RESUMO
2',5'-Oligoadenylate synthetase (OAS) enzymes and RNase-L constitute a major effector arm of interferon (IFN)-mediated antiviral defense. OAS produces a unique oligonucleotide second messenger, 2',5'-oligoadenylate (2-5A), that binds and activates RNase-L. This pathway is down-regulated by virus- and host-encoded enzymes that degrade 2-5A. Phosphodiesterase 12 (PDE12) was the first cellular 2-5A- degrading enzyme to be purified and described at a molecular level. Inhibition of PDE12 may up-regulate the OAS/RNase-L pathway in response to viral infection resulting in increased resistance to a variety of viral pathogens. We generated a PDE12-null cell line, HeLaΔPDE12, using transcription activator-like effector nuclease-mediated gene inactivation. This cell line has increased 2-5A levels in response to IFN and poly(I-C), a double-stranded RNA mimic compared with the parental cell line. Moreover, HeLaΔPDE12 cells were resistant to viral pathogens, including encephalomyocarditis virus, human rhinovirus, and respiratory syncytial virus. Based on these results, we used DNA-encoded chemical library screening to identify starting points for inhibitor lead optimization. Compounds derived from this effort raise 2-5A levels and exhibit antiviral activity comparable with the effects observed with PDE12 gene inactivation. The crystal structure of PDE12 complexed with an inhibitor was solved providing insights into the structure-activity relationships of inhibitor potency and selectivity.
Assuntos
2',5'-Oligoadenilato Sintetase/imunologia , Antivirais/farmacologia , Endorribonucleases/imunologia , Exorribonucleases/química , Imunidade Inata , Bibliotecas de Moléculas Pequenas/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , Nucleotídeos de Adenina/imunologia , Nucleotídeos de Adenina/metabolismo , Antivirais/síntese química , Cristalografia por Raios X , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/metabolismo , Endorribonucleases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/genética , Exorribonucleases/imunologia , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , Interferon-alfa/farmacologia , Modelos Moleculares , Oligorribonucleotídeos/imunologia , Oligorribonucleotídeos/metabolismo , Poli I-C/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/metabolismo , Rhinovirus/genética , Rhinovirus/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Relação Estrutura-AtividadeRESUMO
2,3,5-Trisubstituted pyridines have been designed as potent AKT inhibitors that are selective against ROCK1 based on the comparison between AKT and ROCK1 structures. Substitution at the 2-position of the core pyridine is the key element to provide selectivity against ROCK1. An X-ray co-crystal structure of 9p in PKA supports the proposed rationale of ROCK1 selectivity.
Assuntos
Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Piridinas/química , Quinases Associadas a rho/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Humanos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/síntese química , Piridinas/farmacologia , Relação Estrutura-Atividade , Quinases Associadas a rho/metabolismoRESUMO
Insulin-like growth factor 1 receptor (IGF-1R) is an attractive target for anti-cancer therapy due to its anti-apoptotic effect on tumor cells, but inhibition of insulin receptor (IR) may have undesired metabolic consequences. The primary sequences of the ATP substrate-binding sites of these receptors are identical and the crystal structures of the activated kinase domains are correspondingly similar. Thus, most small-molecule inhibitors described to date are equally potent against the activated kinase domains of IGF-1R and IR. In contrast, the non-phosphorylated kinase domains of these receptors have several structural features that may accommodate differences in binding affinity for kinase inhibitors. We used a cell-based assay measuring IGF-1R autophosphorylation as an inhibitor screen, and identified a potent purine derivative that is selective compared to IR. Surprisingly, the compound is a weak inhibitor of the activated IGF-1R tyrosine kinase domain. Biochemical and structural studies are presented that indicate the compound preferentially binds to the ATP site of non-phosphorylated IGF-1R compared to phosphorylated IGF-1R. The potential selectivity and potency advantages of this binding mode are discussed.
Assuntos
Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/efeitos dos fármacos , Adipócitos/efeitos dos fármacos , Animais , Sítios de Ligação , Humanos , Concentração Inibidora 50 , Camundongos , Células NIH 3T3 , Fosforilação , Fosfotransferases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Aniline 'headgroups' were synthesized and incorporated into an alkynyl thienopyrimidine series of EGFR and ErbB-2 inhibitors. Potent inhibition of enzyme activity and cellular proliferation was observed. In certain instances, protein binding was reduced and oral exposure was found to be somewhat improved relative to compounds containing the reference aniline.
Assuntos
Compostos de Anilina/síntese química , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Pirimidinas/síntese química , Receptor ErbB-2/antagonistas & inibidores , Administração Oral , Compostos de Anilina/administração & dosagem , Compostos de Anilina/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Receptores ErbB/metabolismo , Inibidores do Crescimento/administração & dosagem , Inibidores do Crescimento/síntese química , Inibidores do Crescimento/farmacologia , Humanos , Camundongos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Receptor ErbB-2/metabolismoRESUMO
A novel class of pyrrolidinyl-acetyleneic thieno[3,2-d]pyrimidines has been identified which potently inhibit the EGFR and ErbB-2 receptor tyrosine kinases. Synthetic modifications of the pyrrolidine carbamate moiety result in a range of effects on enzyme and cellular potency. In addition, the impact of the absolute stereochemical configuration on cellular potency and oral mouse pharmacokinetics is described.
Assuntos
Antineoplásicos/química , Receptores ErbB/antagonistas & inibidores , Pirimidinas/farmacologia , Pirrolidinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Administração Oral , Animais , Camundongos , Farmacocinética , Pirimidinas/síntese química , Pirrolidinas/síntese química , Relação Estrutura-AtividadeRESUMO
Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC(50) values less than 1 microM against human tumor cells in vitro.
Assuntos
Antineoplásicos/farmacologia , Química Farmacêutica/métodos , Receptores ErbB/química , Pirimidinas/química , Receptor ErbB-2/antagonistas & inibidores , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacologia , Humanos , Concentração Inibidora 50 , Lapatinib , Modelos Químicos , Conformação Molecular , Quinazolinas/farmacologiaRESUMO
A novel class of substituted pyrrolidinyl-acetylenic thieno[3,2-d]pyrimidines has been identified that are potent and selective inhibitors of both EGFR/ErbB-2 receptor tyrosine kinases. The inhibitors are found to display a range of enzyme and cellular potency and also to display a varying level of covalent modification of the kinase targets. Selected molecules, including compound 15h, were found to be potent in enzymatic and cellular assays while also demonstrating exposure in the mouse from an oral dose.
Assuntos
Receptores ErbB/antagonistas & inibidores , Pirimidinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Animais , Linhagem Celular , Camundongos , Ligação Proteica , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Overexpression of AKT has an antiapoptotic effect in many cell types, and expression of dominant negative AKT blocks the ability of a variety of growth factors to promote survival. Therefore, inhibitors of AKT kinase activity might be useful as monotherapy for the treatment of tumors with activated AKT. Herein, we describe our lead optimization studies culminating in the discovery of compound 3g (GSK690693). Compound 3g is a novel ATP competitive, pan-AKT kinase inhibitor with IC 50 values of 2, 13, and 9 nM against AKT1, 2, and 3, respectively. An X-ray cocrystal structure was solved with 3g and the kinase domain of AKT2, confirming that 3g bound in the ATP binding pocket. Compound 3g potently inhibits intracellular AKT activity as measured by the inhibition of the phosphorylation levels of GSK3beta. Intraperitoneal administration of 3g in immunocompromised mice results in the inhibition of GSK3beta phosphorylation and tumor growth in human breast carcinoma (BT474) xenografts.
Assuntos
Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Feminino , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Camundongos SCID , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Especificidade por SubstratoRESUMO
Akt kinases 1, 2, and 3 are important regulators of cell survival and have been shown to be constitutively active in a variety of human tumors. GSK690693 is a novel ATP-competitive, low-nanomolar pan-Akt kinase inhibitor. It is selective for the Akt isoforms versus the majority of kinases in other families; however, it does inhibit additional members of the AGC kinase family. It causes dose-dependent reductions in the phosphorylation state of multiple proteins downstream of Akt, including GSK3 beta, PRAS40, and Forkhead. GSK690693 inhibited proliferation and induced apoptosis in a subset of tumor cells with potency consistent with intracellular inhibition of Akt kinase activity. In immune-compromised mice implanted with human BT474 breast carcinoma xenografts, a single i.p. administration of GSK690693 inhibited GSK3 beta phosphorylation in a dose- and time-dependent manner. After a single dose of GSK690693, >3 micromol/L drug concentration in BT474 tumor xenografts correlated with a sustained decrease in GSK3 beta phosphorylation. Consistent with the role of Akt in insulin signaling, treatment with GSK690693 resulted in acute and transient increases in blood glucose level. Daily administration of GSK690693 produced significant antitumor activity in mice bearing established human SKOV-3 ovarian, LNCaP prostate, and BT474 and HCC-1954 breast carcinoma xenografts. Immunohistochemical analysis of tumor xenografts after repeat dosing with GSK690693 showed reductions in phosphorylated Akt substrates in vivo. These results support further evaluation of GSK690693 as an anticancer agent.
Assuntos
Antineoplásicos/farmacologia , Oxidiazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Feminino , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Oxidiazóis/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678-1020) with a structurally related propargylic amine (8). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure-activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.
Assuntos
Alcinos/metabolismo , Compostos de Anilina/metabolismo , Receptores ErbB/metabolismo , Modelos Moleculares , Pirimidinas/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Feminino , Isatina/análogos & derivados , Isatina/metabolismo , Espectrometria de Massas , Camundongos , Camundongos SCID , Estrutura Molecular , Pirimidinas/toxicidade , Receptores Proteína Tirosina Quinases/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The goal of this study was to characterize the effects of non-small cell lung carcinoma (NSCLC)-associated mutations in epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2) on interactions with the dual tyrosine kinase inhibitor lapatinib. Biochemical studies show that commonly observed variants of EGFR [G719C, G719S, L858R, L861Q, and Delta746-750 (del15)] are enzyme activating, increasing the tyrosine kinase V(max) and increasing the K(m)((app)) for ATP. The point mutations G719C and L861Q had minor effects on lapatinib K(i)s, whereas EGFR mutations L858R and del15 had a higher K(i) for lapatinib than wild-type EGFR. Structural analysis of wild-type EGFR-lapatinib complexes and modeling of the EGFR mutants were consistent with these data, suggesting that loss of structural flexibility and possible stabilization of the active-like conformation could interfere with lapatinib binding, particularly to the EGFR deletion mutants. Furthermore, EGFR deletion mutants were relatively resistant to lapatinib-mediated inhibition of receptor autophosphorylation in recombinant cells expressing the variants, whereas EGFR point mutations had a modest or no effect. Of note, EGFR T790M, a receptor variant found in patients with gefitinib-resistant NSCLC, was also resistant to lapatinib-mediated inhibition of receptor autophosphorylation. Two HER2 insertional variants found in NSCLC were less sensitive to lapatinib inhibition than two HER2 point mutants. The effects of lapatinib on the proliferation of human NSCLC tumor cell lines expressing wild-type or variant EGFR and HER2 cannot be explained solely on the basis of the biochemical activity or receptor autophosphorylation in recombinant cells. These data suggest that cell line genetic heterogeneity and/or multiple determinants modulate the role played by EGFR/HER2 in regulating cell proliferation.
Assuntos
Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Genes erbB-2 , Polimorfismo de Nucleotídeo Único/fisiologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Animais , Células CHO , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/efeitos dos fármacos , Cricetinae , Cricetulus , Receptores ErbB/química , Gefitinibe , Humanos , Lapatinib , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/genética , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Receptor ErbB-2/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Synthetic modifications on a 6-furanylquinazoline scaffold to optimize the dual ErbB-1/ErbB-2 tyrosine kinase inhibition afforded consistent SAR whereby a 4-(3-fluorobenzyloxy)-3-haloanilino provided the best enzyme potency and cellular selectivity. Changes made to the 6-furanyl group had little impact on the enzyme activity, but appeared to dramatically affect the cellular efficacy. The discovery of lapatinib emerged from this work.
Assuntos
Receptores ErbB/antagonistas & inibidores , Furanos/química , Furanos/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Furanos/síntese química , Humanos , Concentração Inibidora 50 , Lapatinib , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Quinazolinas/síntese química , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Relação Estrutura-AtividadeRESUMO
Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appended to this ring was favored. Selected molecules in the series demonstrated some activity against human tumor cell lines.
Assuntos
Alcinos/química , Receptores ErbB/antagonistas & inibidores , Pirimidinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Estrutura Molecular , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
The discovery, synthesis, potential binding mode, and in vitro kinase profile of several pyrido[1',2':1,5]pyrazolo[3,4-d]pyrimidines as potent kinase inhibitors are discussed.
Assuntos
Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/efeitos dos fármacos , Pirimidinas/síntese química , Pirimidinas/farmacologia , Humanos , Ligação de Hidrogênio , Estrutura Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
GW572016 (Lapatinib) is a tyrosine kinase inhibitor in clinical development for cancer that is a potent dual inhibitor of epidermal growth factor receptor (EGFR, ErbB-1) and ErbB-2. We determined the crystal structure of EGFR bound to GW572016. The compound is bound to an inactive-like conformation of EGFR that is very different from the active-like structure bound by the selective EGFR inhibitor OSI-774 (Tarceva) described previously. Surprisingly, we found that GW572016 has a very slow off-rate from the purified intracellular domains of EGFR and ErbB-2 compared with OSI-774 and another EGFR selective inhibitor, ZD-1839 (Iressa). Treatment of tumor cells with these inhibitors results in down-regulation of receptor tyrosine phosphorylation. We evaluated the duration of the drug effect after washing away free compound and found that the rate of recovery of receptor phosphorylation in the tumor cells reflected the inhibitor off-rate from the purified intracellular domain. The slow off-rate of GW572016 correlates with a prolonged down-regulation of receptor tyrosine phosphorylation in tumor cells. The differences in the off-rates of these drugs and the ability of GW572016 to inhibit ErbB-2 can be explained by the enzyme-inhibitor structures.
Assuntos
Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Quinazolinas/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , Cloridrato de Erlotinib , Humanos , Cinética , Lapatinib , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Oncogênicas v-erbB/antagonistas & inibidores , Conformação Proteica , Estrutura Secundária de Proteína , Quinazolinas/metabolismo , Quinazolinas/farmacologia , Especificidade por SubstratoRESUMO
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.
Assuntos
Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Receptor trkA/antagonistas & inibidores , Quinases relacionadas a CDC2 e CDC28/antagonistas & inibidores , Quinase 2 Dependente de Ciclina , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Indóis/síntese química , Indóis/química , Modelos Moleculares , Estrutura Molecular , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
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.
Assuntos
Antineoplásicos/síntese química , Receptores ErbB/antagonistas & inibidores , Quinazolinas/síntese química , Quinazolinas/farmacocinética , Receptor ErbB-2/antagonistas & inibidores , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais/tratamento farmacológico , Quinazolinas/farmacologia , Relação Estrutura-Atividade , Transplante HeterólogoRESUMO
Epidermal growth factor receptor (EGFR), ErbB-2, and ErbB-4 are members of the type 1 receptor tyrosine kinase family. Overexpression of these receptors, especially ErbB-2 and EGFR, has been implicated in multiple forms of cancer. Inhibitors of EGFR tyrosine kinase activity are being evaluated clinically for cancer therapy. The potency and selectivity of these inhibitors may affect the efficacy and toxicity of therapy. Here we describe the expression, purification, and biochemical comparison of EGFR, ErbB-2, and ErbB-4 intracellular domains. Despite their high degree of sequence homology, the three enzymes have significantly different catalytic properties and substrate kinetics. For example, the catalytic activity of ErbB-2 is less stable than that of EGFR. ErbB-2 uses ATP-Mg as a substrate inefficiently compared with EGFR and ErbB-4. The three enzymes have very similar substrate preferences for three optimized peptide substrates, but differences in substrate synergies were observed. We have used the biochemical and kinetic parameters determined from these studies to develop an assay system that accurately measures inhibitor potency and selectivity between the type 1 receptor family. We report that the selectivity profile of molecules in the 4-anilinoquinazoline series can be modified through specific aniline substitutions. Moreover, these compounds have activity in whole cells that reflect the potency and selectivity of target inhibition determined with this assay system.