RESUMEN

The protein kinase checkpoint kinase 1 (Chk1) has been implicated as a key regulator of cell cycle progression and DNA repair, and inhibitors of Chk1 (e.g., UCN-01 and EXEL-9844) potentiate the cytotoxic actions of chemotherapeutic drugs in tumor cells. We have examined the ability of PD-321852, a small-molecule Chk1 inhibitor, to potentiate gemcitabine-induced clonogenic death in a panel of pancreatic cancer cell lines and evaluated the relationship between endpoints associated with Chk1 inhibition and chemosensitization. Gemcitabine chemosensitization by minimally toxic concentrations of PD-321852 ranged from minimal (<3-fold change in survival) in Panc1 cells to >30-fold in MiaPaCa2 cells. PD-321852 inhibited Chk1 in all cell lines as evidenced by stabilization of Cdc25A; in combination with gemcitabine, a synergistic loss of Chk1 protein was observed in the more sensitized cell lines. Gemcitabine chemosensitization, however, did not correlate with abrogation of the S-M or G2-M checkpoint; PD-321852 did not induce premature mitotic entry in gemcitabine-treated BxPC3 or M-Panc96 cells, which were sensitized to gemcitabine 6.2- and 4.6-fold, respectively. In the more sensitized cells lines, PD-321852 not only inhibited gemcitabine-induced Rad51 focus formation and the recovery from gemcitabine-induced replication stress, as evidenced by persistence of gamma-H2AX, but also depleted these cells of Rad51 protein. Our data suggest the inhibition of this Chk1-mediated Rad51 response to gemcitabine-induced replication stress is an important factor in determining gemcitabine chemosensitization by Chk1 inhibition in pancreatic cancer cells.

Asunto(s)

Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Biocatálisis , Carbazoles/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Daño del ADN , Desoxicitidina/farmacología , Humanos , Neoplasias Pancreáticas/genética , Fosforilación/efectos de los fármacos , Gemcitabina

RESUMEN

Pyrrolo[3,4-c]carbazoles bearing solubilising basic side chains at the 8-position retain potent Wee1 and Chk1 inhibitory properties in isolated enzyme assays, and evidence of G2/M checkpoint abrogation in several cellular assays. Co-crystal structure studies confirm that the primary binding to the Wee1 enzyme is as described previously, with the C-8 side chains residing in an area of bulk tolerance.

Asunto(s)

Carbazoles/síntesis química , Carbazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Técnicas Químicas Combinatorias , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Carbazoles/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad

RESUMEN

A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones were prepared from N-substituted (5-methoxyphenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC(50) 0.057 microM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules. HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.

Asunto(s)

Carbazoles/síntesis química , Carbazoles/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Carbazoles/química , Células HT29 , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad

RESUMEN

High-throughput screening has identified a novel class of inhibitors of the checkpoint kinase Wee1, which have potential for use in cancer chemotherapy. These inhibitors are based on a 4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione template and have been shown by X-ray crystallography to bind at the ATP site of the enzyme. An extensive study of the effects of substitution around this template has been carried out, which has identified substituents which lead to improvements in potency and selectivity for Wee1. While retention of the maleimide ring and pendant 4-phenyl group is necessary for potency, replacement of the carbazole nitrogen by oxygen is well tolerated and results in improved Wee1 selectivity against the related checkpoint kinase Chk1. Wee1 potency and selectivity are also enhanced by the incorporation of lipophilic functionality at the 2'-position of the 4-phenyl ring, and Wee1 selectivity against Chk1 is favored by C3-C5 alkyl substitution of the carbazole nitrogen. These studies provide a basis for the design of active analogues of the pyrrolocarbazole lead with improved physical properties.

Asunto(s)

Derivados del Benceno/síntesis química , Carbazoles/síntesis química , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/química , Pirroles/síntesis química , Derivados del Benceno/química , Carbazoles/química , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Proteínas Quinasas/química , Pirroles/química , Relación Estructura-Actividad

RESUMEN

A series of 2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones were synthesized and evaluated for their inhibitory properties against the non-receptor kinase c-Src and the G2/M checkpoint kinase Wee1. Overall, the compounds were 10-100-fold more potent inhibitors of c-Src than Wee1, and variation of substituents on the 6-phenyl ring did not markedly alter this preference. Solubilizing substituents off the 2-anilino ring in many cases increased Wee1 activity, thus lowering this preference to about 10-fold. 5-Alkyl substituted analogs were generally Wee1 selective, but at the expense of absolute potency.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Compuestos de Anilina/química , Proteína Tirosina Quinasa CSK , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores Enzimáticos/síntesis química , Humanos , Concentración 50 Inhibidora , Naftiridinas/química , Proteínas Nucleares/metabolismo , Proteínas Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Pirimidinonas/química , Solubilidad , Relación Estructura-Actividad , Familia-src Quinasas

RESUMEN

PURPOSE: To determine maximum tolerated dose of CI-994, a novel oral histone deacetylase inhibitor, in combination with carboplatin and paclitaxel in patients with advanced solid tumors. PATIENTS AND METHODS: Patients with advanced solid tumors who had received two or fewer prior chemotherapy regimens were eligible for trial. Five cohorts of patients were treated with escalating doses (4-6 mg/m2) and alternative schedules (7 days or 14 days) of CI-994. Dose escalation of paclitaxel was performed to achieve tolerability of CI-994 with a paclitaxel dose of 225 mg/m2 when administered in combination with carboplatin. Pharmacokinetic assessment of CI-994 was performed by using liquid chromatography/mass spectrometry. Histone deacetylation inhibition was determined by Western blot analysis. RESULTS: A total of 30 patients (median age 58 years) were entered into five treatment cohorts. Maximum tolerated dose of CI-994 was determined to be 4 mg/m2 administered for 7 consecutive days following paclitaxel at a dose of 225 mg/m2 and carboplatin at an area under the curve (AUC) of 6 every 21 days. Neutropenia, thrombocytopenia, and grade 3 respiratory insufficiency limited further dose escalation of CI-994. Pharmacokinetics showed that CI-994 absorption and disposition were unaffected by carboplatin and paclitaxel coadministration. Association between histone H3 acetylation levels and disease response was suggested. A subset of patients with lymphocyte H3 acetylation levels at least 1.5-fold times baseline all achieved either a clinical response or stable disease. All evaluable patients with progressive disease (PD) had H3 acetylation levels <1.5-fold times baseline. Twenty-four of the 30 patients received greater than one cycle of treatment. Five of these patients achieved a partial response (3 nonsmall cell lung cancer, 1 colorectal cancer, and 1 unknown primary) and 2 patients achieved a complete response (esophageal and bladder cancer). CONCLUSION: The combination of CI-994 at a dose of 4 mg/m2 administered orally for 7 consecutive days can be safely coadministered with paclitaxel at a dose of 225 mg/m2 and carboplatin at an AUC of 6 on day 1 of a 21-day cycle. Evidence of antitumor activity is suggested and may correlate with histone modulation.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Acetilación , Adenocarcinoma/secundario , Administración Oral , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Benzamidas , Carboplatino/administración & dosificación , Cromatografía Líquida de Alta Presión , Estudios de Cohortes , Quimioterapia Combinada , Femenino , Histonas/metabolismo , Humanos , Masculino , Espectrometría de Masas , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/patología , Paclitaxel/administración & dosificación , Fenilendiaminas/administración & dosificación , Resultado del Tratamiento

RESUMEN

Inhibition of the constitutively active Bcr-abl tyrosine kinase(TK) by STI571 has proven to be a highly effective treatment for chronic myelogenous leukemia (CML). However, STI571 is only transiently effective in blast crisis, and drug resistance emerges by amplification of or development of mutational changes in Bcr-abl. We have screened a family of TK inhibitors of the pyrido [2,3-d]pyrimidine class, unrelated to STI571, and describe here a compound with substantial activity against STI-resistant mutant Bcr-abl proteins. This compound, PD166326, is a dual specificity TK inhibitor and inhibits src and abl in vitro with IC(50)s of 6 and 8 nM respectively. PD166326 inhibits the growth of K562 cells with IC(50) of 300 pM, leading to apoptotic G(1) arrest, whereas non-Bcr-abl cell types require >1000 times higher concentrations. We tested the effects of PD166326 on two of the clinically observed STI571-resistant Bcr-abl mutants. PD166326 potently inhibits the E255K mutant Bcr-abl protein and the growth of Bcr-ablE255K-driven cells. The T315I mutant Bcr-abl protein, which is mutated within the ATP-binding pocket, is resistant to PD166326; however, the growth of Bcr-ablT315I-driven cells is partially sensitive to this compound, likely through the inhibition of Bcr-abl effector pathways. These findings show that TK drug resistance is a structure-specific phenomenon and can be overcome by TK inhibitors of other structural classes, suggesting new approaches for future anticancer drug development. PD166326 is a prototype of a new generation of anti-Bcr-abl compounds with picomolar potency and substantial activity against STI571-resistant mutants.

Asunto(s)

Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Proteínas de Fusión bcr-abl/metabolismo , Mutación , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirimidinas/uso terapéutico , Benzamidas , Western Blotting , Ciclo Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Células K562 , Piridinas/farmacología , Pirimidinas/farmacología

RESUMEN

CI-994 or N-acetyldinaline [4-(acetylamino)-N-(2-amino-phenyl) benzamide] is an antitumor cytostatic agent currently undergoing clinical trial. Although several changes in cellular metabolism induced by the drug have been characterized, the primary molecular mechanism of its antitumor activity has been previously unknown. Here, we show that CI-994 is a histone deacetylase (HDAC) inhibitor that causes histone hyperacetylation in living cells. In assays of isolated enzymes, CI-994 inhibited HDAC-1 and HDAC-2 in a concentration-dependent fashion but had no effect on the activity of the prototypical histone acetyltransferase GCN5. Acetylated histone H3-specific Western blots were used to monitor histone acetylation in HCT-8 colon carcinoma cells treated with CI-994 in vitro. CI-994 induced hyperacetylation of H3 in a time- and dose-dependent fashion. H3 hyperacetylation was detectable as early as 30 min after the addition of CI-994 to cells. These data demonstrate that inhibition of HDAC is an early event in cells treated with CI-994 and suggest that this inhibition is mechanistically related to the antitumor activity of this compound.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Histonas/química , Fenilendiaminas/farmacología , Acetilación , Benzamidas , Western Blotting , Línea Celular Tumoral , Núcleo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas , Histonas/metabolismo , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Pruebas de Precipitina , Factores de Tiempo

RESUMEN

Chronic myelogenous leukemia (CML) is a myeloproliferative disease characterized by the BCR-ABL genetic translocation and constitutive activation of the Abl tyrosine kinase. Among members of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors, Stat5 is activated by the Bcr-Abl kinase and is implicated in the pathogenesis of CML. We recently identified PD180970 as a new and highly potent inhibitor of Bcr-Abl kinase. In this study, we show that blocking Bcr-Abl kinase activity using PD180970 in the human K562 CML cell line resulted in inhibition of Stat5 DNA-binding activity with an IC(50) of 5 nM. Furthermore, abrogation of Abl kinase-mediated Stat5 activation suppressed cell proliferation and induced apoptosis in K562 cells, but not in the Bcr-Abl-negative myeloid cell lines, HEL 92.1.7 and HL-60. Dominant-negative Stat5 protein expressed from a vaccinia virus vector also induced apoptosis of K562 cells, consistent with earlier studies that demonstrated an essential role of Stat5 signaling in growth and survival of CML cells. RNA and protein analyses revealed several candidate target genes of Stat5, including Bcl-x, Mcl-1, c-Myc and cyclin D2, which were down-regulated after treatment with PD180970. In addition, PD180970 inhibited Stat5 DNA-binding activity in cultured primary leukemic cells derived from CML patients. To detect activated Stat5 in CML patient specimens, we developed an immunocytochemical assay that can be used as a molecular end-point assay to monitor inhibition of Bcr-Abl signaling. Moreover, PD180970 blocked Stat5 signaling and induced apoptosis of STI-571 (Gleevec, Imatinib)-resistant Bcr-Abl-positive cells. Together, these results suggest that the mechanism of action of PD180970 involves inhibition of Bcr-Abl-mediated Stat5 signaling and provide further evidence that compounds in this structural class may represent potential therapeutic agents for CML.

Asunto(s)

División Celular/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Proteínas de la Leche , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridonas/farmacología , Pirimidinas/farmacología , Transactivadores/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Apoptosis/genética , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Fusión bcr-abl , Fase G1 , Humanos , Inmunohistoquímica , Factor de Transcripción STAT5 , Transactivadores/metabolismo , Células Tumorales Cultivadas

RESUMEN

Src is a non-receptor protein tyrosine kinase, the expression and activity of which is increased in >80% of human colon cancers with respect to normal colonic epithelium. Previous studies from this and other laboratories have demonstrated that Src activity contributes to tumorigenicity of established colon adenocarcinoma cell lines. Src participates in the regulation of many signal transduction pathways, among which are those leading to cellular survival. In this study, we addressed the potential role of Src activation to a specific aspect of tumor cell survival, resistance to detachment-induced apoptosis (anoikis). Using five colon tumor cell lines with different biologic properties and genetic alterations, we demonstrate that expression and activity of Src corresponds with resistance to anoikis. Enforced expression of activated Src in subclones of SW480 cells (of low intrinsic Src expression and activity) increases resistance to anoikis; whereas decreased Src expression in HT29 cells (of high Src expression and activity) by transfection with anti-sense Src expression vectors increases susceptibility to anoikis. In contrast, increasing or decreasing Src expression had no effect on susceptibility to staurosporine-induced apoptosis in attached cells. PD173955, a Src family-specific tyrosine kinase inhibitor, increases the susceptibility of HT29 cells to anoikis in a dose- and time-dependent manner. Increasing Src expression and activity led to increased phosphorylation of Akt, a mediator of cellular survival pathways, whereas decreasing Src activity led to decreased Akt phosphorylation. In colon tumor cells with high Src activity, the PI3 kinase inhibitor LY 294002 sensitized cells to anoikis. These results suggest that Src activation may contribute to colon tumor progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis.

Asunto(s)

Adenocarcinoma/patología , Anoicis/fisiología , Neoplasias del Colon/patología , Proteínas de Neoplasias/fisiología , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Sustitución de Aminoácidos , Apoptosis/efectos de los fármacos , Caspasa 3 , Caspasas/metabolismo , Adhesión Celular , Ciclo Celular , Cromonas/farmacología , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Progresión de la Enfermedad , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Genes src , Humanos , Morfolinas/farmacología , Mutación Missense , Metástasis de la Neoplasia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Piridonas/farmacología , Pirimidinas/farmacología , Proteínas Recombinantes de Fusión/fisiología , Estaurosporina/farmacología , Transfección , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Proteína bcl-X

RESUMEN

Activation of protein tyrosine kinases is prevalent in human cancers and previous studies have demonstrated that Stat3 signaling is a point of convergence for many of these tyrosine kinases. Moreover, a critical role for constitutive activation of Stat3 in tumor cell proliferation and survival has been established in diverse cancers. However, the oncogenic signaling pathways in melanoma cells remain to be fully defined. In this study, we demonstrate that Stat3 is constitutively activated in a majority of human melanoma cell lines and tumor specimens examined. Blocking Src tyrosine kinase activity, but not EGF receptor or JAK family kinases, leads to inhibition of Stat3 signaling in melanoma cell lines. Consistent with a role of Src in the pathogenesis of melanoma, we show that c-Src tyrosine kinase is activated in melanoma cell lines. Significantly, melanoma cells undergo apoptosis when either Src kinase activity or Stat3 signaling is inhibited. Blockade of Src or Stat3 is also accompanied by down-regulation of expression of the anti-apoptotic genes, Bcl-x(L) and Mcl-1. These findings demonstrate that Src-activated Stat3 signaling is important for the growth and survival of melanoma tumor cells.

Asunto(s)

Proteínas de Unión al ADN/fisiología , Melanoma/patología , Transactivadores/fisiología , Familia-src Quinasas/fisiología , Apoptosis , División Celular , ADN/metabolismo , Activación Enzimática , Receptores ErbB/fisiología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/fisiología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Piridonas/farmacología , Pirimidinas/farmacología , Factor de Transcripción STAT3 , Células Tumorales Cultivadas , Proteína bcl-X

RESUMEN

Protein kinase C delta (PKC delta) is tyrosine-phosphorylated and catalytically inactive in mouse keratinocytes transformed by a ras oncogene. In several other model systems, Src kinases are upstream regulators of PKC delta. To examine this relationship in epidermal carcinogenesis, v-ras transformed mouse keratinocytes were treated with a selective Src kinase inhibitor (PD 173958). PD 173958 decreased autophosphorylation of Src, Fyn, and Lyn kinases and prevented tyrosine phosphorylation of the Src kinase substrate p120. PD 173958 also prevented PKC delta tyrosine phosphorylation and activated PKC delta as detected by membrane translocation. Expression of keratinocyte differentiation markers increased in PD 173958-treated v-ras-keratinocytes, and fluid-filled domes emerged, indicative of tight junction formation. Antisense PKC delta or bryostatin 1 inhibited dome formation, while overexpression of PKC delta in the presence of PD 173958 enhanced the formation of domes. Plasmids encoding phenylalanine mutants of PKC delta tyrosine residues 64 and 565 induced domes in the absence of PD 173958, while phenylalanine mutants of tyrosine residues 52, 155, and 187 were inactive. Thus, Src kinase mediated post-translational modification of PKC delta on specific tyrosine residues in ras-transformed mouse keratinocytes inactivates PKC delta and contributes to alterations in the differentiated phenotype and tight junction formation associated with neoplasia.

Asunto(s)

Isoenzimas/metabolismo , Queratinocitos/enzimología , Proteína Quinasa C/metabolismo , Neoplasias Cutáneas/enzimología , Tirosina/metabolismo , Familia-src Quinasas/metabolismo , Animales , Antineoplásicos/farmacología , Brioestatinas , Diferenciación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Immunoblotting , Lactonas/farmacología , Macrólidos , Ratones , Ratones Endogámicos BALB C , Oligonucleótidos Antisentido/farmacología , Fenotipo , Fenilalanina/química , Fosforilación , Pruebas de Precipitina , Unión Proteica , Proteína Quinasa C-delta , Piridinas/farmacología , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Transfección