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Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab. Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed. Results: Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9. Conclusion: Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms.
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BACKGROUND: Lymphocyte-activation gene 3 (LAG-3) is an inhibitory immunoreceptor that negatively regulates T-cell activation. This paper presents preclinical characterization of the LAG-3 inhibitor, ieramilimab (LAG525), and phase I data for the treatment of patients with advanced/metastatic solid tumors with ieramilimab ±the anti-programmed cell death-1 antibody, spartalizumab. METHODS: Eligible patients had advanced/metastatic solid tumors and progressed after, or were unsuitable for, standard-of-care therapy, including checkpoint inhibitors in some cases. Patients received ieramilimab ±spartalizumab across various dose-escalation schedules. The primary objective was to assess the maximum tolerated dose (MTD) or recommended phase II dose (RP2D). RESULTS: In total, 255 patients were allocated to single-agent ieramilimab (n=134) and combination (n=121) treatment arms. The majority (98%) had received prior antineoplastic therapy (median, 3). Four patients experienced dose-limiting toxicities in each treatment arm across various dosing cohorts. No MTD was reached. The RP2D on a 3-week schedule was declared as 400 mg ieramilimab plus 300 mg spartalizumab and, on a 4-week schedule (once every 4 weeks; Q4W), as 800 mg ieramilimab plus 400 mg spartalizumab; tumor target (LAG-3) suppression with 600 mg ieramilimab Q4W was predicted to be similar to the Q4W, RP2D schedule. Treatment-related adverse events (TRAEs) occurred in 75 (56%) and 84 (69%) patients in the single-agent and combination arms, respectively. Most common TRAEs were fatigue, gastrointestinal, and skin disorders, and were of mild severity; seven patients experienced at least one treatment-related serious adverse event in the single-agent (5%) and combination group (5.8%). Antitumor activity was observed in the combination arm, with 3 (2%) complete responses and 10 (8%) partial responses in a mixed population of tumor types. In the combination arm, eight patients (6.6%) experienced stable disease for 6 months or longer versus six patients (4.5%) in the single-agent arm. Responding patients trended towards having higher levels of immune gene expression, including CD8 and LAG3, in tumor tissue at baseline. CONCLUSIONS: Ieramilimab was well tolerated as monotherapy and in combination with spartalizumab. The toxicity profile of ieramilimab in combination with spartalizumab was comparable to that of spartalizumab alone. Modest antitumor activity was seen with combination treatment. TRIAL REGISTRATION NUMBER: NCT02460224.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/farmacología , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Persona de Mediana Edad , Adulto JovenRESUMEN
CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.
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Anticuerpos Biespecíficos/farmacología , Antineoplásicos/farmacología , Complejo CD3/inmunología , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Línea Celular Tumoral , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Inmunoterapia , Interferón gamma/farmacología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The emergence of multi-drug-resistant strains of Staphylococcus epidermidis emphasizes the need to develop new antibiotics. The unique and essential role of the peptide deformylase (PDF) in catalysing the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria makes it an attractive antibacterial drug target. In the present study, both deformylase homologues from S. epidermidis (SePDF-1 and SePDF-2) were cloned and expressed, and their enzymic activities were characterized. Co(2+)-substituted SePDF-1 exhibited much higher enzymic activity (k(cat)/K(m) 6.3 × 10(4) M(-1) s(-1)) than those of Ni(2+)- and Zn(2+)-substituted SePDF-1, and SePDF-1 showed much weaker binding ability towards Ni(2+) than towards Co(2+) and Zn(2+), which is different from PDF in Staphylococcus aureus (SaPDF), although they share 80â% amino-acid sequence identity. The determined crystal structure of SePDF-1 was similar to that of (SaPDF), except for differences in the metal-binding sites. The other deformylase homologue, SePDF-2, was shown to have no peptide deformylase activity; the function of SePDF-2 needs to be further investigated.
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Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Staphylococcus epidermidis/enzimología , Amidohidrolasas/genética , Secuencias de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Clonación Molecular , Cobalto/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Níquel/metabolismo , Homología de Secuencia de Aminoácido , Staphylococcus epidermidis/genética , Zinc/metabolismoRESUMEN
AIM: Peroxisome proliferator-activated receptor gamma (PPARgamma) is a therapeutic target for obesity, cancer and diabetes mellitus. In order to develop potent lead compounds for obesity treatment, we screened a natural product library for novel PPARgamma antagonists with inhibitory effects on adipocyte differentiation. METHODS: Surface plasmon resonance (SPR) technology and cell-based transactivation assay were used to screen for PPARgamma antagonists. To investigate the antagonistic mechanism of the active compound, we measured its effect on PPARgamma/RXRalpha heterodimerization and PPARgamma co-activator recruitment using yeast two-hybrid assay, Gal4/UAS cell-based assay and SPR based assay. The 3T3-L1 cell differentiation assay was used to evaluate the effect of the active compound on adipocyte differentiation. RESULTS: A new thiophene-acetylene type of natural product, 7-chloroarctinone-b (CAB), isolated from the roots of Rhaponticum uniflorum, was discovered as a novel PPARgamma antagonist capable of inhibiting rosiglitazone-induced PPARgamma transcriptional activity. SPR analysis suggested that CAB bound tightly to PPARgamma and considerably antagonized the potent PPARgamma agonist rosiglitazone-stimulated PPARgamma-LBD/RXRalpha-LBD binding. Gal4/UAS and yeast two-hybrid assays were used to evaluate the antagonistic activity of CAB on rosiglitazone-induced recruitment of the coactivator for PPARgamma. CAB could efficiently antagonize both hormone and rosiglitazone-induced adipocyte differentiation in cell culture. CONCLUSION: CAB shows antagonistic activity to PPARgamma and can block the adipocyte differentiation, indicating it may be of potential use as a lead therapeutic compound for obesity.
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Adipocitos/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Tiofenos/farmacología , Células 3T3-L1/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Animales , Diferenciación Celular/efectos de los fármacos , Dimerización , Humanos , Ratones , Resonancia por Plasmón de Superficie , Activación Transcripcional/efectos de los fármacos , Técnicas del Sistema de Dos HíbridosRESUMEN
AIM: To search for novel inhibitors of human polo-like kinase 1 (Plk1), which plays important roles in various aspects of mitotic progression and is believed as a promising anti-cancer drug target, and further investigate the potential inhibition mechanism of active compounds against Plk1, thus developing potent anti-tumor lead compounds. METHODS: Surface plasmon resonance (SPR) technology-based assay and enzymatic inhibition assay were used to screen Plk1 inhibitors. Sulphorhodamine B (SRB)-based assay, flow cytometry, confocal microscopy and Western blotting were used to further identify the potent Plk1 inhibitor. To investigate the inhibitory mechanism of the active compound against Plk1, enzymatic inhibition assay, SPR and yeast two-hybrid technology-based assays were used. RESULTS: Aristolactam AIIIa was identified as a new type of Plk1 inhibitors, targeting the Polo Box domain (PBD) which is another efficient tactic for exploring Plk1 inhibitors. Further studies indicated that it could block the proliferations of HeLa, A549, HGC and the HCT-8/V cells (clinical Navelbine-resistant cancer cell), induce mitotic arrest of HeLa cells at G2/M phase with spindle abnormalities and promote apoptosis in HeLa cells. The results from SPR and yeast two-hybrid technology-based assays suggested that it could target both the catalytic domain of Plk1 (CD) and PBD and enhance the CD/PBD interaction. CONCLUSION: Our current work is expected to shed light on the potential anti-tumor mechanism of Aristolactam AIIIa, and this natural product might be possibly used as a lead compound for further developing anti-tumor drugs.
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Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Animales , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Dominio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/enzimología , Relación Dosis-Respuesta a Droga , Células HT29 , Células HeLa , Humanos , Alcaloides Indólicos , Concentración 50 Inhibidora , Lactamas , Leucemia L1210/tratamiento farmacológico , Leucemia L1210/enzimología , Ligandos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Ratones , Estructura Molecular , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Huso Acromático/efectos de los fármacos , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/enzimología , Resonancia por Plasmón de Superficie , Técnicas del Sistema de Dos Híbridos , Quinasa Tipo Polo 1RESUMEN
Axin is a key scaffolding protein responsible for the formation of the ß-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/ß-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/ß-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/ß-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/ß-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/ß-catenin signaling.
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Proteína Axina/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , beta Catenina/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Animales , Proteína Axina/genética , Línea Celular , Línea Celular Tumoral , Citometría de Flujo , Células HCT116 , Humanos , Inmunoprecipitación , Ratones , Osteoblastos/metabolismo , Estabilidad Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Peptidasa Específica de Ubiquitina 7/genética , Ubiquitinación/genética , Ubiquitinación/fisiología , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiología , beta Catenina/genéticaRESUMEN
3C-like protease (3CL pro) plays pivotal roles in the life cycle of severe acute respiratory syndrome coronavirus (SARS-CoV) and only the dimeric protease is proposed as the functional form. Guided by the crystal structure and molecular dynamics simulations, we performed systematic mutation analyses to identify residues critical for 3CL pro dimerization and activity in this study. Seven residues on the dimer interface were selected for evaluating their contributions to dimer stability and catalytic activity by biophysical and biochemical methods. These residues are involved in dimerization through hydrogen bonding and broadly located in the N-terminal finger, the alpha-helix A' of domain I, and the oxyanion loop near the S1 substrate-binding subsite in domain II. We revealed that all seven single mutated proteases still have the dimeric species but the monomer-dimer equilibria of these mutants vary from each other, implying that these residues might contribute differently to the dimer stability. Such a conclusion could be further verified by the results that the proteolytic activities of these mutants also decrease to varying degrees. The present study would help us better understand the dimerization-activity relationship of SARS-CoV 3CL pro and afford potential information for designing anti-viral compounds targeting the dimer interface of the protease.
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Cisteína Endopeptidasas/química , Proteínas Virales/química , Secuencia de Bases , Catálisis , Cromatografía en Gel , Clonación Molecular , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Cartilla de ADN , Dimerización , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Espectrometría de Fluorescencia , Espectrofotometría Ultravioleta , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The contributions of structural biology to drug discovery have expanded over the last 20 years from structure-based ligand optimization to a broad range of clinically relevant topics including the understanding of disease, target discovery, screening for new types of ligands, discovery of new modes of action, addressing clinical challenges such as side effects or resistance, and providing data to support drug registration. This expansion of scope is due to breakthroughs in the technology, which allow structural information to be obtained rapidly and for more complex molecular systems, but also due to the combination of different technologies such as X-ray, NMR, and other biophysical methods, which allows one to get a more complete molecular understanding of disease and ways to treat it. In this review, we provide examples of the types of impact molecular structure information can have in the clinic for both low molecular weight and biologic drug discovery and describe several case studies from our own work to illustrate some of these contributions.
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Descubrimiento de Drogas , Animales , Productos Biológicos/química , Productos Biológicos/uso terapéutico , Humanos , Inmunoterapia , Estructura Molecular , Neoplasias/metabolismo , Neoplasias/terapia , Conformación Proteica , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A member 2 (SMARCA2), also known as Brahma homologue (BRM), is a Snf2-family DNA-dependent ATPase. BRM and its close homologue Brahma-related gene 1 (BRG1), also known as SMARCA4, are mutually exclusive ATPases of the large ATP-dependent SWI/SNF chromatin-remodeling complexes involved in transcriptional regulation of gene expression. No small molecules have been reported that modulate SWI/SNF chromatin-remodeling activity via inhibition of its ATPase activity, an important goal given the well-established dependence of BRG1-deficient cancers on BRM. Here, we describe allosteric dual BRM and BRG1 inhibitors that downregulate BRM-dependent gene expression and show antiproliferative activity in a BRG1-mutant-lung-tumor xenograft model upon oral administration. These compounds represent useful tools for understanding the functions of BRM in BRG1-loss-of-function settings and should enable probing the role of SWI/SNF functions more broadly in different cancer contexts and those of other diseases.
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Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , ADN Helicasas/genética , Diseño de Fármacos , Mutación , Proteínas Nucleares/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Administración Oral , Animales , Antineoplásicos/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Relación Estructura-Actividad , Factores de Transcripción/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Malonyl-CoA: acyl carrier protein transacylase (MCAT) is a critical enzyme responsible for the transfer of the malonyl moiety to holo-acyl carrier protein (ACP) forming the malonyl-ACP intermediates in the initiation step of type II fatty acid synthesis (FAS II) in bacteria. MCAT has been considered as an attractive drug target in the discovery of antibacterial agents. In this study, the crystal structure of MCAT from Helicobacter pylori (Hp) at 2.5 angstroms resolution is reported, and the interaction of HpMCAT with HpACP is extensively investigated by using computational docking, GST-pull-down, and surface plasmon resonance (SPR) technology-based assays. The crystal structure results reveal that HpMCAT has a compact folding composed of a large subdomain with a similar core as in alpha/beta hydrolases, and a similar ferredoxin-like small subdomain as in acylphosphatases. The docking result suggests two positively charged areas near the entrance of the active site of HpMCAT as the ACP-binding region. Binding assay research shows that HpMCAT demonstrates a moderately binding ability against HpACP. The solved 3D structure of HpMCAT is expected to supply useful information for the structure-based discovery of novel inhibitors against MCAT, and the quantitative study of HpMCAT interaction with HpACP is hoped to give helpful hints in the understanding of the detailed catalytic mechanisms for HpMCAT.
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S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Proteínas Bacterianas/metabolismo , Helicobacter pylori/enzimología , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/química , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X/métodos , Helicobacter pylori/genética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Resonancia por Plasmón de SuperficieRESUMEN
Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.
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Antineoplásicos/uso terapéutico , Cadherinas/antagonistas & inhibidores , Neoplasias Renales/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Antineoplásicos/farmacología , Cadherinas/genética , Cadherinas/metabolismo , Femenino , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Macaca fascicularis , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Ratas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Colonization of human stomach by the bacterium Helicobacter pylori is a major causative factor for gastrointestinal illnesses and gastric cancer. However, the discovery of anti-H. pylori agents is a difficult task due to lack of mature protein targets. Therefore, identifying new molecular targets for developing new drugs against H. pylori is obviously necessary. In this study, the in-house potential drug target database (PDTD, http://www.dddc.ac.cn/tarfisdock/) was searched by the reverse docking approach using an active natural product (compound 1) discovered by anti-H. pylori screening as a probe. Homology search revealed that, among the 15 candidates discovered by reverse docking, only diaminopimelate decarboxylase (DC) and peptide deformylase (PDF) have homologous proteins in the genome of H. pylori. Enzymatic assay demonstrated compound 1 and its derivative compound 2 are the potent inhibitors against H. pylori PDF (HpPDF) with IC50 values of 10.8 and 1.25 microM, respectively. X-ray crystal structures of HpPDF and the complexes of HpPDF with 1 and 2 were determined for the first time, indicating that these two inhibitors bind well with HpPDF binding pocket. All these results indicate that HpPDF is a potential target for screening new anti-H. pylori agents. In addition, compounds 1 and 2 were predicted to bind to HpPDF with relatively high selectivity, suggesting they can be used as leads for developing new anti-H. pylori agents. The results demonstrated that our strategy, reverse docking in conjunction with bioassay and structural biology, is effective and can be used as a complementary approach of functional genomics and chemical biology in target identification.
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Amidohidrolasas/química , Cristalografía por Rayos X/métodos , Helicobacter pylori/enzimología , Amidohidrolasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Notch receptors are transmembrane proteins that undergo activating proteolysis in response to ligand stimulation. A negative regulatory region (NRR) maintains receptor quiescence by preventing protease cleavage prior to ligand binding. We report here the X-ray structure of the NRR of autoinhibited human Notch3, and compare it with the Notch1 and Notch2 NRRs. The overall architecture of the autoinhibited conformation, in which three LIN12-Notch repeat (LNR) modules wrap around a heterodimerization domain, is preserved in Notch3, but the autoinhibited conformation of the Notch3 NRR is less stable. The Notch3 NRR uses a highly conserved surface on the third LNR module to form a dimer in the crystal. Similar homotypic interfaces exist in Notch1 and Notch2. Together, these studies reveal distinguishing structural features associated with increased basal activity of Notch3, demonstrate increased ligand-independent signaling for disease-associated mutations that map to the Notch3 NRR, and identify a conserved dimerization interface present in multiple Notch receptors.
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Receptores Notch/química , Línea Celular Tumoral , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteolisis , Receptor Notch3 , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value.
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Factor de Transcripción STAT3/química , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Cristalografía por Rayos X , Factor Inhibidor de Leucemia/fisiología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/fisiología , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Shikimate dehydrogenase (SDH) catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway. In this study, we determined the kinetic properties and crystal structures of Staphylococcus epidermidis SDH (SeSDH) both in its ligand-free form and in complex with shikimate. SeSDH has a k(cat) of 22.8 s(-1) and a K(m) of 73 mum towards shikimate, and a K(m) of 100 microM towards NADP. The overall folding of SeSDH comprises the N-terminal alpha/beta domain for substrate binding and the C-terminal Rossmann fold for NADP binding. The active site is within a large groove between the two domains. Residue Tyr211, normally regarded as important for substrate binding, does not interact with shikimate in the binary SeSDH-shikimate complex structure. However, the Y211F mutation leads to a significant decrease in k(cat) and a minor increase in the K(m) for shikimate. The results indicate that the main function of Tyr211 may be to stabilize the catalytic intermediate during catalysis. The NADP-binding domain of SeSDH is less conserved. The usually long helix specifically recognizing the adenine ribose phosphate is substituted with a short 3(10) helix in the NADP-binding domain. Moreover, the interdomain angle of SeSDH is the widest among all known SDH structures, indicating an inactive 'open' state of the SeSDH structure. Thus, a 'closing' process might occur upon NADP binding to bring the cofactor close to the substrate for catalysis.
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Oxidorreductasas de Alcohol/química , Modelos Moleculares , Staphylococcus epidermidis/enzimología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutación , NADP/metabolismo , Pliegue de Proteína , Ácido Shikímico/metabolismoRESUMEN
The 3C-like protease of SARS coronavirus (SARS-CoV 3CL(pro)) is vital for SARS-CoV replication and is a promising drug target. It has been extensively proved that only the dimeric enzyme is active. Here we discovered that two adjacent mutations (Ser139_Ala and Phe140_Ala) on the dimer interface resulted in completely different crystal structures of the enzyme, demonstrating the distinct roles of these two residues in maintaining the active conformation of SARS-CoV 3CL(pro). S139A is a monomer that is structurally similar to the two reported monomers G11A and R298A. However, this mutant still retains a small fraction of dimer in solution, which might account for its remaining activity. F140A is a dimer with the most collapsed active pocket discovered so far, well-reflecting the stabilizing role of this residue. Moreover, a plausible dimerization mechanism was also deduced from structural analysis. Our work is expected to provide insight on the dimerization-function relationship of SARS-CoV 3CL(pro).
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Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Modelos Moleculares , Mutación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Proteínas Virales/química , Proteínas Virales/genética , Proteasas 3C de Coronavirus , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/metabolismo , Dimerización , Estructura Terciaria de Proteína/genética , Relación Estructura-Actividad , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
SARS-CoV 3C-like protease (3CL(pro)) is an attractive target for anti-severe acute respiratory syndrome (SARS) drug discovery, and its dimerization has been extensively proved to be indispensable for enzymatic activity. However, the reason why the dissociated monomer is inactive still remains unclear due to the absence of the monomer structure. In this study, we showed that mutation of the dimer-interface residue Gly-11 to alanine entirely abolished the activity of SARS-CoV 3CL(pro). Subsequently, we determined the crystal structure of this mutant and discovered a complete crystallographic dimer dissociation of SARS-CoV 3CL(pro). The mutation might shorten the alpha-helix A' of domain I and cause a mis-oriented N-terminal finger that could not correctly squeeze into the pocket of another monomer during dimerization, thus destabilizing the dimer structure. Several structural features essential for catalysis and substrate recognition are severely impaired in the G11A monomer. Moreover, domain III rotates dramatically against the chymotrypsin fold compared with the dimer, from which we proposed a putative dimerization model for SARS-CoV 3CL(pro). As the first reported monomer structure for SARS-CoV 3CL(pro), the crystal structure of G11A mutant might provide insight into the dimerization mechanism of the protease and supply direct structural evidence for the incompetence of the dissociated monomer.
Asunto(s)
Cisteína Endopeptidasas/química , Glicina/genética , Mutación , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Proteínas Virales/química , Dicroismo Circular , Simulación por Computador , Proteasas 3C de Coronavirus , Cristalografía por Rayos X , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Dimerización , Modelos Moleculares , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Alanine racemase (Alr) is an important enzyme that catalyzes the interconversion of L-alanine and D-alanine, an essential building block in the peptidoglycan biosynthesis. For the small size of the Alr active site, its conserved substrate entryway has been proposed as a potential choice for drug design. In this work, we fully analyzed the crystal structures of the native, the D-cycloserine-bound, and four mutants (P219A, E221A, E221K, and E221P) of biosynthetic Alr from Escherichia coli (EcAlr) and studied the potential roles in substrate orientation for the key residues involved in the substrate entryway in conjunction with the enzymatic assays. Structurally, it was discovered that EcAlr is similar to the Pseudomonas aeruginosa catabolic Alr in both overall and active site geometries. Mutation of the conserved negatively charged residue aspartate 164 or glutamate 165 at the substrate entryway could obviously reduce the binding affinity of enzyme against the substrate and decrease the turnover numbers in both D- to L-Ala and L- to D-Ala directions, especially when mutated to lysine with the opposite charge. However, mutation of Pro219 or Glu221 had only negligible or a small influence on the enzymatic activity. Together with the enzymatic and structural investigation results, we thus proposed that the negatively charged residues Asp164 and Glu165 around the substrate entryway play an important role in substrate orientation with cooperation of the positively charged Arg280 and Arg300 on the opposite monomer. Our findings are expected to provide some useful structural information for inhibitor design targeting the substrate entryway of Alr.
Asunto(s)
Alanina Racemasa/metabolismo , Ácido Aspártico/química , Escherichia coli/enzimología , Ácido Glutámico/química , Alanina Racemasa/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
beta-Hydroxyacyl-acyl carrier protein dehydratase (FabZ) is an important enzyme for the elongation cycles of both saturated and unsaturated fatty acids biosyntheses in the type II fatty acid biosynthesis system (FAS II) pathway. FabZ has been an essential target for the discovery of compounds effective against pathogenic microbes. In this work, to characterize the catalytic and inhibitory mechanisms of FabZ, the crystal structures of the FabZ of Helicobacter pylori (HpFabZ) and its complexes with two newly discovered inhibitors have been solved. Different from the structures of other bacterial FabZs, HpFabZ contains an extra short two-turn alpha-helix (alpha4) between alpha3 and beta3, which plays an important role in shaping the substrate-binding tunnel. Residue Tyr-100 at the entrance of the tunnel adopts either an open or closed conformation in the crystal structure. The crystal structural characterization, the binding affinity determination, and the enzymatic activity assay of the HpFabZ mutant (Y100A) confirm the importance of Tyr-100 in catalytic activity and substrate binding. Residue Phe-83 at the exit tunnel was also refined in two alternative conformations, leading the tunnel to form an L-shape and U-shape. All these data thus contributed much to understanding the catalytic mechanism of HpFabZ. In addition, the co-crystal structures of HpFabZ with its inhibitors have suggested that the enzymatic activity of HpFabZ could be inhibited either by occupying the entrance of the tunnel or plugging the tunnel to prevent the substrate from accessing the active site. Our study has provided some insights into the catalytic and inhibitory mechanisms of FabZ, thus facilitating antibacterial agent development.