RESUMEN
The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Acriflavina , Animales , Antivirales/química , Antivirales/farmacología , Humanos , Ratones , Simulación del Acoplamiento Molecular , PandemiasAsunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Fenotiazinas/química , Fenotiazinas/farmacología , Regulación Alostérica/efectos de los fármacos , Caspasas/química , Caspasas/metabolismo , Línea Celular , Humanos , Modelos Moleculares , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismoRESUMEN
The CARD11-BCL10-MALT1 (CBM) complex triggers the adaptive immune response in lymphocytes and lymphoma cells. CARD11/CARMA1 acts as a molecular seed inducing BCL10 filaments, but the integration of MALT1 and the assembly of a functional CBM complex has remained elusive. Using cryo-EM we solved the helical structure of the BCL10-MALT1 filament. The structural model of the filament core solved at 4.9 Å resolution identified the interface between the N-terminal MALT1 DD and the BCL10 caspase recruitment domain. The C-terminal MALT1 Ig and paracaspase domains protrude from this core to orchestrate binding of mediators and substrates at the filament periphery. Mutagenesis studies support the importance of the identified BCL10-MALT1 interface for CBM complex assembly, MALT1 protease activation and NF-κB signaling in Jurkat and primary CD4 T-cells. Collectively, we present a model for the assembly and architecture of the CBM signaling complex and how it functions as a signaling hub in T-lymphocytes.
Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B/ultraestructura , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/ultraestructura , Proteína 10 de la LLC-Linfoma de Células B/química , Proteína 10 de la LLC-Linfoma de Células B/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Microscopía por Crioelectrón , Guanilato Ciclasa/metabolismo , Activación de Linfocitos , Modelos Químicos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Conformación ProteicaRESUMEN
Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.
Asunto(s)
Proteína 10 de la LLC-Linfoma de Células B/inmunología , Proteínas Adaptadoras de Señalización CARD/inmunología , Guanilato Ciclasa/inmunología , Activación de Linfocitos , Multimerización de Proteína/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T/inmunología , Proteína 10 de la LLC-Linfoma de Células B/genética , Proteínas Adaptadoras de Señalización CARD/genética , Guanilato Ciclasa/genética , Células HEK293 , Humanos , Células Jurkat , Multimerización de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Linfocitos T/citologíaRESUMEN
MALT1 paracaspase is activated upon antigen receptor stimulation to promote lymphocyte activation. In addition, deregulated MALT1 protease activity drives survival of distinct lymphomas such as the activated B cell type of diffuse large B cell lymphoma (ABC-DLBCL). Here, we designed fluorophore or biotin-coupled activity based-probes (ABP) that covalently modify the active center of MALT1. MALT1-ABPs are exclusively labeling an active modified full length form of MALT1 upon T cell stimulation. Further, despite the CARMA1 requirement for initial MALT1 activation, the MALT1-ABPs show that protease activity is not confined to the high-molecular CARMA1-BCL10-MALT1 (CBM) complex. Using biotin-coupled ABPs, we developed a robust assay for sensitive and selective detection of active MALT1 in cell lines, primary lymphocytes, and DLBCL tumor biopsies. Taken together, MALT1-ABPs represent powerful chemical tools to measure cellular MALT1 activation, determine efficacy of small molecule inhibitors, and classify lymphomas based on MALT1 activity status.
Asunto(s)
Caspasas/metabolismo , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/patología , Sondas Moleculares/química , Proteínas de Neoplasias/metabolismo , Linfocitos T/enzimología , Biotina/química , Western Blotting , Proteínas Adaptadoras de Señalización CARD/metabolismo , Línea Celular , Química Clic , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Guanilato Ciclasa/metabolismo , Humanos , Células Jurkat , Sondas Moleculares/síntesis química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Rodaminas/química , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The Mre11-Rad50-Nbs1 (MRN) complex tethers, processes and signals DNA double-strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of the nuclease domains of Mre11, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results show that Mre11 forms a flexible dimer and suggest that Nbs1 not only is a checkpoint adaptor but also functionally influences Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1-interaction sites and weaken the Mre11-Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.