Evaluation of Indole Esters As Inhibitors of p60c-Src Receptor Tyrosine Kinase and Investigation of the Inhibition Using Receptor Docking Studies

Several indole esters were tested as inhibitors of tyrosine kinase p60c-Src. Compound (4) was found fairly active against the enzyme with IC50=1.34?μM. DOCK methodology was used to asses our inhibitors for their inhibitory potency against tyrosine kinase. The docking results showed that compounds (4), (25) and (26) were bound to the active site of the enzyme Lys 295 of p60c-Src tyrosine kinase. Both activity and docking studies showed a parallel result, with compound (4) having a better interaction with the enzyme active site and also greater activity than the other compounds, indicating a potential role as new lead inhibitor.     

Discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase

We report here the discovery of novel purine derivatives with potent and selective inhibitory activity against c-Src tyrosine kinase by adopting a strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and bioassay. Thirty two compounds were selected and synthesized. All compounds showed potent inhibitory activity against c-Src kinase with IC₅₀ values ranging from 3.14 μM to 0.02 μM. Compound 5i was identified as one of the most potent agent with an IC₅₀ 120 times lower than those of the hits. The high hit rate (100%) and the potency of the new Src kinase inhibitors demonstrated the efficiency of the strategy for the focused library design and virtual screening. The novel active chemical entities reported here should be good leads for further development of purine-based anticancer drugs targeting Src tyrosine kinase.     

Functional characterization of peanut serine/threonine/tyrosine protein kinase: molecular docking and inhibition kinetics with tyrosine kinase inhibitors

Serine/threonine/tyrosine (STY) protein kinase from peanut is developmentally regulated and is induced by abiotic stresses. In addition, STY protein kinase activity is regulated by tyrosine phosphorylation. Kinetic mechanism of plant dual specificity protein kinases is not studied so far. Recombinant STY protein kinase occurs as a monomer in solution as shown by gel filtration chromatography. The relative phosphorylation rate of kinase against increasing enzyme concentrations follows a first-order kinetics indicating an intramolecular phosphorylation mechanism. Moreover, the active recombinant STY protein kinase could not transphosphorylate a kinase-deficient mutant of STY protein kinase. Molecular docking studies revealed that the tyrosine kinase inhibitors bind the protein kinase at the same region as ATP. STY protein kinase activity was inhibited by the tyrosine kinase inhibitors, and the inhibitor potency series against the recombinant STY protein kinase was tyrphostin > genistein > staurosporine. The inhibition constant (K(i)), and the IC(50) value of STY protein kinase for tyrosine kinase inhibitors with ATP and histone are discussed. All the inhibitors competed with ATP. Genistein was an uncompetitive inhibitor with histone, whereas staurosporine and tyrphostin were linear mixed type noncompetitive inhibitors with histone. Molecular docking and kinetic analysis revealed that Y148F mutant of the "ATP-binding loop" and Y297F mutant of the "activation loop" showed a dramatic increase in K(i) values for genistein and tyrphostin with respect to wild-type STY protein kinase. Data presented here provide the direct evidence on the mechanism of inhibition of plant protein kinases by tyrosine kinase inhibitors. This study also suggests that tyrosine kinase inhibitors may be useful in unraveling the plant tyrosine phosphorylation signaling cascades.     

Discovery and SAR of 1,3,4-thiadiazole derivatives as potent Abl tyrosine kinase inhibitors and cytodifferentiating agents

A series of substituted benzoylamino-2-[(4-benzyl)thio]-1,3,4-thiadiazoles has been discovered as potent Abl tyrosine kinase inhibitors. Molecular docking simulations on the Abl tyrosine kinase were conducted in order to rationalize the SAR of the synthesized inhibitors. The most active compound identified from the enzymatic screening (6a) showed interesting inhibitory activity on Imatinib-sensitive murine myeloid 3B clone and Bcr-Abl-independent Imatinib-resistant leukemia cells. Surprisingly, 6a was also proved to act as differentiating inducers in human promyelocytic leukemia cells (HL-60).     

Identification of potent c-Src inhibitors strongly affecting the proliferation of human neuroblastoma cells

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.     

Synthesis and pharmacological evaluation of thieno[2,3-b]pyridine derivatives as novel c-Src inhibitors

Among the recently investigated targets for cancer therapy is the c-Src non-receptor tyrosine kinase. Indeed research around deregulated activity of this enzyme has proven its role in tumor progression, while the beneficial effects of c-Src inhibitors in several pathological models has also been demonstrated. We report here the preparation and pharmacological profile of a novel series of c-Src inhibitors that was elaborated around a 3-amino-thieno[2,3-b]pyridine discovered during an HTS campaign. c-Src enzyme inhibition and c-Src inhibition were investigated in a series of related compounds derived from the initial hit. Molecular modeling as well as X-ray studies on one active compound allowed us to hypothesize on ligand orientation and interactions within the ATP hydrophobic pocket. Design and synthesis of structural analogs then led to new ligands possessing quite efficient enzymatic and c-Src inhibition. The structure-activity elements disclosed in this study shed light on the role played by substituents on the thienopyridine ring as well as the impact of other aromatic moieties in the molecule when interacting with the enzyme.     

c-Src-dependent tyrosine phosphorylation of IKKbeta is involved in tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 expression

The signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced intercellular adhesion molecule-1 (ICAM-1) expression was further studied in human A549 epithelial cells. TNF-alpha- or 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ICAM-1 promoter activity was inhibited by a protein kinase C (PKC) inhibitor (staurosporine), tyrosine kinase inhibitors (genistein and herbimycin A), or an Src-specific tyrosine kinase inhibitor (PP2). TNF-alpha- or TPA-induced IkappaBalpha kinase (IKK) activation was also blocked by these inhibitors, which slightly reversed TNF-alpha-induced but completely reversed TPA-induced IkappaBalpha degradation. c-Src and Lyn, two members of the Src kinase family, were abundantly expressed in A549 cells, and their activation by TNF-alpha or TPA was inhibited by the same inhibitors. Furthermore, the dominant-negative c-Src (KM) mutant inhibited induction of ICAM-1 promoter activity by TNF-alpha or TPA. Overexpression of the constitutively active PKC or wild-type c-Src plasmids induced ICAM-1 promoter activity, this effect being inhibited by the dominant-negative c-Src (KM) or IKKbeta (KM) mutant but not by the nuclear factor-kappaB-inducing kinase (NIK) (KA) mutant. The c-Src (KM) mutant failed to block induction of ICAM-1 promoter activity caused by overexpression of wild-type NIK. In co-immunoprecipitation and immunoblot experiments, IKK was found to be associated with c-Src and to be phosphorylated on tyrosine residues after TNF-alpha or TPA treatment. Two tyrosine residues, Tyr188 and Tyr199, near the activation loop of IKKbeta, were identified as being important for NF-kappaB activation. Substitution of these residues with phenylalanines abolished ICAM-1 promoter activity and c-Src-dependent phosphorylation of IKKbeta induced by TNF-alpha or TPA. These data suggest that, in addition to activating NIK, TNF-alpha also activates PKC-dependent c-Src. These two pathways converge at IKKbeta and go on to activate NF-kappaB, via serine phosphorylation and degradation of IkappaB-alpha, and, finally, to initiate ICAM-1 expression.     

Rational modification of semaxanib and sunitinib for developing a tumor growth inhibitor targeting ATP binding site of tyrosine kinase

Analysis of the crystal structure of tyrosine kinase in complexation with an ATP analogue, supplemented with the molecular docking studies of semaxanib and sunitinib in the ATP binding site of the enzyme enabled us to make design of a series of tyrosine kinase inhibitors. The combination of pyrrole and indolinone in one molecule and placement of appropriate substituent thereof made the molecule compatible for the hydrophobic sub-pocket of the enzyme. Screening of the compounds over 60 cell line panel of human tumor cell lines identified compound 3a that exhibited GI₅₀ 35?nM and 63?nM against MCF7 and MDA-MB-468 cell lines of breast cancer.     

Evaluation of the combination mode and features of p38 MAPK inhibitors: construction of different pharmacophore models and molecular docking

Abnormal expression of tumour necrosis factor-α (TNF-α) can lead to various pathological reactions, such as arthritis, psoriasis, krone disease, etc. p38 mitogen-activated protein kinase (p38 MAPK) is an important signal transduction enzyme that plays important roles in influencing the release of intracellular TNF-α factor. It is very meaningful to study the targeting kinase with specific inhibitors in the treatment of related diseases. In order to achieve a deeper insight, it is necessary to analyse the structural characteristics and the action mode of the p38 MAPK inhibitors in the active site. In the study, a ligand-based common feature pharmacophore model and the receptor structure-based pharmacophore model were constructed, respectively. Their common chemical features consisted of the hydrophobic groups (H) and the hydrogen bond acceptors (A), and kept the consistency of spatial structure distribution. Then, the molecular docking and molecular dynamics simulation were performed with the eight training set compounds. The binding characteristics of molecules binding were described in the topological region of the active site. Finally, the structure–activity relationship (SAR) was obtained by analysing docking results with the different pharmacophore models. This research leads to the proposal of an interaction model in the p38 MAPK active site and provides guidance for the screening and design of more potent and selective p38 MAPK inhibitors.     

Structural basis for the recognition of c-Src by its inactivator Csk

The catalytic activity of the Src family of tyrosine kinases is suppressed by phosphorylation on a tyrosine residue located near the C terminus (Tyr 527 in c-Src), which is catalyzed by C-terminal Src Kinase (Csk). Given the promiscuity of most tyrosine kinases, it is remarkable that the C-terminal tails of the Src family kinases are the only known targets of Csk. We have determined the crystal structure of a complex between the kinase domains of Csk and c-Src at 2.9 A resolution, revealing that interactions between these kinases position the C-terminal tail of c-Src at the edge of the active site of Csk. Csk cannot phosphorylate substrates that lack this docking mechanism because the conventional substrate binding site used by most tyrosine kinases to recognize substrates is destabilized in Csk by a deletion in the activation loop.     

Agonist-dependent phosphorylation of the G protein-coupled receptor kinase 2 (GRK2) by Src tyrosine kinase

GRK2 is a member of the G protein-coupled receptor kinase (GRK) family, which phosphorylates the activated form of a variety of G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. It has been recently reported that stimulation of the mitogen-activated protein kinase cascade by GPCRs involves tyrosine phosphorylation of docking proteins mediated by members of the Src tyrosine kinase family. In this report, we have investigated the possible role of c-Src in modulating GRK2 function. We demonstrate that c-Src can directly phosphorylate GRK2 on tyrosine residues, as shown by in vitro experiments with purified proteins. The phosphorylation reaction exhibits an apparent K(m) for GRK2 of 12 nM, thus suggesting a physiological relevance in living cells. Consistently, overexpression of the constitutively active c-Src Y527F mutant in COS-7 cells leads to tyrosine phosphorylation of co-expressed GRK2. In addition, GRK2 can be detected in phosphotyrosine immunoprecipitates from HEK-293 cells transiently transfected with this Src mutant. Interestingly, phosphotyrosine immunoblots reveal a rapid and transient increase in GRK2 phosphorylation upon agonist stimulation of beta(2)-adrenergic receptors co-transfected with GRK2 and wild type c-Src in COS-7 cells. This tyrosine phosphorylation is maximal within 5 min of isoproterenol stimulation and reaches values of approximately 5-fold over basal conditions. Furthermore, GRK2 phosphorylation on tyrosine residues promotes an increased kinase activity toward its substrates. Our results suggest that GRK2 phosphorylation by c-Src is inherent to GPCR activation and put forward a new mechanism for the regulation of GPCR signaling.     

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.     

Synthesis and biological evaluation of benzamides and benzamidines as selective inhibitors of VEGFR tyrosine kinases

A series of benzamidines and benzamides was synthesized as selective inhibitors of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, and tested for inhibitory activity toward autophosphorylation by the enzyme assay. Selective inhibition of VEGFR-2 tyrosine kinase was observed in the salicylic amide 4e and the anthranilic amidine 5a, and their percent inhibitions of VEGFR-2 tyrosine kinase were 44-60% at a 10 microM concentration of compounds. The salicylic amide 4a showed inhibition of both VEGFR-1 and VEGFR-2 tyrosine kinases at a 10 microM concentration.     

Tyrosine kinase inhibition effects of novel Pyrazolo[1,5-a]pyrimidines and Pyrido[2,3-d]pyrimidines ligand: Synthesis,biological screening and molecular modeling studies

Tyrosine kinases are one of the most critical mediators in the signaling path way. Late studies have proved the part of tyrosine kinases in the pathophysiology of cancer diseases. This current research paper has focused on investigating the novel Pyrazolo[1,5-a]pyrimidines and Pyrido[2,3-d]pyrimidines as a small molecules that can inhibit tyrosine kinase in cancer cells. NCI protocol was applied to test the antitumor activity of such compounds. Leukemia and renal cancer cell lines proved to be sensitive to some derivatives such as 6b–d, 9a and 11 with GI% values ranging from 30.4 to 41.3%. In addition, compound 11 proved to be the most active against MCF-7 with GI% 62.5. The synthesized compounds were also evaluated for their inhibitory effects against EGFR kinase enzyme. Compound 9b proved to be the most active one among the synthesized series with inhibition % value of 81.72 at 25 nM concentration and IC₅₀ 8.4 nM which is very close to the reference drug Sorafenib. In vitro cytotoxicity test was also performed using the MCF-7 breast cell line. Computer modeling using the active site of tyrosine kinase as a template and the most active tyrosine kinase inhibitors were calculated. Docking studies of the synthesized compounds into the active site of EGFR kinase domain showed good agreement with the obtained biological results.     

Pyrazolo pyrimidine-type inhibitors of SRC family tyrosine kinases promote ovarian steroid-induced differentiation of human endometrial stromal cells in vitro

Reversible protein tyrosine phosphorylation, coordinately controlled by protein tyrosine kinases and phosphatases, is a critical element in signal transduction pathways regulating a wide variety of biological processes, including cell growth, differentiation, and tumorigenesis. We have previously reported that c-Src belonging to the Src family tyrosine kinase (SFK) becomes dephosphorylated at tyrosine 530 (Y530) and thereby activated during progestin-induced differentiation of human endometrial stromal cells (i.e., decidualization). In this study, to elucidate the role of decidual c-Src activation, we examined whether 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), both potent and selective SFK inhibitors, affected the ovarian steroid-induced decidualization in vitro. Unexpectedly, PP1 paradoxically increased the kinase activity of decidual c-Src together with dephosphorylation of Y530 in the presence of ovarian steroids. Concomitantly, PP1 enhanced morphological and functional decidualization, as determined by induction of decidualization markers, such as insulin-like growth factor binding protein-1 and prolactin. PP2 also advanced decidualization along with up-regulation of the active form of c-Src whose Y-530 was dephosphorylated. In contrast to PP1 and PP2, herbimycin A, a tyrosine kinase inhibitor with less specificity for SFKs, showed little enhancing effect on the expression of both IGFBP-1 and active c-Src. These results suggest that SFKs, including c-Src, may play a significant role in stromal cell differentiation, providing a clue for a possible therapeutic strategy to modulate endometrial function by targeting signaling pathway(s) involving SFKs.     

Lauryl gallate inhibits the activity of protein tyrosine kinase c-Src purified from human platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu,Na,Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

Lauryl Gallate Inhibits the Activity of Protein Tyrosine Kinase c-Src Purified from Human Platelets

The inhibitory effect of gallic acid (3,4,5-trihydroxybenzoic acid), and its ester derivatives methyl, propyl, octyl and lauryl has been tested on the tyrosine kinase activity of affinity purified c-Src from human platelets, using the artificial substrate Poly (Glu.Na, Tyr) 4:1. When tested as inhibitor of the autophosphorylation of the enzyme and the phosphorylation of the protein tyrosine phosphatase SHP-1 by c-Src, lauryl gallate was found to be a more potent inhibitor than other widely used protein tyrosine kinase (PTK) inhibitors such as genistein and herbimycin A. However, lauryl gallate did not inhibit the activity of the serine threonine kinases protein kinase A (PKA) and casein kinase II (CKII) from rat brain.     

Insilico docking study of compounds elucidated from helicteres isora fruits with ampkinase- insulin receptor

Insulin receptor (IR) proteins were essential intracellular signaling peptides in the insulin action cascade. Insulin receptor substrate proteins (IRS-1and IRS-2) serve and regulate the insulin level in the normal insulin action. The broad role of IRS-1 and IRS-2 in cell growth and survival reveals a common regulatory pathway linking development, somatic growth, fertility, neuronal proliferation, and aging to the core mechanisms used by vertebrates for nutrient sensing. Such type of proteins were cyclic adenosine monophosphate-activated protein kinase, this proteins play a key role in the insulin response and regulation. Type -2 Diabetes mellitus occurs during prolonged periods of peripheral insulin resistance due to inactivation of IRS proteins. The compounds isolated from the medicinal plants were safer than synthetic drugs and possess high bio activity. In the present study, four compounds were elucidated from fruits of Helicteres isora. The elucidated compounds were evaluated for the antidiabetic activity using in silico docking study. The receptor was analyzed for the active site and pocket finder tools. The aminoacids such as Phenylalanine, Lysine, Glutamic acid and Asparigine were predicted as active site binding residues. Docking studies were done through Autodock 4 software. All the compounds from fruits of Helicteres isora showed good docking profiles with AMP Kinase, except compound-3 (1,2,3,4-tetrahydro-1,5,6,8-tetramethyl-7-(2-methylprop-1-enylnaphthalene-4-ylpivalate). Finally the result from the study demonstrates that the HS-1, HS-2 and HS-4 posses potent anti diabetic activity against type-2 diabetes mellitus through drug action on AMP kinase cascade system.     

Interferon-gamma-induced epithelial ICAM-1 expression and monocyte adhesion. Involvement of protein kinase C-dependent c-Src tyrosine kinase activation pathway.

Interferon-gamma (IFN-gamma) induced intercellular adhesion molecule-1 (ICAM-1) expression in human NCI-H292 epithelial cells, as shown by enzyme-linked immunosorbent assay and immunofluorescence staining. The enhanced ICAM-1 expression resulted in increased adhesion of U937 cells to NCI-H292 cells. Tyrosine kinase inhibitors (genistein or herbimycin), Src family inhibitor (PP2), or a phosphatidylinositol-phospholipase C inhibitor (U73122) attenuated the IFN-gamma-induced ICAM-1 expression. Protein kinase C (PKC) inhibitors (staurosporine or Ro 31-8220) also inhibited IFN-gamma-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression; this effect was inhibited by tyrosine kinase or Src inhibitor. ICAM-1 promoter activity was enhanced by IFN-gamma and TPA in cells transfected with pIC339-Luc, containing the downstream NF-kappaB and gamma-activated site (GAS) sites, but not in cells transfected with GAS-deletion mutant, pIC135 (DeltaAP2). Electrophoretic gel mobility shift assay demonstrated that GAS-binding complexes in IFN-gamma-stimulated cells contained STAT1alpha. The IFN-gamma-induced ICAM-1 promoter activity was inhibited by tyrosine kinase inhibitors, a phosphatidylinositol-phospholipase C inhibitor, or PKC inhibitors, and the TPA-induced ICAM-1 promoter activity was also inhibited by tyrosine kinase inhibitors. Cotransfection with a PLC-gamma2 mutant inhibited IFN-gamma- but not TPA-induced ICAM-1 promoter activity. However, cotransfection with dominant negative mutants of PKCalpha or c-Src inhibited both IFN-gamma- and TPA-induced ICAM-1 promoter activity. The ICAM-1 promoter activity was stimulated by cotransfection with wild type PLC-gamma2, PKCalpha, c-Src, JAK1, or STAT1. An immunocomplex kinase assay showed that both IFN-gamma and TPA activated c-Src and Lyn activities and that these effects were inhibited by staurosporine and herbimycin. Thus, in NCI-H292 epithelial cells, IFN-gamma activates PLC-gamma2 via an upstream tyrosine kinase to induce activation of PKC-alpha and c-Src or Lyn, resulting in activation of STAT1alpha, and GAS in the ICAM-1 promoter, followed by initiation of ICAM-1 expression and monocyte adhesion.