RESUMEN
Protein arginine methyltransferases (PRMTs) play important roles in several cellular processes, including signaling, gene regulation, and transport of proteins and nucleic acids, to impact growth, differentiation, proliferation, and development. PRMT5 symmetrically di-methylates the two-terminal ω-guanidino nitrogens of arginine residues on substrate proteins. PRMT5 acts as part of a multimeric complex in concert with a variety of partner proteins that regulate its function and specificity. A core component of these complexes is the WD40 protein MEP50/WDR77/p44, which mediates interactions with binding partners and substrates. We have determined the crystal structure of human PRMT5 in complex with MEP50 (methylosome protein 50), bound to an S-adenosylmethionine analog and a peptide substrate derived from histone H4. The structure of the surprising hetero-octameric complex reveals the close interaction between the seven-bladed ß-propeller MEP50 and the N-terminal domain of PRMT5, and delineates the structural elements of substrate recognition.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteína-Arginina N-Metiltransferasas/química , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
B and T lymphocyte attenuator (BTLA) is an attractive target for a new class of therapeutics that attempt to rebalance the immune system by agonizing checkpoint inhibitory receptors (CIRs). Herpesvirus entry mediator (HVEM) binds BTLA in both trans- and cis-orientations. We report here the development and structural characterization of three humanized BTLA agonist antibodies, 22B3, 25F7, and 23C8. We determined the crystal structures of the antibody-BTLA complexes, showing that these antibodies bind distinct and non-overlapping epitopes of BTLA. While all three antibodies activate BTLA, 22B3 mimics HVEM binding to BTLA and shows the strongest agonistic activity in functional cell assays and in an imiquimod-induced mouse model of psoriasis. 22B3 is also capable of modulating HVEM signaling through the BTLA-HVEM cis-interaction. The data obtained from crystal structures, biochemical assays, and functional studies provide a mechanistic model of HVEM and BTLA organization on the cell surface and informed the discovery of a highly active BTLA agonist.
Asunto(s)
Receptores Inmunológicos , Linfocitos T , Ratones , Animales , Linfocitos T/metabolismo , Receptores Inmunológicos/metabolismo , Anticuerpos/metabolismoRESUMEN
Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Biología Computacional/métodos , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Biespecíficos/química , Rastreo Diferencial de Calorimetría , Citotoxicidad Inmunológica , Inmunoglobulina G/metabolismo , Ratones , Mutación/genética , Polisacáridos/metabolismo , Desnaturalización Proteica , Estabilidad Proteica , Subunidades de Proteína/metabolismo , Receptores de Antígenos de Linfocitos T/química , Proteínas Recombinantes/metabolismo , Solubilidad , TemperaturaRESUMEN
Loss-of-function mutations in the retinoblastoma gene RB1 are common in several treatment-refractory cancers such as small-cell lung cancer and triple-negative breast cancer. To identify drugs synthetic lethal with RB1 mutation (RB1 mut), we tested 36 cell-cycle inhibitors using a cancer cell panel profiling approach optimized to discern cytotoxic from cytostatic effects. Inhibitors of the Aurora kinases AURKA and AURKB showed the strongest RB1 association in this assay. LY3295668, an AURKA inhibitor with over 1,000-fold selectivity versus AURKB, is distinguished by minimal toxicity to bone marrow cells at concentrations active against RB1 mut cancer cells and leads to durable regression of RB1 mut tumor xenografts at exposures that are well tolerated in rodents. Genetic suppression screens identified enforcers of the spindle-assembly checkpoint (SAC) as essential for LY3295668 cytotoxicity in RB1-deficient cancers and suggest a model in which a primed SAC creates a unique dependency on AURKA for mitotic exit and survival. SIGNIFICANCE: The identification of a synthetic lethal interaction between RB1 and AURKA inhibition, and the discovery of a drug that can be dosed continuously to achieve uninterrupted inhibition of AURKA kinase activity without myelosuppression, suggest a new approach for the treatment of RB1-deficient malignancies, including patients progressing on CDK4/6 inhibitors.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Proteínas de Unión a Retinoblastoma/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas de Unión a Retinoblastoma/genética , Transducción de Señal , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Alkyne 40, 5-(2-amino-4-chloro-7-((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-2-methylpent-4-yn-2-ol (EC144), is a second generation inhibitor of heat shock protein 90 (Hsp90) and is substantially more potent in vitro and in vivo than the first generation inhibitor 14 (BIIB021) that completed phase II clinical trials. Alkyne 40 is more potent than 14 in an Hsp90α binding assay (IC(50) = 1.1 vs 5.1 nM) as well as in its ability to degrade Her-2 in MCF-7 cells (EC(50) = 14 vs 38 nM). In a mouse model of gastric tumors (N87), 40 stops tumor growth at 5 mg/kg and causes partial tumor regressions at 10 mg/kg (po, qd × 5). Under the same conditions, 14 stops tumor growth only at 120 mg/kg, and does not induce partial regressions. Thus, alkyne 40 is approximately 20-fold more efficacious than 14 in mice.