TrkA kinase inhibitors from a library of modified and isosteric Staurosporine aglycone

An immobilized Staurosporine aglycone isostere where one of the indole nitrogen atoms was replaced by carbon has been sequentially functionalized to generate compounds inhibiting TrkA kinase. In the first phase, initial screening of a library of C13-hydroxymethyl-7-oxo-indenopyrrolocarbazoles resulted in several potent compounds, one of which was further optimized to generate the corresponding carbamates on solid phase. Some of the major carbamate diastereomers were found to be several-fold more potent than their alcohol parents. Synthesis, SAR analysis, kinase selectivity, and anti-tumor properties of a TrkA inhibitor (12a) are discussed.     

An isoform-selective inhibitor of tropomyosin receptor kinase A behaves as molecular glue

The production of TrkA-selective inhibitors is considerably difficult because the kinase domains of TrkA and its isoforms TrkB/C have highly homologous amino acid sequences. Here we describe the structural basis for the acquisition of selectivity for a isoform-selective TrkA inhibitor, namely compound V1. The X-ray structure revealed that V1 acts as a molecular glue to stabilize the symmetrical dimer of the TrkA kinase domains. V1 binds to the ATP-binding site and simultaneously engages in the dimeric interface of TrkA. The region of the dimeric interface in TrkA is not conserved in TrkB/C; thus, dimer formation may be a novel mechanism for the production of selective TrkA inhibitors. The biochemical and biophysical assay results confirmed that V1 selectively inhibited TrkA and induced the dimer formation of TrkA, but not TrkB. The binding pocket at the TrkA dimer interface can be used for the production of new isoform-selective TrkA inhibitors.     

P-donor ligand containing ruthenium half-sandwich complexes as protein kinase inhibitors

Metal complexes have emerged as promising and novel scaffolds for the design of enzyme inhibitors. Reported herein are the design, synthesis, and evaluation of protein kinase inhibition properties of pyridocarbazole half-sandwich complexes containing P-donor ligands. The nature of the monodentate P-donor ligand has a strong effect on protein kinase binding properties, most likely due to a direct interaction with the glycine-rich loop in the ATP-binding site. We furthermore discovered that PMe₃ pyridocarbazole complexes are interesting lead structures for the design of potent inhibitors for the protein kinase TrkA for which we obtained a nanomolar organometallic inhibitor.     

Proteasome inhibition induces neurite outgrowth through posttranslational modification of TrkA receptor

The ubiquitin-proteasome pathway regulates many biological processes, including protein degradation, receptor endocytosis, protein sorting, subnuclear trafficking and neuronal differentiation. While proteasome inhibition is known to induce neurite outgrowth, the signaling mechanisms that mediate these effects have not been defined. In this study, we investigated the underlying mechanisms that link proteasome inhibition with neurite generation. We found that the proteasome inhibitors, MG132 and lactacystin, induced neurite outgrowth and also activated extracellular signal-regulated kinase/mitogen activated protein kinase and phosphatidylinositol-3-kinase/AKT pathways. These proteasome inhibitors also induced phosphorylation and ubiquitination of TrkA receptors, indicating that proteasome inhibition activates the major pathways of TrkA signaling. However, in contrast to nerve growth factor stimulation, which induces internalization of surface TrkA receptors, proteasome inhibitor-induced neurite outgrowth did not require TrkA receptor internalization. These results indicate that the ubiquitin-proteasome system regulates neurite formation through posttranslational modification of TrkA receptors.     

Identification of 2-amino-5-(thioaryl)thiazoles as inhibitors of nerve growth factor receptor TrkA

2-Amino-5-(thioaryl)thiazoles are potent inhibitors of TrkA (e.g., 20h, TrkA IC(50)=0.6 nM) that show anti-proliferative effect in cellular assays. A proposed inhibitor binding mode to TrkA active site is consistent with key SAR observations.     

Discovery and in vitro evaluation of potent TrkA kinase inhibitors: oxindole and aza-oxindoles

The discovery, synthesis, potential binding mode, and in vitro kinase profile of 3-(3-bromo-4-hydroxy-5-(2'-methoxyphenyl)-benzylidene)-5-bromo-1,3-dihydro-pyrrolo[2,3-b]pyridin-2-one, 3-[(1-methyl-1H-indol-3-yl)methylene]-1,3-dihydro-2H-pyrrolo[3,2-b]-pyridin-2-one as potent TrkA inhibitors are discussed.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

Apoptotic cell death in TrkA-overexpressing cells: kinetic regulation of ERK phosphorylation and caspase-7 activation

The TrkA tyrosine kinase is activated by autophosphorylation in response to NGF, and plays an important role in cell survival, differentiation, and apoptosis. To investigate its role in cell fate determination, we produced stable TrkA-inducible SK-N-MC and U2OS cell lines using the Tet-On system. Interestingly, TrkA overexpression induced substantial cell death even in the absence of NGF, by stimulating ERK phosphorylation and caspase-7 activation leading to PARP cleavage. TrkA-mediated cell death was shown by the annexin-V binding assay to be, at least in part, apoptotic in both SK-N-MC and U2OS cells. Furthermore, the truncated form (p18) of Bax accumulated in the TrkA-induced cells, suggesting that TrkA induces mitochondria-mediated apoptosis. NGF treatment augmented the cell death induced by TrkA overexpression. This TrkA-induced cell death was blocked by the tyrosine kinase inhibitors, K-252a and GW441756. Moreover, TrkA overexpression inhibited long-term proliferation of both the neuronal SK-N-MC cells and the non-neuronal U2OS cells, suggesting a potential role of TrkA as a tumor suppressor.     

The Crystal Structures of TrkA and TrkB Suggest Key Regions for Achieving Selective Inhibition

The Trk family of neurotrophin receptors, which includes the three highly homologous proteins TrkA, TrkB and TrkC, is strongly associated with central and peripheral nervous system processes. Trk proteins are also of interest in oncology, since Trk activation has been observed in several cancer types. While Trk kinases are attractive oncology targets, selectivity might be more of an issue than for other kinases due to potential CNS side effects if several Trk kinases are simultaneously targeted. In order to address this issue, we present here the first structures of human TrkA and TrkB kinase domains and three complexes between TrkB and Trk inhibitors. These structures reveal different conformations of the kinase domain and suggest new regions of selectivity among the Trk family.     

The neurotrophin receptor, gp75, forms a complex with the receptor tyrosine kinase TrkA

The high-affinity NGF receptor is thought to be a complex of two receptors , gp75 and the tyrosine kinase TrkA, but direct biochemical evidence for such an association had been lacking. In this report, we demonstrate the existence of such a gp75-TrkA complex by a copatching technique. Gp75 on the surface of intact cells is patched with an anti- gp75 antibody and fluorescent secondary antibody, the cells are then fixed to prevent further antibody-induced redistributions, and the distribution of TrkA is probed with and anti-TrkA antibody and fluorescent secondary antibody. We utilize a baculovirus-insect cell expression of wild-type and mutated NGF receptors. TrkA and gp75 copatch in both the absence and presence of NGF. The association is specific, since gp75 does not copatch with other tyrosine kinase receptors, including TrkB, platelet-derived growth factor receptor- beta, and Torso (Tor). To determine which domains of TrkA are required for copatching, we used a series of TrkA-Tor chimeric receptors and show that the extracellular domain of TrkA is sufficient for copatching with gp75. A chimeric receptor with TrkA transmembrane and intracellular domains show partial copatching with gp75. Deletion of the intracellular domain of gp75 decreases but does not eliminate copatching. A point mutation which inactivates the TrkA kinase has no effect on copatching, indicating that this enzymatic activity is not required for association with gp75. Hence, although interactions between the gp75 and TrkA extracellular domains are sufficient for complex formation, interactions involving other receptor domains also play a role.     

Design,synthesis and SAR of substituted indoles as selective TrkA inhibitors

A series of substituted indoles were examined as selective inhibitors of tropomyosin-related kinase receptor A (TrkA), a therapeutic target for the treatment of pain. An SAR optimization campaign based on ALIS screening lead compound 1 is reported.     

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.     

The effect of new potent selective inhibitors of protein kinase C on the neutrophil respiratory burst

New potent inhibitors of protein kinase C were found to inhibit protein kinase C isolated from rat brain and human neutrophils, with a large degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. These novel compounds were potent inhibitors of the fluoride, diC8- and formyl-methionyl-leucyl-phenylalanine-mediated respiratory bursts in intact neutrophils. The opsonized zymosan-stimulated burst was only marginally affected by the compounds. These results differ from those obtained in studies with H7 and CI, (which are less potent and less specific protein kinase C inhibitors) and are consistent with the hypothesis that protein kinase C has a role in the transduction mechanism for the neutrophil oxidative burst stimulated with fluoride, formyl-methionyl-leucyl-phenylalanine and diC8.     

Trypanosome trans-sialidase targets TrkA tyrosine kinase receptor and induces receptor internalization and activation

Trypanosome trans-sialidase (TS) is a sialic acid-transferring enzyme that hydrolyzes alpha2,3-linked sialic acids and transfers them to acceptor molecules. Here we show that a highly purified recombinant TS derived from T. cruzi parasites targets TrkA receptors on TrkA-expressing PC12 cells and colocalizes with TrkA internalization and phosphorylation (pTrkA). Maackia amurensis lectin II (MAL-II) and Sambucus nigra lectin (SNA) block TS binding to TrkA-PC12 cells in a dose-dependent manner with subsequent inhibition of TS colocalization with pTrkA. Cells treated with lectins alone do not express pTrkA. The catalytically inactive mutant TSDeltaAsp98-Glu also binds to TrkA-expressing cells, but is unable to induce pTrkA. TrkA-PC12 cells treated with a purified recombinant alpha2,3-neuraminidase (Streptococcus pneumoniae) express pTrkA. Wild-type TS but not the mutant TSDeltaAsp98-Glu promotes neurite outgrowth in TrkA-expressing PC12 cells. In contrast, these effects are not observed in TrkA deficient PC12nnr5 cells but are reestablished in PC12nnr5 cells stably transfected with TrkA and are significantly blocked by inhibitors of tyrosine kinase (K-252a) and MAP/MEK protein kinase (PD98059). Together these observations suggest for the first time that hydrolysis of sialyl alpha2,3-linked beta-galactosyl residues of TrkA receptors plays an important role in TrkA receptor activation, sufficient to promote cell differentiation (neurite outgrowth) independent of nerve growth factor.     

The Csk homologous kinase associates with TrkA receptors and is involved in neurite outgrowth of PC12 cells.

Csk homologous kinase (CHK), a member of the Csk regulatory tyrosine kinase family, is expressed primarily in brain and hematopoietic cells. The role of CHK in the nervous system is as yet unknown. Using PC12 cells as a model system of neuronal cells, we show that CHK participates in signaling mediated by TrkA receptors. CHK was found to be associated with tyrosine-phosphorylated TrkA receptors in PC12 cells upon stimulation with NGF. Binding assays and far Western blotting analysis, using glutathione S-transferase fusion proteins containing the Src homology 2 (SH2) and SH3 domains of CHK, demonstrate that the SH2 domain of CHK binds directly to the tyrosine-phosphorylated TrkA receptors. Site-directed mutagenesis of TrkA cDNA, as well as phosphopeptide inhibition of the in vitro interaction of the CHK-SH2 domain or native CHK with TrkA receptors, indicated that the residue Tyr-785 on TrkA is required for its binding to the CHK-SH2 domain upon NGF stimulation. In addition, overexpression of CHK resulted in enhanced activation of the mitogen-activated protein kinase pathway upon NGF stimulation, and microinjection of anti-CHK antibodies, but not anti-Csk antibodies, inhibited neurite outgrowth of PC12 cells in response to NGF. Thus, CHK is a novel signaling molecule that participates in TrkA signaling, associates directly with TrkA receptors upon NGF stimulation, and is involved in neurite outgrowth of PC12 cells in response to NGF.     

Nerve Growth Factor Receptor TrkA,a New Receptor in Insulin Signaling Pathway in PC12 Cells

TrkA is a cell surface transmembrane receptor tyrosine kinase for nerve growth factor (NGF). TrkA has an NPXY motif and kinase regulatory loop similar to insulin receptor (INSR) suggesting that NGF→TrkA signaling might overlap with insulin→INSR signaling. During insulin or NGF stimulation TrkA, insulin receptor substrate-1 (IRS-1), INSR (and presumably other proteins) forms a complex in PC12 cells. In PC12 cells, tyrosine phosphorylation of INSR and IRS-1 is dependent upon the functional TrkA kinase domain. Moreover, expression of TrkA kinase-inactive mutant blocked the activation of Akt and Erk5 in response to insulin or NGF. Based on these data, we propose that TrkA participates in insulin signaling pathway in PC12 cells.     

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.     

Nerve growth factor signals through TrkA,phosphatidylinositol 3-kinase,and Rac1 to inactivate RhoA during the initiation of neuronal differentiation of PC12 cells

In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.     

The discovery of highly selective erbB2 (Her2) inhibitors for the treatment of cancer

The synthesis and biological evaluation of potent and selective inhibitors of the erbB2 kinase is presented. Based on the 4-anilinoquinazoline chemotype, the syntheses of several new series of erbB2 inhibitors are described with quinazoline and pyrido[4,3-d]pyrimidine cores. The vast majority of these compounds are found to be >100x selective over the closely related EGFR kinase. Two lead compounds are further shown to have low clearance and moderate bioavailability in rat.