RESUMEN
PROTACs have shown promise as a new class of therapy, with a unique mechanism of action orthogonal to traditional small molecules that are used to regulate protein activity. Their novel MOA utilizing the body's natural protein degradation machinery degrades a protein of interest rather than inhibiting its function. This strategy has several advantages over conventional small-molecule inhibitors, e.g., higher sensitivity, less off-target effects, and greater target space. However, unlocking the potential of PROTACs necessitates drug discovery techniques that can support the complexity of the novel MOA. In this chapter, we describe the application of MicroScale Thermophoresis (MST) and Temperature-Related Intensity Change (TRIC) to characterize both the binary and ternary binding of PROTACs with target proteins and ubiquitin ligases along with an efficient determination of the cooperativity of the ternary complex formation. The assay development and experimental procedure to characterize the well-described BET PROTAC MZ1 show how MST and TRIC can be applied as a fast and highly sensitive method for PROTAC discovery.
Asunto(s)
Desarrollo de Medicamentos , Ubiquitina-Proteína Ligasas , Proteolisis , Temperatura , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
KRAS is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.See related commentary by Zhao et al., p. 17.This article is highlighted in the In This Issue feature, p. 1.
Asunto(s)
Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Línea Celular Tumoral , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos , Mutación , Nucleótidos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genéticaRESUMEN
Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1S727 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Quinasa 8 Dependiente de Ciclina/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Células Asesinas Naturales/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Melanoma Experimental/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Animales , Apoptosis , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Proliferación Celular , Citotoxicidad Inmunológica/inmunología , Femenino , Humanos , Células Asesinas Naturales/efectos de los fármacos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Melanoma Experimental/enzimología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Fosforilación , Factor de Transcripción STAT1/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The epidermal growth factor receptor (EGFR), when carrying an activating mutation like del19 or L858R, acts as an oncogenic driver in a subset of lung tumors. While tumor responses to tyrosine kinase inhibitors (TKIs) are accompanied by marked tumor shrinkage, the response is usually not durable. Most patients relapse within two years of therapy often due to acquisition of an additional mutation in EGFR kinase domain that confers resistance to TKIs. Crucially, oncogenic EGFR harboring both resistance mutations, T790M and C797S, can no longer be inhibited by currently approved EGFR TKIs. Here, we describe the discovery of BI-4020, which is a noncovalent, wild-type EGFR sparing, macrocyclic TKI. BI-4020 potently inhibits the above-described EGFR variants and induces tumor regressions in a cross-resistant EGFRdel19â¯T790Mâ¯C797S xenograft model. Key was the identification of a highly selective but moderately potent benzimidazole followed by complete rigidification of the molecule through macrocyclization.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Antineoplásicos/farmacocinética , Bencimidazoles/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cristalografía por Rayos X , Ciclización , Entropía , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/química , Receptores ErbB/genética , Femenino , Hepatocitos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Mutación , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD+)-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD+.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Fosfoglicerato-Deshidrogenasa/antagonistas & inhibidores , Serina/antagonistas & inhibidores , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Indoles/síntesis química , Indoles/química , Modelos Moleculares , Estructura Molecular , Fosfoglicerato-Deshidrogenasa/metabolismo , Serina/biosíntesis , Relación Estructura-ActividadRESUMEN
The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be "undruggable," between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.
Asunto(s)
Descubrimiento de Drogas , Preparaciones Farmacéuticas/química , Proteínas Proto-Oncogénicas p21(ras)/química , Guanosina Trifosfato/metabolismo , Humanos , Modelos Moleculares , Nanopartículas/químicaRESUMEN
Here, we report the fragment-based discovery of BI-9321, a potent, selective and cellular active antagonist of the NSD3-PWWP1 domain. The human NSD3 protein is encoded by the WHSC1L1 gene located in the 8p11-p12 amplicon, frequently amplified in breast and squamous lung cancer. Recently, it was demonstrated that the PWWP1 domain of NSD3 is required for the viability of acute myeloid leukemia cells. To further elucidate the relevance of NSD3 in cancer biology, we developed a chemical probe, BI-9321, targeting the methyl-lysine binding site of the PWWP1 domain with sub-micromolar in vitro activity and cellular target engagement at 1 µM. As a single agent, BI-9321 downregulates Myc messenger RNA expression and reduces proliferation in MOLM-13 cells. This first-in-class chemical probe BI-9321, together with the negative control BI-9466, will greatly facilitate the elucidation of the underexplored biological function of PWWP domains.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Proteínas Nucleares/antagonistas & inhibidores , Sistemas CRISPR-Cas , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Regulación de la Expresión Génica/efectos de los fármacos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismoRESUMEN
In the version of this article originally published, several lines of text in the last paragraph of the right column on page 1 of the PDF were transposed into the bottom paragraph of the left column. The affected text of the left column should read "The ATP-dependent activities of the BAF (SWI/SNF) chromatin remodeling complexes affect the positioning of nucleosomes on DNA and thereby many cellular processes related to chromatin structure, including transcription, DNA repair and decatenation of chromosomes during mitosis12,13." The affected text of the right column should read "SMARCA2/4BD inhibitors are thus precluded from use for the treatment of SMARCA4 mutant cancers but could provide attractive ligands for PROTAC conjugation. Small molecules binding to other bromodomains have been successfully converted into PROTACs by conjugating them with structures capable of binding to the E3 ligases von Hippel-Lindau (VHL) or cereblon5,6,10,11,25,26,27." The errors have been corrected in the PDF version of the paper.
RESUMEN
Targeting subunits of BAF/PBAF chromatin remodeling complexes has been proposed as an approach to exploit cancer vulnerabilities. Here, we develop proteolysis targeting chimera (PROTAC) degraders of the BAF ATPase subunits SMARCA2 and SMARCA4 using a bromodomain ligand and recruitment of the E3 ubiquitin ligase VHL. High-resolution ternary complex crystal structures and biophysical investigation guided rational and efficient optimization toward ACBI1, a potent and cooperative degrader of SMARCA2, SMARCA4 and PBRM1. ACBI1 induced anti-proliferative effects and cell death caused by SMARCA2 depletion in SMARCA4 mutant cancer cells, and in acute myeloid leukemia cells dependent on SMARCA4 ATPase activity. These findings exemplify a successful biophysics- and structure-based PROTAC design approach to degrade high profile drug targets, and pave the way toward new therapeutics for the treatment of tumors sensitive to the loss of BAF complex ATPases.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Estructura Molecular , Proteínas Nucleares/metabolismoRESUMEN
Focal adhesion tyrosine kinase (PTK2) is often overexpressed in human hepatocellular carcinoma (HCC), and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity. However, the clinical relevance of targeting PTK2 still remains to be proven. Here, we present two highly selective and functional PTK2 proteolysis-targeting chimeras utilizing von Hippel-Lindau and cereblon ligands to hijack E3 ligases for PTK2 degradation. BI-3663 (cereblon-based) degrades PTK2 with a median DC50 of 30 nM to >80% across a panel of 11 HCC cell lines. Despite effective PTK2 degradation, these compounds did not phenocopy the reported antiproliferative effects of PTK2 depletion in any of the cell lines tested. By disclosing these compounds, we hope to provide valuable tools for the study of PTK2 degradation across different biological systems.
Asunto(s)
Quinasa 1 de Adhesión Focal/efectos de los fármacos , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/metabolismo , Línea Celular Tumoral , Proliferación Celular , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Humanos , Ligandos , Proteolisis , Interferencia de ARNRESUMEN
Protein ubiquitylation is a dynamic post-translational modification that can be reversed by deubiquitylating enzymes (DUBs). It is unclear how the small number (â¼100) of DUBs present in mammalian cells regulate the thousands of different ubiquitylation events. Here, we analysed annotated transcripts of human DUBs and found â¼300 ribosome-associated transcripts annotated as protein coding, which thus increases the total number of DUBs. By using USP35, a poorly studied DUB, as a case study, we provide evidence that alternative isoforms contribute to the functional expansion of DUBs. We show that there are two different USP35 isoforms that localise to different intracellular compartments and have distinct functions. Our results reveal that isoform 1 is an anti-apoptotic factor that inhibits staurosporine- and TNF-related apoptosis-inducing ligand (TRAIL; also known as TNFSF10)-induced apoptosis. In contrast, USP35 isoform 2 is an integral membrane protein of the endoplasmic reticulum (ER) that is also present at lipid droplets. Manipulations of isoform 2 levels cause rapid ER stress, likely through deregulation of lipid homeostasis, and lead to cell death. Our work highlights how alternative isoforms provide functional expansion of DUBs and sets directions for future research.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Endopeptidasas/metabolismo , Isoformas de Proteínas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Apoptosis , Endopeptidasas/genética , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Isoformas de Proteínas/genética , Transporte de Proteínas , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has attracted interest as a target for pharmacological intervention in malignant diseases. Here, we describe BI 853520, a novel ATP-competitive inhibitor distinguished by high potency and selectivity. In vitro, the compound inhibits FAK autophosphorylation in PC-3 prostate carcinoma cells with an IC50 of 1 nmol/L and blocks anchorage-independent proliferation of PC-3 cells with an EC50 of 3 nmol/L, whereas cells grown in conventional surface culture are 1000-fold less sensitive. In mice, the compound shows long half-life, high volume of distribution and high oral bioavailability; oral dosing of immunodeficient mice bearing subcutaneous PC-3 prostate adenocarcinoma xenografts resulted in rapid, long-lasting repression of FAK autophosphorylation in tumor tissue. Daily oral administration of BI 853520 to nude mice at doses of 50 mg/kg was well tolerated for prolonged periods of time. In a diverse panel of 16 subcutaneous adenocarcinoma xenograft models in nude mice, drug treatment resulted in a broad spectrum of outcomes, ranging from group median tumor growth inhibition values >100% and tumor regression in subsets of animals to complete lack of sensitivity. Biomarker analysis indicated that high sensitivity is linked to a mesenchymal tumor phenotype, initially defined by loss of E-cadherin expression and subsequently substantiated by gene set enrichment analysis. Further, we obtained microRNA expression profiles for 13 models and observed that hsa-miR-200c-3p expression is strongly correlated with efficacy (R2 = 0.889). BI 853520 is undergoing evaluation in early clinical trials.
RESUMEN
The transcription factor BCL6 is a known driver of oncogenesis in lymphoid malignancies, including diffuse large B cell lymphoma (DLBCL). Disruption of its interaction with transcriptional repressors interferes with the oncogenic effects of BCL6. We used a structure-based drug design to develop highly potent compounds that block this interaction. A subset of these inhibitors also causes rapid ubiquitylation and degradation of BCL6 in cells. These compounds display significantly stronger induction of expression of BCL6-repressed genes and anti-proliferative effects than compounds that merely inhibit co-repressor interactions. This work establishes the BTB domain as a highly druggable structure, paving the way for the use of other members of this protein family as drug targets. The magnitude of effects elicited by this class of BCL6-degrading compounds exceeds that of our equipotent non-degrading inhibitors, suggesting opportunities for the development of BCL6-based lymphoma therapeutics.
Asunto(s)
Proteolisis , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Cinética , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Dominios Proteicos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-6/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Ubiquitinación/efectos de los fármacosRESUMEN
Although the MAPK pathway is frequently deregulated in cancer, inhibitors targeting RAF or MEK have so far shown clinical activity only in BRAF- and NRAS-mutant melanoma. Improvements in efficacy may be possible by combining inhibition of mitogenic signal transduction with inhibition of cell-cycle progression. We have studied the preclinical pharmacology of BI 847325, an ATP-competitive dual inhibitor of MEK and Aurora kinases. Potent inhibition of MEK1/2 and Aurora A/B kinases by BI 847325 was demonstrated in enzymatic and cellular assays. Equipotent effects were observed in BRAF-mutant cells, whereas in KRAS-mutant cells, MEK inhibition required higher concentrations than Aurora kinase inhibition. Daily oral administration of BI 847325 at 10 mg/kg showed efficacy in both BRAF- and KRAS-mutant xenograft models. Biomarker analysis suggested that this effect was primarily due to inhibition of MEK in BRAF-mutant models but of Aurora kinase in KRAS-mutant models. Inhibition of both MEK and Aurora kinase in KRAS-mutant tumors was observed when BI 847325 was administered once weekly at 70 mg/kg. Our studies indicate that BI 847325 is effective in in vitro and in vivo models of cancers with BRAF and KRAS mutation. These preclinical data are discussed in the light of the results of a recently completed clinical phase I trial assessing safety, tolerability, pharmacokinetics, and efficacy of BI 847325 in patients with cancer. Mol Cancer Ther; 15(10); 2388-98. ©2016 AACR.
Asunto(s)
Antineoplásicos/farmacología , Aurora Quinasas/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Aurora Quinasas/química , Aurora Quinasas/metabolismo , Unión Competitiva , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/química , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Moleculares , Conformación Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.
Asunto(s)
Antineoplásicos/farmacología , Diseño de Fármacos , Piridonas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Piridonas/síntesis química , Piridonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of the IGF1R, INSRA, and INSRB receptor tyrosine kinases represents an attractive approach of pharmacologic intervention in cancer, owing to the roles of the IGF1R and INSRA in promoting cell proliferation and survival. However, the central role of the INSRB isoform in glucose homeostasis suggests that prolonged inhibition of this kinase could result in metabolic toxicity. We describe here the profile of the novel compound BI 885578, a potent and selective ATP-competitive IGF1R/INSR tyrosine kinase inhibitor distinguished by rapid intestinal absorption and a short in vivo half-life as a result of rapid metabolic clearance. BI 885578, administered daily per os, displayed an acceptable tolerability profile in mice at doses that significantly reduced the growth of xenografted human GEO and CL-14 colon carcinoma tumors. We found that treatment with BI 885578 is accompanied by increases in circulating glucose and insulin levels, which in turn leads to compensatory hyperphosphorylation of muscle INSRs and subsequent normalization of blood glucose within a few hours. In contrast, the normalization of IGF1R and INSR phosphorylation in GEO tumors occurs at a much slower rate. In accordance with this, BI 885578 led to a prolonged inhibition of cell proliferation and induction of apoptosis in GEO tumors. We propose that the remarkable therapeutic window observed for BI 885578 is achieved by virtue of the distinctive pharmacokinetic properties of the compound, capitalizing on the physiologic mechanisms of glucose homeostasis and differential levels of IGF1R and INSR expression in tumors and normal tissues.
Asunto(s)
Antígenos CD/biosíntesis , Neoplasias del Colon/tratamiento farmacológico , Homeostasis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/administración & dosificación , Pirazoles/administración & dosificación , Quinazolinas/administración & dosificación , Receptor de Insulina/biosíntesis , Receptores de Somatomedina/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Humanos , Ratones , Receptor IGF Tipo 1 , Receptor de Insulina/antagonistas & inhibidores , Receptores de Somatomedina/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Deregulation of the ErbB (proto-oncogene B of the avian erythroblastosis virus AEV-H strain) receptor network is well recognized as an oncogenic driver in epithelial cancers. Several targeted drugs have been developed, including antibodies and small-molecule kinase inhibitors, each of them characterized by distinct patterns of ErbB receptor interactions. Understanding the precise pharmacological properties of these compounds is important for optimal use in clinical practice. Afatinib [BIBW 2992; N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butenamide] is an ATP-competitive anilinoquinazoline derivative harboring a reactive acrylamide group. It was designed to covalently bind and irreversibly block enzymatically active ErbB receptor family members. Here, we show by X-ray crystallography the covalent binding of afatinib to wild-type epidermal growth factor receptor (EGFR) and by mass spectrometry the covalent interaction with EGFR, EGFRL858R/T790M, human epidermal growth factor receptor 2 (HER2), and ErbB-4. Afatinib potently inhibits the enymatic activity of ErbB-4 (EC50=1 nM) and the proliferation of cancer cell lines driven by multiple ErbB receptor aberrations at concentrations below 100 nM. N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)-2-butanamide (BI 37781), a close analog of afatinib lacking the acrylamide group and thus incapable of covalent bond formation, had similar potency on cells driven by EGFR or EGFRL858R, but less or no detectable activity on cells expressing EGFRL858R/T790M HER2 or ErbB-4. These results stress the importance of the acrylamide group and show that afatinib differs from approved ErbB targeting agents by irreversibly inhibiting the kinase activity of all ErbB family members. They provide a mechanistic rationale for the distinct pharmacological features of this compound and explain the clinical activity seen in some patients who are resistant to antibody or kinase inhibitor therapy because of secondary mutations or ErbB receptor "reprogramming."
Asunto(s)
Genes erbB/efectos de los fármacos , Quinazolinas/metabolismo , Quinazolinas/farmacología , Afatinib , Animales , Proliferación Celular , Células Cultivadas , Cristalografía , Interpretación Estadística de Datos , Receptores ErbB/metabolismo , Genes erbB/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Fosfotransferasas/antagonistas & inhibidores , Conformación Proteica , Proto-Oncogenes Mas , Quinazolinas/química , Receptor ErbB-2/antagonistas & inhibidores , Análisis Espectral , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
Inhibition of tumor angiogenesis through blockade of the vascular endothelial growth factor (VEGF) signaling pathway is a novel treatment modality in oncology. Preclinical findings suggest that long-term clinical outcomes may improve with blockade of additional proangiogenic receptor tyrosine kinases: platelet-derived growth factor receptors (PDGFR) and fibroblast growth factor receptors (FGFR). BIBF 1120 is an indolinone derivative potently blocking VEGF receptor (VEGFR), PDGFR and FGFR kinase activity in enzymatic assays (IC(50), 20-100 nmol/L). BIBF 1120 inhibits mitogen-activated protein kinase and Akt signaling pathways in three cell types contributing to angiogenesis, endothelial cells, pericytes, and smooth muscle cells, resulting in inhibition of cell proliferation (EC(50), 10-80 nmol/L) and apoptosis. In all tumor models tested thus far, including human tumor xenografts growing in nude mice and a syngeneic rat tumor model, BIBF 1120 is highly active at well-tolerated doses (25-100 mg/kg daily p.o.), as measured by magnetic resonance imaging of tumor perfusion after 3 days, reducing vessel density and vessel integrity after 5 days, and inducing profound growth inhibition. A distinct pharmacodynamic feature of BIBF 1120 in cell culture is sustained pathway inhibition (up to 32 hours after 1-hour treatment), suggesting slow receptor off-kinetics. Although BIBF 1120 is rapidly metabolized in vivo by methylester cleavage, resulting in a short mean residence time, once daily oral dosing is fully efficacious in xenograft models. These distinctive pharmacokinetic and pharmacodynamic properties may help explain clinical observations with BIBF 1120, currently entering phase III clinical development.
Asunto(s)
Factores de Coagulación Sanguínea/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Indoles/uso terapéutico , Neoplasias Experimentales/tratamiento farmacológico , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Femenino , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Ratones , Ratones Desnudos , Estructura Molecular , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Pericitos/citología , Pericitos/efectos de los fármacos , Pericitos/metabolismo , Ratas , Ratas Endogámicas F344 , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal , Distribución Tisular , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.