Discovery of novel EGFR tyrosine kinase inhibitors by structure-based virtual screening

By using of structure-based virtual screening, 13 novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors were discovered from 197,116 compounds in the SPECS database here. Among them, 8 compounds significantly inhibited EGFR kinase activity with IC(50) values lower than 10 μM. 3-{[1-(3-Chloro-4-fluorophenyl)-3,5-dioxo-4-pyrazolidinylidene]methyl}phenyl 2-thiophenecarboxylate (13), particularly, was the most potent inhibitor possessing the IC(50) value of 3.5 μM. The docking studies also provide some useful information that the docking models of the 13 compounds are beneficial to find a new path for designing novel EGFR inhibitors.     

Novel inhibitors of epidermal growth factor receptor: (4-(Arylamino)-7H-pyrrolo[2,3-d]pyrimidin-6-yl)(1H-indol-2-yl)methanones and (1H-indol-2-yl)(4-(phenylamino)thieno[2,3-d]pyrimidin-6-yl)methanones

Several members of the quinazoline class of known tyrosine kinase inhibitors are approved anticancer agents, often showing selectivity for receptors of the HER/ErbB-family. Combining structural elements of this class with the bisindolylmethanone-structure led to a series of novel compounds. These compounds inhibited EGFR in the nanomolar range. Moreover, inhibition of EGFR autophosphorylation in intact A431 cells was shown, with IC₅₀ values ranging form 0.3–1 μM for compound 42, and 0.1–0.3 μM for 45. In a panel of 42 human tumor cell lines the sensitivity profile of the novel compounds was shown to be similar to that of the quinazoline class of tyrosine kinase inhibitors lapatinib and erlotinib (Tarceva®).     

Synthesis and biological evaluation of novel tricyclic oxazine and oxazepine fused quinazolines. Part 1: Erlotinib analogs

Two series of novel tricyclic oxazine and oxazepine fused quinazolines have been designed and synthesized. The in vitro antitumor effect of the title compounds was screened on N87, A431, H1975, BT474 and Calu-3 cell lines. Compared to erlotinib and gefitinib, compounds 1a–1h were found to demonstrate more potent antitumor activities. Several derivatives could counteract EGF-induced phosphorylation of EGFR in cells, and their potency was comparable to the reference compounds. Compounds 1a–1h were chosen for further evaluation of EGFR and HER2 in vitro kinase inhibitory activity. Compounds 1b–1f, 1h effectively inhibited the in vitro kinase activity of EGFR and HER2 with similar efficacy as erlotinib and gefitinib.     

Enhancement of EGFR tyrosine kinase inhibition by C–C multiple bonds-containing anilinoquinazolines

A series of 4-anilinoquinazolines with C–C multiple bond substitutions at the 6-position were synthesized and investigated for their potential to inhibit epidermal growth factor receptor (EGFR) tyrosine kinase activity. Among the compounds synthesized, alkyne 6d and allenes 7d and 7f significantly inhibited EGFR tyrosine kinase activity. These compounds inhibited EGF-mediated phosphorylation of EGFR in A431 cells, resulting in cell-cycle arrest and apoptosis induction. The C–C multiple bonds substituted at the C-6 position of the anilinoquinazoline framework were essential for the significant inhibitory activity. Compounds with long carbon chains (n = 3–6), such as 6c–f, 7c–f, 11, and 12, displayed prolonged inhibitory activity.     

Discovery and biological evaluation of potent dual ErbB-2/EGFR tyrosine kinase inhibitors: 6-thiazolylquinazolines

We have identified a novel class of 6-thiazolylquinazolines as potent and selective inhibitors of both ErbB-2 and EGFR tyrosine kinase activity, with IC(50) values in the nanomolar range. These compounds inhibited the growth of both EGFR (HN5) and ErbB-2 (BT474) over-expressing human tumor cell lines in vitro. Using xenograft models of the same cell lines, we found that the compounds given orally inhibited in vivo tumor growth significantly compared with control animals.     

Lyso-GM3, its dimer,and multimer: their synthesis,and their effect on epidermal growth factor-induced receptor tyrosine kinase

Glycosphingolipids, particularly gangliosides, are known to modulate growth factor receptor tyrosine kinase. A well-documented example is the inhibitory effect of GM3 on kinase associated with epidermal growth factor receptor (EGFR) in human epidermoid carcinoma A431 cells. Lyso-GM3 was detected as a minor component in A431 cells, and may function as an auxiliary factor in GM3-dependent inhibition of EGFR. We studied the inhibitory effect of chemically synthesized GM3, lyso-GM3, and its derivatives, on EGFR function, based on their interaction in membrane microdomain, with the following major findings: (1) GM3, EGFR, and caveolin coexist, but tetraspanins CD9 and CD82 are essentially absent, within the same low-density membrane fraction, separated by sucrose density gradient ultracentrifugation. (2) Strong interaction between EGFR and GM3 was indicated by increasing binding of EGFR to GM3-coated polystyrene beads, in a GM3 dose-dependent manner. Confocal microscopy results suggested that three components in the microdomain (GM3, EGFR, and caveolin) are closely associated. (3) Lyso-GM3 or lyso-GM3 dimer strongly inhibited EGFR kinase activity, in a dose-dependent manner, while lyso-GM3 trimer and tetramer did not. >50 μM lyso-GM3 was cytolytic, while >50 μM lyso-GM3 dimer was not cytolytic, yet inhibited EGFR kinase strongly. Thus, lyso-GM3 and its dimer exert an auxiliary effect on GM3-induced inhibition of EGFR kinase and cell growth, and lyso-GM3 dimer may be a good candidate for pharmacological inhibitor of epidermal tumor growth.     

Synthesis and evaluation of 2,7-diamino-thiazolo[4,5-d] pyrimidine analogues as anti-tumor epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors

2,7-Diamino-thiazolo[4,5-d]pyrimidine analogues were synthesized as novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. Representative compounds showed potent and selective EGFR inhibitory activities and inhibited in vitro cellular proliferation in EGFR-overexpressing human tumor cells. The synthesis and preliminary biological, physical, and pharmacokinetic evaluation of these thiazolopyrimidine compounds are reported.     

A cell-based high-throughput screen for epidermal growth factor receptor pathway inhibitors

Epidermal growth factor receptor (EGFR) is a valid drug target for development of target-based therapeutics against non-small-cell lung cancer. In this study, we established a high-throughput cell-based assay to screen for compounds that may inhibit EGFR activation and/or EGFR-mediated downstream signaling pathway. This drug screening platform is based on the characterization of an EGFR-transfected 32D cell line (32D-EGFR). The expression of EGFR in 32D cells allowed cell proliferation in the presence of either epidermal growth factor (EGF) or interleukin 3 (IL-3) and provided a system for both screening and counterscreening of EGFR pathway-inhibitory compounds. After the completion of primary and secondary screenings in which 32D-EGFR cells were grown under the stimulation of either EGF or IL-3, 9 of 20,000 compounds were found to selectively inhibit the EGF-dependent proliferation, but not the IL-3-dependent proliferation, of 32D-EGFR cells. Subsequent analysis showed that 3 compounds of the 9 initial hits directly inhibited the kinase activity of recombinant EGFR in vitro and the phosphorylation of EGFR in H1299 cells transfected with EGFR. Thus, this 32D-EGFR assay system provides a promising approach for identifying novel EGFR and EGFR signaling pathway inhibitors with potential antitumor activity.     

Tricyclic azepine derivatives: pyrimido[4,5-b]-1,4-benzoxazepines as a novel class of epidermal growth factor receptor kinase inhibitors

A novel class of pyrimido[4,5-b]-1,4-benzoxazepines is described as inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase. Two compounds display potent EGFR inhibitory activity of less than 1 microM in cellular phosphorylation assays (IC(50) 0.47-0.69 microM) and are highly selective against a small kinase panel. Such compounds demonstrate anti-EGFR activity within a class that is different from any known EGFR inhibitor scaffolds. They also provide a basis for the design of kinase inhibitors with the desired selectivity profile.     

Synthesis and biological evaluation of substituted 6-alkynyl-4-anilinoquinazoline derivatives as potent EGFR inhibitors

A series of C-6 or C-3' alkynyl-substituted 4-anilinoquinazoline derivatives was prepared straightforwardly by a Sonogashira reaction of the corresponding bromo-substituted 4-anilinoquinazolines. Bioactive assay of these compounds for in vitro EGFR kinase inhibition demonstrated that the novel 6-hydroxypropynyl-4-anilinoquinazoline 5e was a very potent EGFR kinase inhibitor with an IC(50) of 14 nM.     

Synthesis and SAR of potent EGFR/erbB2 dual inhibitors

A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2 and EGFR kinase activity. The IC(50) values of the best compounds were below 0.10 uM. Further, several of these compounds inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.     

The protein tyrosine phosphatase TCPTP suppresses the tumorigenicity of glioblastoma cells expressing a mutant epidermal growth factor receptor

Glioblastoma multiforme (GBM) is the most aggressive type of glioma and GBMs frequently contain amplifications or mutations of the EGFR gene. The most common mutation results in a truncated receptor tyrosine kinase known as Delta EGFR that signals constitutively and promotes GBM growth. Here, we report that the 45-kDa variant of the protein tyrosine phosphatase TCPTP (TC45) can recognize Delta EGFR as a cellular substrate. TC45 dephosphorylated Delta EGFR in U87MG glioblastoma cells and inhibited mitogen-activated protein kinase ERK2 and phosphatidylinositol 3-kinase signaling. In contrast, the substrate-trapping TC45-D182A mutant, which is capable of forming stable complexes with TC45 substrates, suppressed the activation of ERK2 but not phosphatidylinositol 3-kinase. TC45 inhibited the proliferation and anchorage-independent growth of Delta EGFR cells but TC45-D182A only inhibited cellular proliferation. Notably, neither TC45 nor TC45-D182A inhibited the proliferation of U87MG cells that did not express Delta EGFR. Delta EGFR activity was necessary for the activation of ERK2, and pharmacological inhibition of ERK2 inhibited the proliferation of Delta EGFR-expressing U87MG cells. Expression of either TC45 or TC45-D182A also suppressed the growth of Delta EGFR-expressing U87MG cells in vivo and prolonged the survival of mice implanted intracerebrally with these tumor cells. These results indicate that TC45 can inhibit the Delta EGFR-mediated activation of ERK2 and suppress the tumorigenicity of Delta EGFR-expressing glioblastoma cells in vivo.     

Optimization of Substituted 6-Salicyl-4-Anilinoquinazoline Derivatives as Dual EGFR/HER2 Tyrosine Kinase Inhibitors

4-Anilinoquinazolines as an important class of protein kinase inhibitor are widely investigated for epidermal growth factor receptor (EGFR) tyrosine kinase or epidermal growth factor receptor 2 (HER2) inhibition. A series of novel 6-salicyl-4-anilinoquinazoline derivatives 9–27 were prepared and evaluated for their EGFR/HER2 tyrosine kinase inhibitory activity as well as their antiproliferative properties on three variant cancer cell lines (A431, MCF-7, and A549). The bioassay results showed most of the designed compounds exhibited moderate to potent in vitro inhibitory activity in the enzymatic and cellular assays, of which compound 21 revealed the most potent dual EGFR/HER2 inhibitory activity, with IC₅₀ values of 0.12 µM and 0.096 µM, respectively, comparable to the control compounds Erlotinib and Lapatinib. Furthermore, the kinase selectivity profile of 21 was accessed and demonstrated its good selectivity over the majority of the close kinase targets. Docking simulation was performed to position compound 21 into the EGFR/HER2 active site to determine the probable binding pose. These new findings along with molecular docking observations could provide an important basis for further development of compound 21 as a potent EGFR/HER2 dual kinase inhibitor.     

Hit discovery of 4-amino-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide: A novel EGFR inhibitor from a designed small library

Searching for hit compounds within the huge chemical space resembles the attempt to find a needle in a haystack. Cheminformatics-guided selection of few representative molecules of a rationally designed virtual combinatorial library is a powerful tool to confront this challenge, speed up hit identification and cut off costs. Herein, this approach has been applied to identify hit compounds with novel scaffolds able to inhibit EGFR kinase. From a generated virtual library, six 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were selected, synthesized and evaluated as hit EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC₅₀ 1.05–5.37 μM. Cell-based assay of the most potent EGFR inhibitor hit (10ac) confirmed its cytotoxicity against different cancerous cells. In spite of no EGFR, HER2 or VEGFR1 inhibition was elicited by 4-aryloxy-5-(thio)ureidopyrimidine derivatives, cell-based evaluation suggested them as antiproliferative hits acting by other mechanism(s). Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more optimized ligands.     

TGF-alpha-driven tumor growth is inhibited by an EGF receptor tyrosine kinase inhibitor.

The simultaneous presence of the EGFR and its ligand TGF-alpha in human tumor tissues suggests that autocrine TGF-alpha stimulation drives tumor growth. Here we show that autocrine TGF-alpha stimulation does cause increased tumor growth in vivo, an effect that was proven to be mediated via EGFR activation, and that this TGF-alpha/EGFR autocrine loop was accessible to an EGFR specific tyrosine kinase inhibitor. Clones of the EGFR expressing glioma cell line U-1242 MG were transfected with TGF-alpha cDNA using a tetracycline-inhibitory system for gene expression. TGF-alpha expression was inhibited by the presence of tetracycline, and subcutaneous tumors forming from cell lines injected into nude mice could be inhibited by feeding mice tetracycline. We confirmed that TGF-alpha mRNA and protein were present in these tumors and that, subsequently, the endogenous EGFR was activated. Tumor growth could be inhibited by an EGFR specific tyrosine kinase inhibitor of the type 4-(3-chloroanilino)-6,7-dimethoxy-quinazoline, administered daily by intraperitoneal injection, thereby interrupting the autocrine loop.     

Discovery of 5-(methylthio)pyrimidine derivatives as L858R/T790M mutant selective epidermal growth factor receptor (EGFR) inhibitors

To overcome the drug-resistance of first generation EGFR inhibitors and the nonselective toxicities of second generation inhibitors among NSCLC patients, a series of 5-(methylthio)pyrimidine derivatives were discovered as novel EGFR inhibitors, which harbored not only potent enzymatic and antiproliferative activities against EGFR^L858R/T790M mutants, but good selectivity over wide-type form of the receptor. This goal was achieved by employing structure-based drug design and traditional optimization strategies, based on WZ4002 and CO1686. These derivatives inhibited the enzymatic activity of EGFR^L858R/T790M mutants with IC₅₀ values in subnanomolar ranges, while exhibiting hundreds of fold less potency on EGFR^WT. These compounds also strongly inhibited the proliferation of H1975 non-small cell lung cancer cells bearing EGFR^L858R/T790M, while being significantly less toxic to A431 human epithelial carcinoma cells with overexpressed EGFR^WT. The EGFR kinase inhibitory and antiproliferative activities were further validated by Western blot analysis for activation of EGFR and the downstream signaling in cancer cells.     

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551-563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C(12) aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063-1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G(2)/M phase. Enhanced G(2)/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.     

Ligand-independent phosphorylation of Y869 (Y845) links mutant EGFR signaling to stat-mediated gene expression

Activating mutants of EGFR have been identified in a subset of non-small-cell lung cancers. To investigate mutant-driven signaling, we focused on Y869, a residue in the same activation loop where the L858R and L861Q mutations are located. We observed ligand-independent phosphorylation of Y869 in 32D cells EGFR(L858R) and EGFR(L861Q). The EGFR tyrosine kinase inhibitor (TKI) erlotinib inhibited Y869 P-EGFR in intact cells as well as in a cell-free kinase reaction. Expression of kinase domain of EGFR(L858R) and EGFR(L861Q) exhibited auto-phosphorylation of Y869; this was inhibited by EGFR TKIs but not by Src kinase inhibitor. P-Y859 of EGFR-mediated downstream component, STAT5, was also analyzed. Y694 P-STAT5 was eliminated by erlotinib treatment. Analysis of immune-complexes showed constitutive association of mutant EGFRs with STAT5 and Src which was unaffected by erlotinib or PP1. On the other hand, 32D-EGFR(WT) exhibited constitutive STAT5 phosphorylation and association of EGFR with JAK2. In these cells, a JAK2 inhibitor abrogated P-STAT5 whereas mutant EGFRs did not associate with JAK2. Expression of c-myc was regulated by EGFR/STAT5 signaling in cells expressing EGFR(L858R) and EGFR(L861Q). Our results suggest that ligand-independent and Src activity-independent phosphorylation of Y869 in mutant EGFR regulates STAT5 activation and c-myc expression.     

Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.