RESUMEN
The peptidyl-prolyl isomerase, Pin1, is exploited in cancer to activate oncogenes and inactivate tumor suppressors. However, despite considerable efforts, Pin1 has remained an elusive drug target. Here, we screened an electrophilic fragment library to identify covalent inhibitors targeting Pin1's active site Cys113, leading to the development of Sulfopin, a nanomolar Pin1 inhibitor. Sulfopin is highly selective, as validated by two independent chemoproteomics methods, achieves potent cellular and in vivo target engagement and phenocopies Pin1 genetic knockout. Pin1 inhibition had only a modest effect on cancer cell line viability. Nevertheless, Sulfopin induced downregulation of c-Myc target genes, reduced tumor progression and conferred survival benefit in murine and zebrafish models of MYCN-driven neuroblastoma, and in a murine model of pancreatic cancer. Our results demonstrate that Sulfopin is a chemical probe suitable for assessment of Pin1-dependent pharmacology in cells and in vivo, and that Pin1 warrants further investigation as a potential cancer drug target.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.
Asunto(s)
Transporte Axonal , Axones/metabolismo , Cinesinas/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Gránulos Citoplasmáticos/metabolismo , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Proteínas Asociadas a Microtúbulos , Mitocondrias/metabolismo , Mutación/genética , Péptidos/metabolismo , Fosforilación , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.
Asunto(s)
Citotoxicidad Inmunológica , Inmunoterapia , Proteínas de la Membrana/metabolismo , Neoplasias/inmunología , Neoplasias/terapia , Serpinas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Progresión de la Enfermedad , Femenino , Eliminación de Gen , Granzimas/metabolismo , Inmunidad/efectos de los fármacos , Melanoma/patología , Ratones Endogámicos C57BL , Neoplasias/prevención & control , Bibliotecas de Moléculas Pequeñas/farmacología , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is commonly overexpressed in human cancers, including pancreatic ductal adenocarcinoma (PDAC). While Pin1 is dispensable for viability in mice, it is required for activated Ras to induce tumorigenesis, suggesting a role for Pin1 inhibitors in Ras-driven tumors, such as PDAC. We report the development of rationally designed peptide inhibitors that covalently target Cys113, a highly conserved cysteine located in the Pin1 active site. The inhibitors were iteratively optimized for potency, selectivity and cell permeability to give BJP-06-005-3, a versatile tool compound with which to probe Pin1 biology and interrogate its role in cancer. In parallel to inhibitor development, we employed genetic and chemical-genetic strategies to assess the consequences of Pin1 loss in human PDAC cell lines. We demonstrate that Pin1 cooperates with mutant KRAS to promote transformation in PDAC, and that Pin1 inhibition impairs cell viability over time in PDAC cell lines.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/antagonistas & inhibidores , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Animales , Antineoplásicos/química , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Cristalografía por Rayos X , Cisteína/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Peptidilprolil Isomerasa de Interacción con NIMA/química , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Conformación Proteica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Mammalian switch/sucrose non-fermentable (mSWI/SNF) complexes are multi-component machines that remodel chromatin architecture. Dissection of the subunit- and domain-specific contributions to complex activities is needed to advance mechanistic understanding. Here, we examine the molecular, structural, and genome-wide regulatory consequences of recurrent, single-residue mutations in the putative coiled-coil C-terminal domain (CTD) of the SMARCB1 (BAF47) subunit, which cause the intellectual disability disorder Coffin-Siris syndrome (CSS), and are recurrently found in cancers. We find that the SMARCB1 CTD contains a basic α helix that binds directly to the nucleosome acidic patch and that all CSS-associated mutations disrupt this binding. Furthermore, these mutations abrogate mSWI/SNF-mediated nucleosome remodeling activity and enhancer DNA accessibility without changes in genome-wide complex localization. Finally, heterozygous CSS-associated SMARCB1 mutations result in dominant gene regulatory and morphologic changes during iPSC-neuronal differentiation. These studies unmask an evolutionarily conserved structural role for the SMARCB1 CTD that is perturbed in human disease.