Inhibitor‐based affinity probes for the investigation of JAK signaling pathways

The Janus Kinase (JAK) signaling pathway plays a key role for many cellular processes and has recently been correlated with neuronal disorders. In order to understand new links of JAK family members with other signaling pathways, chemical proteomics tools with broad kinase coverage are desirable. A probe that shows outstanding kinase selectivity and allows for the enrichment of up to 133 kinases including many mitogen activated kinase (MAPK) members and JAK kinases has been developed. Furthermore, this probe was applied to establish the selectivity profile of the JAK1/2 inhibitor momelotinib that is currently evaluated in clinical phase 3 studies. These results render this probe a valuable tool for the investigation of JAK and JAK related signaling pathways and the selectivity profiling of kinase inhibitors.     

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.     

Recent advances in JAK3 inhibition: Isoform selectivity by covalent cysteine targeting

Janus kinases (JAKs) are a family of four cytosolic protein kinases with a high degree of structural similarity. Due to its very restricted role in immune regulation, JAK3 was promoted as an excellent target for immunosuppression for more than a decade, but clinical validation of this concept is still elusive. During the last years, speculation arose that kinase activity of JAK1, which cooperates with JAK3 in cytokine receptor signaling, may have a dominant role over the one of JAK3. Until recently, however, this issue could not be appropriately addressed due to a lack of highly isoform-selective tool compounds. With the recent resurgence of covalent drugs, targeting of a specific cysteine that distinguishes JAK3 from other JAK family members became an attractive design option. By applying this strategy, a set of JAK3 inhibitors with excellent selectivity against other JAK isoforms and the kinome was developed during the last three years and used to decipher JAK3-dependent signaling. The data obtained with these tool compounds demonstrates that selective JAK3 inhibition is sufficient to block downstream signaling. Since one of these inhibitors is currently under evaluation in phase II clinical studies against several inflammatory disorders, it will soon become apparent whether selective JAK3 inhibition translates into clinical efficacy.     

Pharmacophore and Virtual Screening of JAK3 inhibitors

Janus kinase 3 (JAK3) is a non-receptor tyrosine kinases family of protein which is comprised of JAK1, JAK2, JAK3 and TYK2. It plays an important role in immune function and lymphoid development and it only resides in the hematopoietic system. Therefore, selective targeting JAK3 is a rational approach in developing new therapeutic molecule. In this study, about 116 JAK3 inhibitors were collected from the literature and were used to build four-point pharmacophore model using Phase (Schrodinger module). The statistically significant pharmacophore hypothesis of AAHR.92 with r2 value of 0.942 was used as 3D query to search against 3D database namely Zincpharmer. A total of 2, 27,483 compounds obtained as hit were subjected to high throughput virtual screening (HTVS module of Schrodinger). Among the hits, ten compounds with good G-score ranging from -12.96 to -11.18 with good binding energy to JAK3 were identified.     

JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR).METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs.RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed that the gatekeeper residues of JAK family kinases are methionine in WT, similar to EGFR T790M, suggesting that TKIs for JAKs may also be effective for EGFR T790M.CONCLUSION: Our findings demonstrate that JAK3 inhibitor VI is a gatekeeper mutant selective TKI and offer a strategy to search for new EGFR T790M inhibitors.     

PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization

Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases. Several of these kinases are oncogenic, and may function in the development of prostate cancer. One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally. PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain. Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers. In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus. Kinase-defective PSK (K57A) was unable to reduce stress fibers. PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.     

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

Involvement of JAK-family tyrosine kinases in hematopoietin receptor signal transduction

A variety of cytokines, hormones and hematopoietic growth factors signal through the hematopoietin family of membrane receptors, which share several structural features, including a Trp-Ser-X-Trp-Ser motif and four paired cysteine residues. The signal transduction mechanisms utilized by these receptors have remained elusive, although tyrosine kinase activation has been one common element. Recently, a role for the cytoplasmic tyrosine kinases of the Janus kinase (JAK) family has been implicated in signalling by these receptors. There are currently three known JAK family kinases, including JAK1, JAK2 and TYK2. This review will focus on the role of such tyrosine kinases in hematopoietin receptor signal transduction, and address the possibility of the involvement also of unidentified Janus kinases.     

Autocrine-mediated activation of STAT3 correlates with cell proliferation in breast carcinoma lines

The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

Magic bullets for protein kinases

A chemical-genetic method for the generation of target-specific protein kinase inhibitors has been developed recently. This strategy utilizes a functionally silent active-site mutation to sensitize a target kinase to inhibition by a small molecule that does not inhibit wild-type kinases. Tyrosine and serine/threonine kinases are equally amenable to the drug-sensitization approach, which has been used to generate selective inhibitors of mutant Src-family kinases, Abl-family kinases, cyclin-dependent kinases, mitogen-activated kinases, p21-activated kinases and Ca(2+)/calmodulin-dependent kinases. The designed inhibitors are specific for the sensitized kinase in a cellular background where the wild-type kinase has been inactivated. By these means, kinase-sensitization has been used systematically to generate and analyze conditional alleles of several yeast protein kinases in vivo.     

Molecular cloning of a chicken JAK homolog from activated T cells

A cDNA encoding a JAK-related protein was isolated from a chicken T cell library by screening with a PCR-generated probe that corresponds to a conserved region in the kinase domain. Sequence analysis reveals an ORF of 3318 nt, encoding a protein with a calculated molecular weight of 123 000. Chicken JAK (cJAK) contains a double catalytic domain that is characteristic of the JAK family of tyrosine kinases. Compared with mammalian JAKs, the kinase domain shows 70% sequence identity with the corresponding region of the mammalian JAKs. Overall, cJAK shows approximately 59% amino acid identity with mammalian JAK3s, and 52% amino acid identity with mammalian JAK2s. cJAK is expressed predominantly in thymus and spleen, with lower levels in kidney, thyroid and liver. cJAK is also expressed at low levels in unstimulated splenic T cells, whereas mRNA levels are increased after activation of the T cells with Con A. The sequence analysis and pattern of expression suggests that this is an avian homolog of JAK3.     

Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase.

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.