RESUMEN
Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.
Asunto(s)
Sinergismo Farmacológico , Inhibidores de Topoisomerasa II , Neoplasias de la Mama Triple Negativas , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Femenino , Humanos , Ratones , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Doxorrubicina/análogos & derivados , Epirrubicina/farmacología , Etopósido/farmacología , Etopósido/uso terapéutico , Polietilenglicoles/farmacología , Sulfonas/farmacología , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Wee1 is a tyrosine kinase that is highly expressed in several cancer types. Wee1 inhibition can lead to suppression of tumor cell proliferation and sensitization of cells to the effects of DNA-damaging agents. AZD1775 is a nonselective Wee1 inhibitor for which myelosuppression has been observed as a dose-limiting toxicity. We have applied structure-based drug design (SBDD) to rapidly generate highly selective Wee1 inhibitors that demonstrate better selectivity than AZD1775 against PLK1, which is known to cause myelosuppression (including thrombocytopenia) when inhibited. While selective Wee1 inhibitors described herein still achieved in vitro antitumor efficacy, thrombocytopenia was still observed in vitro.
RESUMEN
Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential molecular therapeutic agents for the treatment of cancer. Here we describe the identification of novel small molecular weight inhibitors of Hsp90 using a fragment based approach. Fragments were selected by docking, tested in a biochemical assay and the confirmed hits were crystallized. Information gained from X-ray structures of these fragments and other chemotypes was used to drive the fragment evolution process. Optimization of these high µM binders resulted in 3-benzylindazole derivatives with significantly improved affinity and anti-proliferative effects in different human cancer cell lines.
Asunto(s)
Amidas/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Indazoles/química , Bibliotecas de Moléculas Pequeñas/química , Amidas/toxicidad , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Estructura Terciaria de Proteína , Bibliotecas de Moléculas Pequeñas/toxicidad , Relación Estructura-ActividadRESUMEN
RAF kinase plays a critical role in the RAF-MEK-ERK signaling pathway and inhibitors of RAF could be of use for the treatment of various cancer types. We have designed potent RAF-1 inhibitors bearing novel bicyclic heterocycles as key structural elements for the interaction with the hinge region. In both series exploration of the SAR was focussed on the substitution of the phenyl ring, which binds to the induced fit pocket. Overall, it was confirmed that incorporation of lipophilic substituents was needed for potent Raf inhibition and a number of potent analogues were obtained.
Asunto(s)
Bencimidazoles/química , Isoquinolinas/química , Inhibidores de Proteínas Quinasas/síntesis química , Quinasas raf/antagonistas & inhibidores , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Diseño de Fármacos , Isoquinolinas/síntesis química , Isoquinolinas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Relación Estructura-Actividad , Quinasas raf/metabolismoRESUMEN
Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Proteína Quinasa Activada por ADN/antagonistas & inhibidores , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias de Cabeza y Cuello/radioterapia , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Quinazolinas/farmacología , Radiación Ionizante , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Proliferación Celular , Femenino , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/radioterapia , Ratones , Ratones Desnudos , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.
Asunto(s)
Hemopexina/metabolismo , Integrina alfaVbeta3/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloendopeptidasas/metabolismo , Neovascularización Fisiológica/fisiología , Animales , Bovinos , Citocinas/farmacología , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Activación Enzimática , Factor 2 de Crecimiento de Fibroblastos/farmacología , Hemopexina/farmacología , Humanos , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/farmacología , Metaloproteinasas de la Matriz Asociadas a la Membrana , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/biosíntesis , Oligopéptidos/farmacología , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Common strategies to optimize prodrugs use either in vitro or rodent in vivo approaches, which do not consider elimination pathways that do not result in the generation of the desired product or might be misleading because of species differences, respectively. As a step forward, we have incorporated a novel application of hepatocytes into our prodrug optimization strategy to increase the bioavailability of a poorly soluble drug candidate by attaching a charged ester linker. The model involves the incubation of hepatocytes from multiple species in serum-containing medium to mimic formation as well as simultaneous metabolism of both prodrug and active drug. Using this strategy, a correlation between the in vitro AUC and the AUC after intravenous administration was obtained for active drug formation in several species. Moreover, hepatocytes correctly predicted the likelihood of undesired exposure with nonhydrolyzed prodrug. This novel approach enabled us to identify several prodrugs, which showed improved exposure over a wide dose range. Furthermore, a strategy was developed resulting in a decision tree that can be used to determine the applicability of the hepatocyte model in the screening process.
Asunto(s)
Hepatocitos/metabolismo , Profármacos/administración & dosificación , Profármacos/metabolismo , Suero/metabolismo , Animales , Área Bajo la Curva , Disponibilidad Biológica , Perros , Ésteres/química , Femenino , Humanos , Cinética , Macaca fascicularis , Masculino , Ratones , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Profármacos/química , Ratas , Ratas Wistar , SolubilidadRESUMEN
The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES), which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8) is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD) in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD). Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.