RESUMEN
Ribosomes frequently stall during mRNA translation, resulting in the context-dependent activation of quality control pathways to maintain proteostasis. However, surveillance mechanisms that specifically respond to stalled ribosomes with an occluded A site have not been identified. We discovered that the elongation factor-1α (eEF1A) inhibitor, ternatin-4, triggers the ubiquitination and degradation of eEF1A on stalled ribosomes. Using a chemical genetic approach, we unveiled a signaling network comprising two E3 ligases, RNF14 and RNF25, which are required for eEF1A degradation. Quantitative proteomics revealed the RNF14 and RNF25-dependent ubiquitination of eEF1A and a discrete set of ribosomal proteins. The ribosome collision sensor GCN1 plays an essential role by engaging RNF14, which directly ubiquitinates eEF1A. The site-specific, RNF25-dependent ubiquitination of the ribosomal protein RPS27A/eS31 provides a second essential signaling input. Our findings illuminate a ubiquitin signaling network that monitors the ribosomal A site and promotes the degradation of stalled translation factors, including eEF1A and the termination factor eRF1.
Asunto(s)
Proteínas de Unión al ARN , Transactivadores , Proteínas Portadoras/metabolismo , Factores de Elongación de Péptidos/genética , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Humanos , Células HeLa , Células HEK293 , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Factor 1 de Elongación Peptídica/metabolismoRESUMEN
Rapid and accurate mRNA translation requires efficient codon-dependent delivery of the correct aminoacyl-tRNA (aa-tRNA) to the ribosomal A site. In mammals, this fidelity-determining reaction is facilitated by the GTPase elongation factor-1 alpha (eEF1A), which escorts aa-tRNA as an eEF1A(GTP)-aa-tRNA ternary complex into the ribosome. The structurally unrelated cyclic peptides didemnin B and ternatin-4 bind to the eEF1A(GTP)-aa-tRNA ternary complex and inhibit translation but have different effects on protein synthesis in vitro and in vivo. Here, we employ single-molecule fluorescence imaging and cryogenic electron microscopy to determine how these natural products inhibit translational elongation on mammalian ribosomes. By binding to a common site on eEF1A, didemnin B and ternatin-4 trap eEF1A in an intermediate state of aa-tRNA selection, preventing eEF1A release and aa-tRNA accommodation on the ribosome. We also show that didemnin B and ternatin-4 exhibit distinct effects on the dynamics of aa-tRNA selection that inform on observed disparities in their inhibition efficacies and physiological impacts. These integrated findings underscore the value of dynamics measurements in assessing the mechanism of small-molecule inhibition and highlight potential of single-molecule methods to reveal how distinct natural products differentially impact the human translation mechanism.
Asunto(s)
Productos Biológicos , Aminoacil-ARN de Transferencia , Animales , Humanos , Productos Biológicos/metabolismo , Codón/metabolismo , Guanosina Trifosfato/metabolismo , Mamíferos/genética , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/genética , Factor Tu de Elongación Peptídica/metabolismo , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/metabolismo , Ribosomas/metabolismo , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.
Asunto(s)
Lisina , Inhibidores de Proteínas Quinasas , Animales , Cisteína/metabolismo , Lisina/metabolismo , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismoRESUMEN
The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Femenino , Humanos , Masculino , Neoplasias/inmunologíaRESUMEN
Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/metabolismo , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Inyecciones Intravenosas , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.