RESUMEN
PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Interferón Tipo I/metabolismo , Neoplasias/tratamiento farmacológico , Proteínas de Transporte de Nucleósidos/genética , Proteínas de Transporte de Nucleósidos/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Inmunidad Adaptativa/efectos de los fármacos , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Escape del Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anti-apoptotic Bcl-2 family proteins are overexpressed in a wide spectrum of cancers and have become well validated therapeutic targets. Cancer cells display survival dependence on individual or subsets of anti-apoptotic proteins that could be effectively targeted by multimodal inhibitors. We designed a 2,5-substituted benzoic acid scaffold that displayed equipotent binding to Mcl-1 and Bfl-1. Structure-based design was guided by several solved cocrystal structures with Mcl-1, leading to the development of compound 24, which binds both Mcl-1 and Bfl-1 with Ki values of 100 nM and shows appreciable selectivity over Bcl-2/Bcl-xL. The selective binding profile of 24 was translated to on-target cellular activity in model lymphoma cell lines. These studies lay a foundation for developing more advanced dual Mcl-1/Bfl-1 inhibitors that have potential to provide greater single agent efficacy and broader coverage to combat resistance in several types of cancer than selective Mcl-1 inhibitors alone.
Asunto(s)
Antineoplásicos/farmacología , Ácido Benzoico/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ácido Benzoico/química , Línea Celular Tumoral , Humanos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Ratones , Ratones Transgénicos , Antígenos de Histocompatibilidad Menor/metabolismo , Simulación del Acoplamiento Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
FBXW7, a classic tumor suppressor, is a substrate recognition subunit of the Skp1-cullin-F-box (SCF) ubiquitin ligase that targets oncoproteins for ubiquitination and degradation. We recently found that FBXW7 is recruited to DNA damage sites to facilitate nonhomologous end-joining (NHEJ). The detailed underlying molecular mechanism, however, remains elusive. Here we report that the WD40 domain of FBXW7, which is responsible for substrate binding and frequently mutated in human cancers, binds to poly(ADP-ribose) (PAR) immediately following DNA damage and mediates rapid recruitment of FBXW7 to DNA damage sites, whereas ATM-mediated FBXW7 phosphorylation promotes its retention at DNA damage sites. Cancer-associated arginine mutations in the WD40 domain (R465H, R479Q and R505C) abolish both FBXW7 interaction with PAR and recruitment to DNA damage sites, causing inhibition of XRCC4 polyubiquitination and NHEJ. Furthermore, inhibition or silencing of poly(ADP-ribose) polymerase 1 (PARP1) inhibits PAR-mediated recruitment of FBXW7 to the DNA damage sites. Taken together, our study demonstrates that the WD40 domain of FBXW7 is a novel PAR-binding motif that facilitates early recruitment of FBXW7 to DNA damage sites for subsequent NHEJ repair. Abrogation of this ability seen in cancer-derived FBXW7 mutations provides a molecular mechanism for defective DNA repair, eventually leading to genome instability.
Asunto(s)
Reparación del ADN por Unión de Extremidades , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli Adenosina Difosfato Ribosa/metabolismo , Factor de Células Madre/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Daño del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/química , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibroblastos/ultraestructura , Rayos gamma , Células HCT116 , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de la radiación , Células Secretoras de Insulina/ultraestructura , Modelos Moleculares , Mutación , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli Adenosina Difosfato Ribosa/química , Unión Proteica , Dominios Proteicos , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Factor de Células Madre/metabolismo , Ubiquitinación/efectos de la radiaciónRESUMEN
Protein-protein interactions (PPIs) represent important and promising therapeutic targets that are associated with the regulation of various molecular pathways, particularly in cancer. Although they were once considered "undruggable," the recent advances in screening strategies, structure-based design, and elucidating the nature of hot spots on PPI interfaces, have led to the discovery and development of successful small-molecule inhibitors. In this report, we are describing an integrated high-throughput and computational screening approach to enable the discovery of small-molecule PPI inhibitors of the anti-apoptotic protein, Mcl-1. Applying this strategy, followed by biochemical, biophysical, and biological characterization, nineteen new chemical scaffolds were discovered and validated as Mcl-1 inhibitors. A novel series of Mcl-1 inhibitors was designed and synthesized based on the identified difuryl-triazine core scaffold and structure-activity studies were undertaken to improve the binding affinity to Mcl-1. Compounds with improved in vitro binding potency demonstrated on-target activity in cell-based studies. The obtained results demonstrate that structure-based analysis complements the experimental high-throughput screening in identifying novel PPI inhibitor scaffolds and guides follow-up medicinal chemistry efforts. Furthermore, our work provides an example that can be applied to the analysis of available screening data against numerous targets in the PubChem BioAssay Database, leading to the identification of promising lead compounds, fuelling drug discovery pipelines.
Asunto(s)
Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Simulación por Computador , Ensayos de Selección de Medicamentos Antitumorales , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Using a high-throughput screening (HTS) approach, we have identified and validated several small-molecule Mcl-1 inhibitors (SMI). Here, we describe a novel selective Mcl-1 SMI inhibitor, 2 (UMI-77), developed by structure-based chemical modifications of the lead compound 1 (UMI-59). We have characterized the binding of UMI-77 to Mcl-1 by using complementary biochemical, biophysical, and computational methods and determined its antitumor activity against a panel of pancreatic cancer cells and an in vivo xenograft model. UMI-77 binds to the BH3-binding groove of Mcl-1 with Ki of 490 nmol/L, showing selectivity over other members of the antiapoptotic Bcl-2 family. UMI-77 inhibits cell growth and induces apoptosis in pancreatic cancer cells in a time- and dose-dependent manner, accompanied by cytochrome c release and caspase-3 activation. Coimmunoprecipitation experiments revealed that UMI-77 blocks the heterodimerization of Mcl-1/Bax and Mcl-1/Bak in cells, thus antagonizing the Mcl-1 function. The Bax/Bak-dependent induction of apoptosis was further confirmed using murine embryonic fibroblasts that are Bax- and Bak-deficient. In an in vivo BxPC-3 xenograft model, UMI-77 effectively inhibited tumor growth. Western blot analysis in tumor remnants revealed enhancement of proapoptotic markers and significant decrease of survivin. Collectively, these promising findings show the therapeutic potential of Mcl-1 inhibitors against pancreatic cancer and warrant further preclinical investigations.
Asunto(s)
Plomo/administración & dosificación , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Neoplasias Pancreáticas/genética , Sulfonamidas/administración & dosificación , Tioglicolatos/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dimerización , Humanos , Ratones , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Neoplasias Pancreáticas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/química , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/química , Proteína X Asociada a bcl-2/genética , Neoplasias PancreáticasRESUMEN
Kinases present an attractive target for drug development, since they are involved in vital cellular processes and are implicated in a variety of diseases, such as cancer and diabetes. However, obtaining selectivity for a specific kinase over others is difficult since many current kinase inhibitors exclusively target the highly conserved kinase ATP binding domain. Previously, a microarray-based strategy to discover so-called bisubstrate-based inhibitors that target the more specific peptide binding groove in addition to the ATP binding site was described. One attractive feature of this strategy is the opportunity to tune the selectivity of these inhibitors by systematically varying components. In an extension to this previous work, this study explores the potential of this guided selectivity modulation, leading to a series of inhibitors with different selectivity profiles against highly homologous protein kinase C (PKC) isozymes. Of the inhibitors studied, most exhibited improved potency and selectivity compared with their constituent parts. Furthermore, the selectivity was found to be tunable either through modification of the pseudosubstrate peptide (peptide binding groove) or the ATP-competitive part (ATP binding site). In a number of cases, the selectivity of the construct could be predicted from the initial peptide substrate profiling experiment. Since this strategy is applicable to all kinase sets, it could be used to rapidly develop uniquely selective inhibitors.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Química Clic , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Péptidos/síntesis química , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Técnicas de Síntesis en Fase Sólida , Especificidad por SustratoRESUMEN
An antitumor agent thiocoraline is a thiodepsipeptide marine product derived from two Micromonospora sp. strains that inhibits protein synthesis by binding of its key 3-hydroxyquinaldic acid (3HQA) chromophores to duplex DNA. There are at least two potential pathways via which the 3HQA moiety could be biosynthesized from L-Trp. By biochemical characterization and by preparation of knockouts of an adenylation-thiolation enzyme, TioK, and of two type II thioesterases, TioP and TioQ, found in the thiocoraline biosynthetic gene cluster, we gained valuable insight into the pathway followed for the production of 3HQA.
Asunto(s)
Proteínas Bacterianas/genética , Depsipéptidos/biosíntesis , Ácido Graso Sintasas/genética , Familia de Multigenes , Proteínas Recombinantes/genética , Streptomyces/genética , Tioléster Hidrolasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Coenzima A/química , Depsipéptidos/genética , Pruebas de Enzimas , Ácido Graso Sintasas/química , Ácido Graso Sintasas/metabolismo , Técnicas de Inactivación de Genes , Hidrólisis , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Filogenia , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Streptomyces/enzimología , Especificidad por Sustrato , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismoRESUMEN
The bisintercalator natural products are a family of nonribosomal peptides possessing a range of biological properties that include antiviral, antibiotic, and anticancer activities. The name bisintercalator is derived from the ability to directly bind to duplex DNA through two planar intercalating moieties. Although 19 members of this family of compounds have been identified over the past 50 years, the biosynthetic genes responsible for the formation of four of these molecules (thiocoraline, SW-163, triostin A, and echinomycin) were identified only recently. This recent progress opens an avenue towards understanding how Nature produces these bisintercalating products and provides the potential to develop and identify novel potent analogous lead compounds for clinical applications. This review discusses the mode of action of bisintercalators and summarizes recent genetic and biochemical insights into their biosynthetic production, analog formation, and possible mechanisms by which resistance to these compounds is achieved by their producing organisms.