RESUMEN
LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure-based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology.
Asunto(s)
Proteasas ATP-Dependientes/antagonistas & inhibidores , Diseño de Fármacos , Proteínas Mitocondriales/antagonistas & inhibidores , Inhibidores de Proteasas/química , Proteasas ATP-Dependientes/metabolismo , Sitios de Unión , Ácidos Borónicos/química , Ácidos Borónicos/metabolismo , Ácidos Borónicos/farmacología , Bortezomib/química , Bortezomib/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Humanos , Proteínas Mitocondriales/metabolismo , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Relación Estructura-ActividadRESUMEN
Smad proteins transduce signals carried by the transforming growth factor beta (TGF-beta) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-beta signaling was restored as determined by TGF-beta-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-beta1 also resulted in an altered cell morphology that coincided with enhanced beta1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-beta-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-beta signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.
Asunto(s)
Anoicis/fisiología , Neoplasias de la Mama/patología , Proteínas de Unión al ADN/fisiología , Transactivadores/fisiología , Factor de Crecimiento Transformador beta/fisiología , Adenoviridae/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Caspasas/metabolismo , División Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Activación Enzimática , Quinasa 2 de Adhesión Focal , Humanos , Integrina beta1/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Transducción de Señal/fisiología , Proteína Smad4 , Transactivadores/biosíntesis , Transactivadores/genética , Transfección , Células Tumorales CultivadasRESUMEN
Non-small cell lung cancer patients carrying oncogenic EGFR mutations initially respond to EGFR-targeted therapy, but later elicit minimal response due to dose-limiting toxicities and acquired resistance. EGF816 is a novel, irreversible mutant-selective EGFR inhibitor that specifically targets EGFR-activating mutations arising de novo and upon resistance acquisition, while sparing wild-type (WT) EGFR. EGF816 potently inhibited the most common EGFR mutations L858R, Ex19del, and T790M in vitro, which translated into strong tumor regressions in vivo in several patient-derived xenograft models. Notably, EGF816 also demonstrated antitumor activity in an exon 20 insertion mutant model. At levels above efficacious doses, EGF816 treatment led to minimal inhibition of WT EGFR and was well tolerated. In single-dose studies, EGF816 provided sustained inhibition of EGFR phosphorylation, consistent with its ability for irreversible binding. Furthermore, combined treatment with EGF816 and INC280, a cMET inhibitor, resulted in durable antitumor efficacy in a xenograft model that initially developed resistance to first-generation EGFR inhibitors via cMET activation. Thus, we report the first preclinical characterization of EGF816 and provide the groundwork for its current evaluation in phase I/II clinical trials in patients harboring EGFR mutations, including T790M.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Mutación/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Ratas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Cultured primary human cells have been widely used to assess the selectivity of oncolytic viruses as potential anticancer agents. As culture conditions can potentially have a significant impact on virus replication and ultimately cell killing, we evaluated the effects of dl309, a wild-type adenovirus, and dl01 / 07, a conditionally replicating adenovirus mutant, on quiescent and proliferating primary mammary epithelial cells. When primary cells were induced into quiescence, both viruses exhibited similar attenuated cell killing. However, cell killing by dl309 was superior to dl01 / 07 in proliferating primary cells. Analysis of viral effects at the level of entry, E2F activation, DNA replication, and late gene expression indicated that attenuation of dl309 in quiescent cells correlated with decreased expression of viral late genes such as hexon. In contrast, attenuation of dl01 / 07 in quiescent cells correlated with inefficient induction of E2F activity and inability to undergo efficient DNA replication. In proliferating cells, dl309 replicated efficiently, whereas dl01 / 07 still showed attenuated replication. In summary, our results indicate the intrinsic preference of wild-type adenoviruses for killing proliferating cells, which is an attractive feature for using adenoviruses as oncolytic agents. These results also highlight the need for the use of appropriate growth conditions for primary cells in vitro to distinguish subtle differences in cell killing among various oncolytic viruses.
Asunto(s)
Adenoviridae/fisiología , Antineoplásicos/toxicidad , Proteínas de la Cápside/genética , Replicación Viral , Adenoviridae/genética , Células Cultivadas , ADN Viral/análisis , ADN Viral/genética , Células Epiteliales/química , Células Epiteliales/metabolismo , Expresión Génica , Genes Virales , Humanos , Transducción GenéticaRESUMEN
Oncogene addiction due to Myc deregulation has been identified in a variety of tumor types. In order to identify pharmacological agents that cause selective apoptosis in tumors with deregulated Myc expression, we designed a cell-based screening assay based on our Anti-cancer Screening Apoptosis Program (ASAP) technology targeting increased activity in a "Myc-addicted" cancer cell panel. We have identified a novel set of substituted 4-aryl-3-(3-aryl-1-oxo-2-propenyl)-2(1H)-quinolinones that activates apoptosis in cancer cell lines with deregulated Myc, but show low activity in cell lines where Myc is not deregulated. Apoptosis induced by these compounds is rapid, and is associated with a significant downregulation of Myc protein. Selective knockdown of Myc levels in these cells by RNA interference increased sensitivity to apoptosis with compound treatment. By targeting the Myc pathway in Myc-addicted cancer cells, we have identified a novel class of apoptotic inducers that selectively and efficiently target cancer cells with deregulated Myc.