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1.
J Med Chem ; 66(7): 4888-4909, 2023 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-36940470

RESUMEN

Immune activating agents represent a valuable class of therapeutics for the treatment of cancer. An area of active research is expanding the types of these therapeutics that are available to patients via targeting new biological mechanisms. Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of immune signaling and a target of high interest for the treatment of cancer. Herein, we present the discovery and optimization of novel amino-6-aryl pyrrolopyrimidine inhibitors of HPK1 starting from hits identified via virtual screening. Key components of this discovery effort were structure-based drug design aided by analyses of normalized B-factors and optimization of lipophilic efficiency.


Asunto(s)
Proteínas Serina-Treonina Quinasas , Transducción de Señal , Humanos , Proteínas Serina-Treonina Quinasas/metabolismo , Pirroles/farmacología
2.
Nat Cancer ; 3(6): 710-722, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35726063

RESUMEN

Lorlatinib is currently the most advanced, potent and selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor for the treatment of ALK-positive non-small cell lung cancer in the clinic; however, diverse compound ALK mutations driving therapy resistance emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations in patients, following treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. We further identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R versus I1171N/S/T compound ALK mutations. Structural analysis revealed increased potency against compound mutations through improved inhibition of either G1202R or I1171N/S/T mutant kinases. Overall, we propose a classification of heterogenous ALK compound mutations enabling the development of distinct therapeutic strategies for precision targeting following sequential tyrosine kinase inhibitors.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Aminopiridinas , Quinasa de Linfoma Anaplásico/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Humanos , Lactamas , Lactamas Macrocíclicas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles
3.
Mol Cancer Ther ; 21(1): 3-15, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34737197

RESUMEN

Protein arginine methyltransferase 5 (PRMT5) overexpression in hematologic and solid tumors methylates arginine residues on cellular proteins involved in important cancer functions including cell-cycle regulation, mRNA splicing, cell differentiation, cell signaling, and apoptosis. PRMT5 methyltransferase function has been linked with high rates of tumor cell proliferation and decreased overall survival, and PRMT5 inhibitors are currently being explored as an approach for targeting cancer-specific dependencies due to PRMT5 catalytic function. Here, we describe the discovery of potent and selective S-adenosylmethionine (SAM) competitive PRMT5 inhibitors, with in vitro and in vivo characterization of clinical candidate PF-06939999. Acquired resistance mechanisms were explored through the development of drug resistant cell lines. Our data highlight compound-specific resistance mutations in the PRMT5 enzyme that demonstrate structural constraints in the cofactor binding site that prevent emergence of complete resistance to SAM site inhibitors. PRMT5 inhibition by PF-06939999 treatment reduced proliferation of non-small cell lung cancer (NSCLC) cells, with dose-dependent decreases in symmetric dimethyl arginine (SDMA) levels and changes in alternative splicing of numerous pre-mRNAs. Drug sensitivity to PF-06939999 in NSCLC cells associates with cancer pathways including MYC, cell cycle and spliceosome, and with mutations in splicing factors such as RBM10. Translation of efficacy in mouse tumor xenograft models with splicing mutations provides rationale for therapeutic use of PF-06939999 in the treatment of splicing dysregulated NSCLC.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , S-Adenosilmetionina/metabolismo , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular , Resistencia a Medicamentos , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones
4.
Cancer Cell ; 39(10): 1404-1421.e11, 2021 10 11.
Artículo en Inglés | MEDLINE | ID: mdl-34520734

RESUMEN

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.


Asunto(s)
Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Femenino , Humanos , Masculino , Neoplasias/inmunología
5.
J Med Chem ; 64(13): 9056-9077, 2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-34110834

RESUMEN

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.


Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inyecciones Intravenosas , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Células Tumorales Cultivadas
6.
J Biol Chem ; 294(23): 9029-9036, 2019 06 07.
Artículo en Inglés | MEDLINE | ID: mdl-31018963

RESUMEN

Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) is a Ser/Thr kinase that operates via the c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways to dampen the T-cell response and antitumor immunity. Accordingly, selective HPK1 inhibition is considered a means to enhance antitumor immunity. Sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor approved for the management of gastrointestinal stromal tumors (GISTs), renal cell carcinoma (RCC), and pancreatic cancer, has been reported to inhibit HPK1 in vitro In this report, we describe the crystal structures of the native HPK1 kinase domain in both nonphosphorylated and doubly phosphorylated states, in addition to a double phosphomimetic mutant (T165E,S171E), each complexed with sunitinib at 2.17-3.00-Å resolutions. The native nonphosphorylated cocrystal structure revealed an inactive dimer in which the activation loop of each monomer partially occupies the ATP- and substrate-binding sites of the partner monomer. In contrast, the structure of the protein with a doubly phosphorylated activation loop exhibited an active kinase conformation with a greatly reduced monomer-monomer interface. Conversely, the phosphomimetic mutant cocrystal structure disclosed an alternative arrangement in which the activation loops are in an extended domain-swapped configuration. These structural results indicate that HPK1 is a highly dynamic kinase that undergoes trans-regulation via dimer formation and extensive intramolecular and intermolecular remodeling of the activation segment.


Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Sunitinib/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Interleucina-2/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Sunitinib/química , Sunitinib/farmacología , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo
7.
ACS Med Chem Lett ; 9(9): 872-877, 2018 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-30258533

RESUMEN

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that can become oncogenic by activating mutations or overexpression. Full kinetic characterization of both phosphorylated and nonphosphorylated wildtype and mutant ALK kinase domain was done. Our structure-based drug design programs directed at ALK allowed us to interrogate whether X-ray crystallography data could be used to support the hypothesis that activation of ALK by mutation occurs due to increased protein dynamics. Crystallographic B-factors were converted to normalized B-factors, which allowed analysis of wildtype ALK, ALK-C1156Y, and ALK-L1196M. This data suggests that mobility of the P-loop, αC-helix, and activation loop (A-loop) may be important in catalytic activity increases, with or without phosphorylation. Both molecular dynamics simulations and hydrogen-deuterium exchange experimental data corroborated the normalized B-factors data.

8.
ACS Med Chem Lett ; 9(9): 878-883, 2018 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-30258534

RESUMEN

Structure-based drug design (SBDD) is commonly leveraged in rational drug design. Usually, ligand and binding site atomic coordinates from crystallographic data are exploited to optimize potency and selectivity. In addition to traditional, static views of proteins and ligands, we propose using normalized B-factors to study protein dynamics as a part of the drug optimization process. A retrospective case study of crizotinib and lorlatinib bound to both c-ros oncogene 1 kinase (ROS1) and anaplastic lymphoma kinase (ALK) L1196M related normalized B-factors to differences in binding affinity. This analysis showed that ligand binding can have protein-stabilizing effects that start near the ligand but propagate through nearby residues and structural waters to more distal motifs. The potential opportunities for analyzing normalized B-factors in SBDD are also discussed.

9.
N Engl J Med ; 374(1): 54-61, 2016 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-26698910

RESUMEN

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Lactamas Macrocíclicas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/genética , Aminopiridinas , Quinasa de Linfoma Anaplásico , Sitios de Unión , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/secundario , Crizotinib , Femenino , Humanos , Lactamas , Fallo Hepático/etiología , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Estructura Molecular , Pirimidinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Sulfonas/uso terapéutico
10.
Proc Natl Acad Sci U S A ; 112(11): 3493-8, 2015 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-25733882

RESUMEN

Oncogenic c-ros oncogene1 (ROS1) fusion kinases have been identified in a variety of human cancers and are attractive targets for cancer therapy. The MET/ALK/ROS1 inhibitor crizotinib (Xalkori, PF-02341066) has demonstrated promising clinical activity in ROS1 fusion-positive non-small cell lung cancer. However, emerging clinical evidence has shown that patients can develop resistance by acquiring secondary point mutations in ROS1 kinase. In this study we characterized the ROS1 activity of PF-06463922, a novel, orally available, CNS-penetrant, ATP-competitive small-molecule inhibitor of ALK/ROS1. In vitro, PF-06463922 exhibited subnanomolar cellular potency against oncogenic ROS1 fusions and inhibited the crizotinib-refractory ROS1(G2032R) mutation and the ROS1(G2026M) gatekeeper mutation. Compared with crizotinib and the second-generation ALK/ROS1 inhibitors ceritinib and alectinib, PF-06463922 showed significantly improved inhibitory activity against ROS1 kinase. A crystal structure of the PF-06463922-ROS1 kinase complex revealed favorable interactions contributing to the high-affinity binding. In vivo, PF-06463922 showed marked antitumor activity in tumor models expressing FIG-ROS1, CD74-ROS1, and the CD74-ROS1(G2032R) mutation. Furthermore, PF-06463922 demonstrated antitumor activity in a genetically engineered mouse model of FIG-ROS1 glioblastoma. Taken together, our results indicate that PF-06463922 has potential for treating ROS1 fusion-positive cancers, including those requiring agents with CNS-penetrating properties, as well as for overcoming crizotinib resistance driven by ROS1 mutation.


Asunto(s)
Resistencia a Antineoplásicos/genética , Lactamas Macrocíclicas/farmacología , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Pirazoles/farmacología , Piridinas/farmacología , Aminopiridinas , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Proliferación Celular/efectos de los fármacos , Crizotinib , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/patología , Humanos , Lactamas , Lactamas Macrocíclicas/química , Ratones , Modelos Moleculares , Transducción de Señal/efectos de los fármacos
11.
Nature ; 519(7541): 102-5, 2015 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-25686603

RESUMEN

The BCR-ABL1 fusion gene is a driver oncogene in chronic myeloid leukaemia and 30-50% of cases of adult acute lymphoblastic leukaemia. Introduction of ABL1 kinase inhibitors (for example, imatinib) has markedly improved patient survival, but acquired drug resistance remains a challenge. Point mutations in the ABL1 kinase domain weaken inhibitor binding and represent the most common clinical resistance mechanism. The BCR-ABL1 kinase domain gatekeeper mutation Thr315Ile (T315I) confers resistance to all approved ABL1 inhibitors except ponatinib, which has toxicity limitations. Here we combine comprehensive drug sensitivity and resistance profiling of patient cells ex vivo with structural analysis to establish the VEGFR tyrosine kinase inhibitor axitinib as a selective and effective inhibitor for T315I-mutant BCR-ABL1-driven leukaemia. Axitinib potently inhibited BCR-ABL1(T315I), at both biochemical and cellular levels, by binding to the active form of ABL1(T315I) in a mutation-selective binding mode. These findings suggest that the T315I mutation shifts the conformational equilibrium of the kinase in favour of an active (DFG-in) A-loop conformation, which has more optimal binding interactions with axitinib. Treatment of a T315I chronic myeloid leukaemia patient with axitinib resulted in a rapid reduction of T315I-positive cells from bone marrow. Taken together, our findings demonstrate an unexpected opportunity to repurpose axitinib, an anti-angiogenic drug approved for renal cancer, as an inhibitor for ABL1 gatekeeper mutant drug-resistant leukaemia patients. This study shows that wild-type proteins do not always sample the conformations available to disease-relevant mutant proteins and that comprehensive drug testing of patient-derived cells can identify unpredictable, clinically significant drug-repositioning opportunities.


Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/química , Imidazoles/química , Imidazoles/farmacología , Indazoles/química , Indazoles/farmacología , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Axitinib , Línea Celular , Proliferación Celular/efectos de los fármacos , Cristalización , Cristalografía por Rayos X , Reposicionamiento de Medicamentos , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Humanos , Imidazoles/uso terapéutico , Indazoles/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Modelos Moleculares , Conformación Molecular , Fosforilación/efectos de los fármacos , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/genética , Proteínas Proto-Oncogénicas c-abl/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
12.
J Med Chem ; 57(11): 4720-44, 2014 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-24819116

RESUMEN

Although crizotinib demonstrates robust efficacy in anaplastic lymphoma kinase (ALK)-positive non-small-cell lung carcinoma patients, progression during treatment eventually develops. Resistant patient samples revealed a variety of point mutations in the kinase domain of ALK, including the L1196M gatekeeper mutation. In addition, some patients progress due to cancer metastasis in the brain. Using structure-based drug design, lipophilic efficiency, and physical-property-based optimization, highly potent macrocyclic ALK inhibitors were prepared with good absorption, distribution, metabolism, and excretion (ADME), low propensity for p-glycoprotein 1-mediated efflux, and good passive permeability. These structurally unusual macrocyclic inhibitors were potent against wild-type ALK and clinically reported ALK kinase domain mutations. Significant synthetic challenges were overcome, utilizing novel transformations to enable the use of these macrocycles in drug discovery paradigms. This work led to the discovery of 8k (PF-06463922), combining broad-spectrum potency, central nervous system ADME, and a high degree of kinase selectivity.


Asunto(s)
Antineoplásicos/síntesis química , Encéfalo/metabolismo , Lactamas Macrocíclicas/síntesis química , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Aminopiridinas , Quinasa de Linfoma Anaplásico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Cristalografía por Rayos X , Resistencia a Antineoplásicos , Humanos , Lactamas , Lactamas Macrocíclicas/farmacocinética , Lactamas Macrocíclicas/farmacología , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Mutación , Células 3T3 NIH , Pirazoles , Ratas , Proteínas Tirosina Quinasas Receptoras/genética , Estereoisomerismo , Relación Estructura-Actividad
13.
J Med Chem ; 57(4): 1170-87, 2014 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-24432909

RESUMEN

Crizotinib (1), an anaplastic lymphoma kinase (ALK) receptor tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration in 2011, is efficacious in ALK and ROS positive patients. Under pressure of crizotinib treatment, point mutations arise in the kinase domain of ALK, resulting in resistance and progressive disease. The successful application of both structure-based and lipophilic-efficiency-focused drug design resulted in aminopyridine 8e, which was potent across a broad panel of engineered ALK mutant cell lines and showed suitable preclinical pharmacokinetics and robust tumor growth inhibition in a crizotinib-resistant cell line (H3122-L1196M).


Asunto(s)
Resistencia a Antineoplásicos/genética , Mutación Puntual , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Quinasa de Linfoma Anaplásico , Crizotinib , Humanos
14.
J Med Chem ; 56(17): 6651-65, 2013 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-23944843

RESUMEN

The hepatocyte growth factor (HGF)/c-Met signaling axis is deregulated in many cancers and plays important roles in tumor invasive growth and metastasis. An exclusively selective c-Met inhibitor (S)-6-(1-(6-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)ethyl)quinoline (8) was discovered from a highly selective high-throughput screening hit via structure-based drug design and medicinal chemistry lead optimization. Compound 8 had many attractive properties meriting preclinical evaluation. Broad off-target screens identified 8 as a pan-phosphodiesterase (PDE) family inhibitor, which was implicated in a sustained increase in heart rate, increased cardiac output, and decreased contractility indices, as well as myocardial degeneration in in vivo safety evaluations in rats. Compound 8 was terminated as a preclinical candidate because of a narrow therapeutic window in cardio-related safety. The learning from multiparameter lead optimization and strategies to avoid the toxicity attrition at the late stage of drug discovery are discussed.


Asunto(s)
Cardiomiopatías/enzimología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Inhibidores de Fosfodiesterasa/química , Inhibidores de Proteínas Quinasas/química , Quinolinas/química , Ratas , Proteínas Tirosina Quinasas Receptoras/química
15.
N Engl J Med ; 368(25): 2395-401, 2013 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-23724914

RESUMEN

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).


Asunto(s)
Adenocarcinoma/genética , Resistencia a Medicamentos/genética , Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Translocación Genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Crizotinib , Resultado Fatal , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Persona de Mediana Edad , Mutación , Conformación Proteica , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas/química , Relación Estructura-Actividad
16.
Proc Natl Acad Sci U S A ; 109(45): 18281-9, 2012 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-22988103

RESUMEN

Analyses of compounds in clinical development have shown that ligand efficient-molecules with privileged physical properties and low dose are less likely to fail in the various stages of clinical testing, have fewer postapproval withdrawals, and are less likely to receive black box safety warnings. However, detailed side-by-side examination of molecular interactions and properties within single drug classes are lacking. As a class, VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) have changed the landscape of how cancer is treated, particularly in clear cell renal cell carcinoma, which is molecularly linked to the VEGF signaling axis. Despite the clear role of the molecular target, member molecules of this validated drug class exhibit distinct clinical efficacy and safety profiles in comparable renal cell carcinoma clinical studies. The first head-to-head randomized phase III comparative study between active VEGFR TKIs has confirmed significant differences in clinical performance [Rini BI, et al. (2011) Lancet 378:193-1939]. To elucidate how fundamental drug potency-efficiency is achieved and impacts differentiation within the VEGFR TKI class, we determined potencies, time dependence, selectivities, and X-ray structures of the drug-kinase complexes using a VEGFR2 TK construct inclusive of the important juxtamembrane domain. Collectively, the studies elucidate unique drug-kinase interactions that are dependent on distinct juxtamembrane domain conformations, resulting in significant potency and ligand efficiency differences. The identified structural trends are consistent with in vitro measurements, which translate well to clinical performance, underscoring a principle that may be broadly applicable to prospective drug design for optimal in vivo performance.


Asunto(s)
Conformación Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/química , Carcinoma de Células Renales/tratamiento farmacológico , Ensayos Clínicos como Asunto , Cristalografía por Rayos X , Supervivencia sin Enfermedad , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Neoplasias Renales/tratamiento farmacológico , Modelos Moleculares , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Resultado del Tratamiento , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo
17.
J Med Chem ; 55(18): 8091-109, 2012 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-22924734

RESUMEN

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit 6 was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor 10 with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl)-1H-pyrazol-1-yl)ethanol (2, PF-04217903), an extremely potent and exquisitely selective c-MET inhibitor. 2 demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. 2 progressed to clinical evaluation in a Phase I oncology setting.


Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Pirazinas/farmacología , Triazoles/farmacología , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Estabilidad de Medicamentos , Ensayos Analíticos de Alto Rendimiento , Humanos , Indoles/química , Modelos Moleculares , Datos de Secuencia Molecular , Oxindoles , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-met/química , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazinas/química , Pirazinas/metabolismo , Especificidad por Sustrato , Triazoles/química , Triazoles/metabolismo
18.
Mol Cancer Ther ; 11(4): 1036-47, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22389468

RESUMEN

The c-Met pathway has been implicated in a variety of human cancers for its critical role in tumor growth, invasion, and metastasis. PF-04217903 is a novel ATP-competitive small-molecule inhibitor of c-Met kinase. PF-04217903 showed more than 1,000-fold selectivity for c-Met compared with more than 150 kinases, making it one of the most selective c-Met inhibitors described to date. PF-04217903 inhibited tumor cell proliferation, survival, migration/invasion in MET-amplified cell lines in vitro, and showed marked antitumor activity in tumor models harboring either MET gene amplification or a hepatocyte growth factor (HGF)/c-Met autocrine loop at well-tolerated dose levels in vivo. Antitumor efficacy of PF-04217903 was dose-dependent and showed a strong correlation with inhibition of c-Met phosphorylation, downstream signaling, and tumor cell proliferation/survival. In human xenograft models that express relatively high levels of c-Met, complete inhibition of c-Met activity by PF-04217903 only led to partial tumor growth inhibition (38%-46%) in vivo. The combination of PF-04217903 with Recepteur d'origine nantais (RON) short hairpin RNA (shRNA) knockdown in the HT29 model that also expresses activated RON kinase-induced tumor cell apoptosis and resulted in enhanced antitumor efficacy (77%) compared with either PF-04217903 (38%) or RON shRNA alone (56%). PF-04217903 also showed potent antiangiogenic properties in vitro and in vivo. Furthermore, PF-04217903 strongly induced phospho-PDGFRß (platelet-derived growth factor receptor) levels in U87MG xenograft tumors, indicating a possible oncogene switching mechanism in tumor cell signaling as a potential resistance mechanism that might compromise tumor responses to c-Met inhibitors. Collectively, these results show the use of highly selective inhibition of c-Met and provide insight toward targeting tumors exhibiting different mechanisms of c-Met dysregulation.


Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Pirazinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Triazoles/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto
19.
J Med Chem ; 54(18): 6342-63, 2011 Sep 22.
Artículo en Inglés | MEDLINE | ID: mdl-21812414

RESUMEN

Because of the critical roles of aberrant signaling in cancer, both c-MET and ALK receptor tyrosine kinases are attractive oncology targets for therapeutic intervention. The cocrystal structure of 3 (PHA-665752), bound to c-MET kinase domain, revealed a novel ATP site environment, which served as the target to guide parallel, multiattribute drug design. A novel 2-amino-5-aryl-3-benzyloxypyridine series was created to more effectively make the key interactions achieved with 3. In the novel series, the 2-aminopyridine core allowed a 3-benzyloxy group to reach into the same pocket as the 2,6-dichlorophenyl group of 3 via a more direct vector and thus with a better ligand efficiency (LE). Further optimization of the lead series generated the clinical candidate crizotinib (PF-02341066), which demonstrated potent in vitro and in vivo c-MET kinase and ALK inhibition, effective tumor growth inhibition, and good pharmaceutical properties.


Asunto(s)
Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Pirazoles/síntesis química , Piridinas/síntesis química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Quinasa de Linfoma Anaplásico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Crizotinib , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos de Selección de Medicamentos Antitumorales , Transición Epitelial-Mesenquimal , Humanos , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Modelos Moleculares , Conformación Molecular , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Piridinas/farmacología , Estereoisomerismo , Relación Estructura-Actividad
20.
Cancer Res ; 71(3): 1081-91, 2011 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-21266357

RESUMEN

Therapies targeting receptor tyrosine kinases have shown efficacy in molecularly defined subsets of cancers. Unfortunately, cancers invariably develop resistance, and overcoming or preventing resistance will ultimately be key to unleashing their full therapeutic potential. In this study, we examined how cancers become resistant to MET inhibitors, a class of drugs currently under clinical development. We utilized the highly sensitive gastric carcinoma cell line, SNU638, and two related MET inhibitors PHA-665752 and PF-2341066. To our surprise, we observed at least two mechanisms of resistance that arose simultaneously. Both resulted in maintenance of downstream PI3K (phosphoinositide 3-kinase)-AKT and MEK (MAP/ERK kinase)-ERK signaling in the presence of inhibitor. One mechanism, observed by modeling resistance both in vitro and in vivo, involved the acquisition of a mutation in the MET activation loop (Y1230). Structural analysis indicates that this mutation destabilizes the autoinhibitory conformation of MET and abrogates an important aromatic stacking interaction with the inhibitor. The other cause of resistance was activation of the epidermal growth factor receptor (EGFR) pathway due to increased expression of transforming growth factor α. Activation of EGFR bypassed the need for MET signaling to activate downstream signaling in these cells. This resistance could be overcome by combined EGFR and MET inhibition. Thus, therapeutic strategies that combine MET inhibitors capable of inhibiting Y1230 mutant MET in combination with anti-EGFR-based therapies may enhance clinical benefit for patients with MET-addicted cancers. Importantly, these results also underscore the notion that a single cancer can simultaneously develop resistance induced by several mechanisms and highlight the daunting challenges associated with preventing or overcoming resistance.


Asunto(s)
Mutación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Receptores de Factores de Crecimiento/antagonistas & inhibidores , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Animales , Línea Celular Tumoral , Crizotinib , Resistencia a Antineoplásicos , Elafina/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Indoles/farmacología , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Desnudos , Proteína Oncogénica v-akt/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazoles/farmacología , Piridinas/farmacología , Receptores de Factores de Crecimiento/metabolismo , Neoplasias Gástricas/enzimología , Sulfonas/farmacología , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
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