell lymphoma cells.63,74 STAT3 is mutated in 40% of large granular leukemias,75 with its SH2 domain altered at some of the same positions in which it is altered in patients with the hyper-IgE syndrome, albeit with different amino acid substitutions. Constitutive STAT activation, which is common in epithelial cells, hepatocytes and breast cancer, promotes the proliferation and survival of malignant cells as well as tumor-promoting inflammation while reducing antitumor immunity.76-79 The complex role of STAT makes it a challenging An important research topic.
In the past few years, researchers have identified a noncanonical, epigenetic role for JAK signaling in the nucleus in lymphomas and leukemias.62,80In leukemias, JAK2 translocate into the nucleus with the V617F mutation and in primary mediastinal B-cell lymphoma and Hodgkin’s lymphoma with amplification of the JAK2 locus. There, JAK2 phosphorylates histone H3 tail on tyrosine 41 to counteract the formation of heterochromatin and promote gene expression, including the expression of the oncogene MYC in both types of lymphomas.62,80 The JAK2 amplicon in these lymphomas also includes JMJD2C, which encodes a chromatin modifier counteracts the formation of heterochromatin and cooperates with JAK2 to elicit genes epigenetically.62
It has been reported that JAK-STAT signaling is modulated with mechanisms by multiple processes. There are various regulators, which mainly involves cytokine signaling (SOCS) protein inhibitors and inhibitors of activated STAT (PIAS) families and protein tyrosine phosphatases (PTP). It may be important to regulate JAKs and STAT by interactions between modifications of multiple proteins and different JAK-STAT pathways and other cellular signaling pathways, which serve as additional regulatory levels. This review discusses the regulation these mechanisms and the latest insights into the JAK-STAT signal in the immune system.
FIG. # Negative regulation of the JAK–STAT pathway. The Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway is regulated at many levels. JAKs can be negatively regulated by suppressor of cytokine signalling (SOCS) proteins, protein tyrosine phosphatases (PTPs), such as SRC homology 2 (SH2)-domain-containing PTP1 (SHP1), SHP2, CD45 and T-cell PTP (TCPTP), and ubiquitin-mediated protein degradation. SOCS proteins, which are induced by cytokines, act as a negative-feedback loop to switch off the activity of JAKs. Many PTPs participate in the regulation of JAKs. The regulation of JAK2 by ubiquitylation (Ub) has been suggested. The physiological significance of protein ubiquitylation in the regulation of JAKs remains to be determined. JAKs might also be regulated by other proteins, such as the SH2-B family of putative adaptor proteins (not shown). STATs can be negatively regulated by PTPs (such as PTP1B and TCPTP) in the cytoplasm, and by PIAS proteins, as well as PTPs (such as TCPTP and SHP2), in the nucleus. Protein inhibitor of activated STAT (PIAS) proteins interact with STATs in response to cytokine stimulation and they inhibit the transcriptional activity of STATs through distinct mechanisms.
Regulation Of Jaks By The SOCS Proteins
SOCS protein regulates JAK-STAT signaling pathway. The key role of SOCS in the immune system has been adequately validated by biochemical and genetic investigations. The SOCS protein family has eight members: CIS (cytokine-induced SH2 domain protein) and SOCS1-SOCS7 (REFS 15,16). The SH2 domain is contained in SOCS protein, flanking the variable amino terminal domain and the carboxy terminal SOCS cassette 17 (Figure 3a). Among SOCS series, the functions of SOCS1, SOCS2, SOCS3 and CIS in JAK-STAT signaling have been thoroughly investigated. generally, the levels of such SOCS proteins are low in unstimulated cells but they can be rapidly induced by cytokines, thus leading to the inhibition of JAK-STAT signaling and the formation of a classical negative feedback loop. Cytokine signaling is inhibited by SOCS protein by different mechanisms (Fig. 3b). The SH2 domain directly binds to tyrosine phosphorylated JAKs, causing the direct suppression of JAK activity. However, inhibition of JAKs by SOCS3 requires SOCS3 bind to activated receptors 23,24. CIS is shown to inhibit STAT activation rather than influencing JAK by competing with STATs to bind the docking sites of receptor. Finally, the role of SOCS proteins in the signaling proteins degradation by ubiquitin-proteasome pathway has also been proved. The SOCS box can bind to elongins B and C, the components of a ubiquitin E3 ligase complex26,27. So, SOCS proteins possibly act on signaling proteins for degradation
Cytokine regulation of SOCS3 can also be observed post-translational levels. Studies have demonstrated that SOCS3 is rapidly phosphorylated on residues Tyr204 and Tyr221, which are present in conserved SOCS cassettes and are stimulated by JAKs and receptor tyrosine kinases in response to certain cytokines and growth factors. [28, 29] Mutational studies proved that tyrosine phosphorylation of SOCS3 contributes to the degradation of SOCS3 protein, thus regulating the feedback inhibition of JAK-STAT signaling. 【REGULATION OF JAK–STAT SIGNALLING IN THE IMMUNE SYSTEM】
Regulation of Jaks By Protein Tyrosine Phosphatase
It has been noted that PTP regulates JAKs, including SHP1, SHP2, CD45, PTP1B and T-cell PTP (TCPTP). SHP1 and SHP2 are PTPs42 containing the SH2 domain. SHP1 is mainly expressed by hematopoietic cells and has been shown to be physically associated with c-KIT receptor, IL-3 receptor beta chain and erythropoietin receptor (EPOR). Mutations that defective expressing SHP1-binding are allergic to EPO and have extended EPO-induced autophosphorylation of JAK2, indicating the effect of SHP1 on JAK2 dephosphorization.81
SHP1 is also involved in the dephosphorization of JAK1. In SHP1-/-macrophages, IFN-α-induced JAK1, but not TYK2 tyrosine phosphorylation was enhanced. Genetic studies have shown that SHP2 is involved in the negative regulation of JAK1. In SHP2 -/-fibroblasts, IFN-γ stimulated JAK1 tyrosine phosphorylation levels increased.82
CD45 is a highly expressed receptor PTP by hematopoietic cells and plays a key role in antigen receptor signal transduction in T cells and B cells. CD45 can bind and dephosphorylate all JAKs47 directly, and enhanced JAK phosphorylation is observed in CD45-/-cells. Removal of CD45 increases erythrocyte colony formation and antiviral activity, which is consistent with the role of CD45 in the negative regulation of EPO and IFN signaling. However, further research is needed to fully understand the importance of CD45 regulation of cytokine signaling under physiological conditions.
PTP1B and TCPTP-two highly correlated PTPs48- are also believed to dephosphorylate JAK. JAK2 and TYK2, but not JAK1, as the substrate for PTP1B49, and increased JAK2 phosphorylation observed in Ptp1b-/-mouse embryonic fibroblasts. In addition, PTP1B has been involved in the negative regulation of LEPTIN signaling, possibly by targeting JAK2.83,84 However, no immunophenotyped of PTP1B was reported. TCPTP can dephosphorylate JAK1 and JAK3,85 and IFN-γ-induced JAK1, but not JAK2 tyrosine phosphorylation, is observed in TCPTP – / – macrophages. Thus, many PTPs participate in the dephosphorization of JAK (Table 2). Gene targeting studies support the importance of these PTPs.
Regulation Of Jaks By Ubiquitylation And Isgylation
The ubiquitin-proteasome pathway (BOX 1) has been involved in the regulation of JAK-STAT signaling. JAK2 is ubiquitinated both in vivo and in vitro, whereas IL-3 and IFN-γ stimulation enhances the ubiquitination of JAK2. Interestingly, the discovery of JAK2 tyrosine phosphorylation is necessary for its effective ubiquitination. Multi-ubiquitination JAK2 rapid degradation. SOCS1, but not SOCS3 promotes the degradation of ubiquitination JAK2. The SOCS frame of SOCS1 associated with ubiquitination of JAK2 is necessary to interact with prolonged proteins B and C. Thus, the stability of JAK2 appears to be regulated by the SOCS1-mediated ubiquitin-proteasome pathway. Clearly, further research is needed to understand the exact contribution of protein ubiquitination to JAKs regulation under physiological conditions.
Recently, a series of modifications of JAKs has been reported, through conjugation to interferon-stimulated gene 15 (ISG15) – a ubiquitin like protein group (BOX 1). One of the most strongly induced gene products is ISG15, by which type I IFNs, viral infection and lipopolysaccharide (LPS) stimulation.86,87 However, the identity of ISGylation proteins and the biological effects of ISGylation are poorly understood. Several ISGylated proteins, including JAK1 and STAT1, have been identified using high-throughput immunoblot screening assays.88 The biological effects of the ISG gene in the regulation of JAK-STAT signaling have been shown by gene targeting studies. UBP43 (also known as ubiquitin-specific protease 18, USP18) – a member of the ubiquitin-protease family – is an important protease that removes ISG15 from ISGylated protein.89 Defects in UBP43 lead to prolonged STAT1 tyrosine phosphorylation, DNA binding And STAT1-dependent gene activation response to IFN- [beta] stimulation. As a result, Ubp43 – / – mice were allergic to type I IFNs attacks.90 An increased ISG gene for JAK1 and STAT1 has been detected in Ubp43 – / – cells. The exact ISG site of JAK1 or STAT1 has not been determined and it is not known how the activity of JAK1 or STAT1 is regulated by the ISG gene. However, ISGylation appears to be part of the classical positive feedback loop in the JAK-STAT pathway regulation. There may also be other mechanisms for regulating JAKs. For example, the putative protein of the SH2-B family has been involved in the regulation of JAKs. It would be interesting to check the meaning of these adapter molecules in the JAK regulation.91 【REGULATION OF JAK–STAT SIGNALLING IN THE IMMUNE SYSTEM】
In the ATP-binding site, the key amino acids are glu930, tyr931 and ***, in which the imidazole ring and the amino group in the structural scaffold interact with the pocket. AZ’s patent of wo2010038060 reports ** compounds, of which IC50 of the best compound for JAK2 is less than 3 nM, and the company subsequently reported compounds with same scaffold in the later patent WO2010020810. The two compounds have similar activities, but
Two patents filed in 2010 by AstraZeneca claim analogues of AZD1480, with an imidazole ring replacing the pyrazole ring of AZD1480 (see Table 2 for AZD1480) [37,38]. Three types of JAK2 assays were illustrated in the two applications, but the IC50 data of the examples were limited. Stereochemistry at the benzylic center isimportant for activity, similar to the earlier pyrazol-3-yl compounds. One of the enantiomers (not specified) of example 41, a diaminopyrido[3,4-d]pyrimidine, shows a IC50 of under 3 nM for JAK2 but only 2.74 μM for epimer [37]. The values of nearly three orders of magnitude difference is unusual and implies very close interaction with the protein. A similar profile can be found in example 20, a morpholino-substituted pyrimidine (Figure 5) [38].
[Inhibitors of JAK2 and JAK3: an update on the patent literature 2010 — 2012]
Takeda, the pan-JAK inhibitors, are applicated in different series of related aminopyridine / pyrimidine TYK2 inhibitors in the therapy of many autoimmune diseases, each substituted at C4 with a lactone appended with a nitrile. WO2015016206 [88] claims aminopyridines, with example 1 demonstrating TYK2 99% inhibition of at 1 μM concentration of, while earlier filing WO2013146963 [89] finds aminopyrimidines with similar substituents. Here, example 1 also shows 99% TYK2 inhibition. It remains unclear to what extent the selectivity is over other JAK-family members.
In 2013, Steven Magnuson’s team reported the co-crystal structures of pyridin-2-amine compounds with TYK2 and JAK2. The group used pyridine as the starting compound to discuss the structure-activity relationship of R1, R2 and R3 positions respectively, and finally found that compound 46 has the best kinase selectivity (TYK2 4.8 nM, JAK1 84 nM, JAK2 28 nM), physicochemical properties (LogD 3.4, aqueous solubility (pH 7.4) 0.17mg/ml) and pharmacokinetic properties (Clhep (mL/min/kg) (rat, mouse) 21,65, T1/2 (h) (rat, mouse) 1.2, 0.3). [Lead identification of novel and selective TYK2 inhibitors]
In the compound having pyrimidin-2-amine as a skeleton, the amino group and the N1 form a hydrogen bond interaction with Leu932, and the C6 form the second hydrogen bond with Glu930. Many companies have reported JAKs inhibitors of related framework compounds since 2010. Take rigel as an example, Rigel claimed a series of JAK3 compounds related tricyclic carbamate diaminopyrimidine moieties [101] used as JAK2 inhibitors [102]. These indane-fused cyclic carbamates, which are not usual, show either a 5- or 6-membered carbamate ring, however, a range of substitutions are tolerated on the pyrimidine ring (see simplified Markush and 6-membered ring example 40, Figure 23). 10 of the compounds that had different pyrimidine amino-substituents exhibited JAK2 IC50 values lower than 250 nM. Whole cell assays data were not disclosed, despite the fact that the procedures were delineated (Figure 23).
In another Rigel application, it is claimed that similar types of compounds having two anilino groups on the pyrimidine with a distinctive sulfonyl urea group attached to one aniline via a short spacer. The only data given were cellular results in human T cells of healthy volunteers was given (Figure 23) [104].
The United States biotechnology company Exelixis reported the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors in 2012. High throughput screening of their in-house compound library led to the identification of hit 1, from which optimization (shown above) leaded to the discovery of highly potent and selective JAK2 inhibitors. The compound has high selectivity in vitro kinase (JAK2 2.2 nM, JAK3 214.2 nM), and shows high activity of phosphorylation of stat (pstat1, pstat3 386.4 and 695nM, respectively) Advanced lead 10d showed an obvious dose-dependent pharmacodynamic behavior and antitumor effect. According to the desirable profile of 10d (XL019), it was further developed into clinical trials.
[SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.]
Merck reported the co-crystal complexes of compound 28 and JAK1 in 2017. From crystal structure analysis, Glu957 and Leu959 are key amino acids in the hinge region in the ATP-binding pocket. The amino group of the backbone 1H-pyrazole-4-carboxamide can form a hydrogen bond with the carbonyl group of Leu959, and the carbonyl group and the amino group of the amide bond in the skeleton form a hydrogen bond interaction with the amino group of Leu and the carbonyl group of Glu957, respectively. In addition, the pyrazole part can form a hydrophobic interaction with *** amino acid. The detailed kinase activity, physicochemical properties, pharmacokinetic parameters, and pharmacodynamic results of Compound 22 and compound 28 are demonstrated in the literature.
[The Discovery of 3‐((4-Chloro-3-methoxyphenyl)amino)-1-((3R,4S)‐4- cyanotetrahydro‐2H‐pyran-3-yl)‐1H‐pyrazole-4-carboxamide, a Highly Ligand Efficient and Efficacious Janus Kinase 1 Selective Inhibitor with Favorable Pharmacokinetic Properties]
Two filings in early 2010 were disclosed by Merck . 3-amino-4-carboxamidopyrazoles were claimed as JAK inhibitors by the first application[WO 2010014453]. There were only 18 examples of these primary amides having been exemplified, with JAK2 IC50 values ranging from 8 nM to 1 μM. Methylsulfone example 17 had been found to be the most potent with JAK2 IC50 of 8 nM. These show certain selectivity against JAK3 as none had IC50 < 3 μM.
In the analogs with 1H-pyrrolo[2,3-b]pyridine scaffold, a co-crystal complex was reported by Vertex Corporation in 2015, in which Glu930 and Leu932 are key amino acids, which can form three hydrogen bond interactions with the scaffold. In the literature, authors obtained a comparison of compounds of 1H-pyrrolo[2,3-b]pyridine by screening for jak3, which was used as a design strategy to discuss the substituted ring system and groups in detail. Finally, the compound VX-509 was found, which exhibited JAKs kinase activity and pharmacokinetic properties. Also, VX-509 which demonstrates good efficacy in vivo in the rat host versus graft model (HvG). This compound is also reported in WO2013070606. [Discovery of VX-509 (Decernotinib): A Potent and Selective Janus kinase (JAK) 3 Inhibitor for the Treatment of Autoimmune Diseases]