Design and synthesis of a novel tyrosine kinase inhibitor template

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).     

Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.     

Pyrazolone-based anaplastic lymphoma kinase (ALK) inhibitors: control of selectivity by a benzyloxy group

Anaplastic lymphoma kinase (ALK) is transmembrane receptor tyrosine kinase, with oncogenic variants that have been implicated in ALCL, NSCLC and other cancers. Screening of a VEGFR2-biased kinase library resulted in identification of 1 which showed cross-reactivity with ALK. SAR on the indole segment of 1 showed that a subtle structural modification (the ethoxy group of 1 changed to a benzyloxy to generate 5a) enhanced potency (ALK), selectivity for VEGFR2 and IR along with improvement in metabolic stability. From docking studies of ALK versus VEGFR2 kinase, we postulated that the loss of entropy of the VEGFR2 in the bound form with 5a might be the origin of the reduced activity against that protein. Modification of the heterocyclic segment showed that thiazole-bearing pyrazolones preserved enzyme potency, and enhanced inhibition of NPM-ALK autophosphorylation in ALK-positive ALCL cells (Karpas-299). SAR of the benzyloxy group resulted in compounds which demonstrated good cellular potency in Karpas-299 cells. Compound 8 showed best overall profile for the series with broad kinome selectivity and liver micorsome stability. Compound 8 showed reasonable iv PK in rat, but with little oral exposure.     

Activin receptor-like kinase 1 inhibits human microvascular endothelial cell migration: potential roles for JNK and ERK

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific type I receptor of the TGFbeta receptor family that is implicated in angiogenesis and in the pathogenesis of the vascular disease, hereditary hemorrhagic telangiectasia (HHT). In the absence of a specific ligand, ALK1 cellular functions have been mainly studied through the use of a constitutively active form of this receptor (ALK1ca) and are still debated. We previously reported that ALK1ca inhibits proliferation and migration of human endothelial cells suggesting that ALK1 plays an important role in the maturation phase of angiogenesis (Lamouille et al., 2002, Blood 100: 4495-4501). In the present work, we further analyzed the role of ALK1 in the migration of human dermal microvascular endothelial cell (HMVEC-d) and observed that silencing endogenous ALK1 expression with siRNAs accelerates endothelial cell migration in the wound assay. Further, we demonstrate that ALK1-induced inhibition of migration is Smad-independent. Using a panel of kinase inhibitors, we found that HMVEC-d wound closure was completely inhibited by a JNK inhibitor and to a lower degree by an ERK kinase inhibitor. Further, HMVEC-d wounding induced activation of both JNK and ERK, and these were inhibited by ALK1ca expression. Taken together, these results support a significant role for ALK1 as a negative regulator of endothelial cell migration and suggest the implication of JNK and ERK as mediators of this effect.     

Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.     

Anti-apoptotic signaling of pleiotrophin through its receptor,anaplastic lymphoma kinase

The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.     

Insulin gene is a target in activin receptor-like kinase 7 signaling pathway in pancreatic beta-cells

Activins regulate pancreatic development, differentiation and insulin secretion. Activin receptor-like kinase 7 (ALK7) has been identified as a receptor for Nodal and Activin AB and B, and is expressed in pancreatic islets and β-cell lines. In this study, human insulin promoter was activated by Smad2, Smad3 and the pancreatic and duodenal homeobox factor-1 (PDX-1) in the ALK7 pathway. A conserved Smad binding element was related to the promoter activation. Phosphorylated Smad2/Smad3 and PDX-1 were bound to insulin gene with Nodal and Activin AB, and the phosphorylated Smad2/Smad3 interacted with PDX-1. These results indicate that one of the direct target genes of Nodal and Activin AB signals is the insulin gene in pancreatic β-cells and that PDX-1 is directly involved in the ALK7-Smad pathway.     

Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.     

Screening and identification of a novel class of TGF-β type 1 receptor kinase inhibitor

Transforming growth factor β (TGF-β) type I receptor (activin receptor-like kinase 5, ALK5) has been identified as a promising target for fibrotic diseases. To find a novel inhibitor of ALK5, the authors performed a high-throughput screen of a library of 420,000 compounds using dephosphorylated ALK5. From primary hits of 1521 compounds, 555 compounds were confirmed. In total, 124 compounds were then selected for follow-up based on their unique structures and other properties. Repeated concentration-response testing and final interference assays of the above compounds resulted in the discovery of a structurally novel ALK5 inhibitor (compound 8) (N-(thiophen 2-ylmethyl)-3-(3,4,5 trimethoxyphenyl)imidazo[1,2β]pyridazin 6-amine) with a low IC(50) value of 0.7 μM. Compound 8 also inhibited the TGF-β-induced nuclear translocation of SMAD with an EC(50) value of 0.8 μM. Kinetic analysis revealed that compound 8 inhibited ALK5 via mixed-type inhibition, suggesting that it may bind to ALK5 differently than other published adenosine triphosphate site inhibitors.     

Control of ALK (wild type and mutated forms) phosphorylation: Specific role of the phosphatase PTP1B

Phosphorylation of proteins on tyrosine residues is regulated by the activities of protein tyrosine kinases and protein tyrosine phosphatases. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) essentially and transiently expressed during development of the central and peripheral nervous systems. ALK has been identified as a major neuroblastoma predisposition gene and activating mutations have been identified in a subset of sporadic neuroblastoma tumors. We previously established that the mutated receptors were essentially retained in the endoplasmic reticulum/Golgi compartments due to their constitutive activity. Intriguingly we demonstrated a stronger phosphorylation for the minor pool of receptor addressed to the plasma membrane. We decided to investigate the potential involvement of tyrosine phosphatase in dephosphorylation of this intracellular pool. In this study we first showed that general inhibition of tyrosine phosphatases resulted in a dramatic increase of the tyrosine phosphorylation of the wild type but also of the mutated receptors. This increase not only required the intrinsic kinase activity of the ALK receptor but also involved the Src tyrosine kinase family. Second we provided strong evidences that the endoplasmic reticulum associated phosphatase PTP1B is key player in the control of ALK phosphorylation. Our data shed a new light on the biological significance of the basal phosphorylation levels of both wild type and mutated ALK receptors and could be essential to further understand their roles in malignancies.     

Unique substrate specificity of anaplastic lymphoma kinase (ALK): development of phosphoacceptor peptides for the assay of ALK activity

The anaplastic lymphoma kinase (ALK), whose constitutively active fusion proteins are responsible for 5-10% of non-Hodgkin's lymphomas, shares with the other members of the insulin receptor kinase (IRK) subfamily an activation loop (A-loop) with the triple tyrosine motif Y-x-x-x-Y-Y. However, the amino acid sequence of the ALK A-loop differs significantly from the sequences of both the IRK A-loop and the consensus A-loop for this kinase subfamily. A major difference is the presence of a unique "RAS" triplet between the first and second tyrosines of the ALK A-loop, which in IRK is replaced by "ETD". Here we show that a peptide reproducing the A-loop of ALK is readily phosphorylated by ALK, while a homologous IRK A-loop peptide is not unless its "ETD" triplet is substituted by "RAS". Phosphorylation occurs almost exclusively at the first tyrosine of the Y-x-x-x-Y-Y motif, as judged by Edman analysis of the phosphoradiolabeled product. Consequently, a peptide in which the first tyrosine had been replaced by phenylalanine (FYY) was almost unaffected by ALK. In contrast, a peptide in which the second and third tyrosines had been replaced by phenylalanine (YFF) was phosphorylated more rapidly than the parent peptide (YYY). A number of substitutions in the YFF peptide outlined the importance of Ile and Arg at positions n - 1 and n + 6 in addition to the central triplet, to ensure efficient phosphorylation by ALK. Such a peculiar substrate specificity allows the specific monitoring of ALK activity in crude extracts of NPM-ALK positive cells, using the YFF peptide, which is only marginally phosphorylated by a number of other tyrosine kinases.     

BioID-Screening Identifies PEAK1 and SHP2 as Components of the ALK Proximitome in Neuroblastoma Cells

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase (RTK) that is mutated in approximately 10% of pediatric neuroblastoma (NB). To shed light on ALK-driven signaling processes, we employed BioID-based in vivo proximity labeling to identify molecules that interact intracellularly with ALK. NB-derived SK-N-AS and SK-N-BE(2) cells expressing inducible ALK-BirA* fusion proteins were generated and stimulated with ALKAL ligands in the presence and absence of the ALK tyrosine kinase inhibitor (TKI) lorlatinib. LC/MS-MS analysis identified multiple proteins, including PEAK1 and SHP2, which were validated as ALK interactors in NB cells. Further analysis of the ALK-SHP2 interaction confirmed that the ALK-SHP2 interaction as well as SHP2-Y542 phosphorylation was dependent on ALK activation. Use of the SHP2 inhibitors, SHP099 and RMC-4550, resulted in inhibition of cell growth in ALK-driven NB cells. In addition, we noted a strong synergistic effect of combined ALK and SHP2 inhibition that was specific to ALK-driven NB cells, suggesting a potential therapeutic option for ALK-driven NB.     

Transforming growth factor-beta1 protects against pulmonary artery endothelial cell apoptosis via ALK5

Transforming growth factor (TGF)-beta1 has been reported to cause endothelial cell apoptosis. However, conflicting data have also demonstrated that TGF-beta1 promotes endothelial cell survival. In this study, the effect of TGF-beta1 on apoptosis of cultured bovine pulmonary artery endothelial cells (PAEC) induced by multiple stimuli was investigated. TGF-beta1 protected against apoptosis of bovine PAEC induced by serum deprivation or the VEGF receptor inhibitor SU-5416, but not by UV light exposure or TNFalpha. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9, an effect that was abolished by TGF-beta1. Furthermore, serum deprivation and inhibition of VEGF receptors significantly decreased the protein level of Bcl-2, an effect that was also abrogated by TGF-beta1. In addition, the baseline level of Bcl-2 was enhanced by TGF-beta1 and reduced by inhibition of activin receptor-like kinase 5 (ALK5), a TGF-beta1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of bovine PAEC. These results suggest that TGF-beta1 signaling is critical for maintenance of bovine PAEC survival. Finally, the protective effects of TGF-beta1 on bovine PAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. Also, TGF-beta1 activated SMAD2 and SMAD1/5, an effect that was abolished by ALK5 inhibition. The results of this study suggest that TGF-beta1 protects against bovine PAEC apoptosis, possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of the mitochondria-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-beta1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.     

Activation of the orphan receptor tyrosine kinase ALK by zinc

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.     

ALK is a Novel Dependence Receptor: Potential Implications in Development and Cancer

ALK (anaplastic lymphoma kinase) is a transmembrane receptor tyrosine kinase, initially discovered as part of the NPM-ALK fusion protein, resulting from a chromosomal rearrangement frequently associated with anaplastic large cell lymphomas. The native ALK protein is normally expressed in the developing and, at a weaker level, adult nervous system. We recently demonstrated that ALK is a novel dependence receptor. As such, in the absence of ligand, the ALK receptor is kinase inactive and its expression results in enhanced apoptosis, whereas kinase activation, due to a ligand or constitutive as in NPM-ALK, decreases apoptosis. Unligated/kinase unactivated ALK receptor facilitates apoptosis via its own cleavage by caspases, a phenomenon allowing the exposure of a proapoptotic juxta-membrane intra-cellular domain. This review summarizes the biological significance of the ALK receptor in cancer and development, in perspective with its dependence receptor function. The dual function of ALK in the physiology of development is illustrated in the visual system of Drosophila. In this part of the nervous system, ALK in the presence of ligand appears essential for axonal guidance, whereas in the absence of ligand, ALK expression can lead to developmental neuronal apoptosis. ALK is also found expressed in neural crest-derived tumors such as human neuroblastomas or glioblastomas but its role is not fully elucidated. However, an excessive or constitutive ALK tyrosine kinase activation can lead to deregulation of cell proliferation and survival, therefore to human cancers such as lymphomas and inflammatory myofibroblastic tumors. Our observations could have important implications in the therapy of ALK-positive tumors harboring the chimeric or wild type ALK protein.     

Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.

Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.     

Detection of anaplastic lymphoma kinase (ALK) gene rearrangement in non-small cell lung cancer and related issues in ALK inhibitor therapy: a literature review

Anaplastic lymphoma kinase (ALK) encodes a receptor tyrosine kinase, and ALK gene rearrangement (ALK+) is implicated in the oncogenesis of non-small cell lung carcinomas (NSCLCs), especially adenocarcinomas. The ALK inhibitor crizotinib was approved in August 2011 by the US Food and Drug Administration (FDA) for treating late-stage NSCLCs that are ALK+, with a companion fluorescent in situ hybridization (FISH) test using the Vysis ALK Break Apart FISH Probe Kit. This review covers pertinent issues in ALK testing, including approaches to select target patients for the test, pros and cons of different detection methods, and mechanisms as well as monitoring of acquired crizotinib resistance in ALK+ NSCLCs.