RESUMO
We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.
Assuntos
Inibidores Enzimáticos/farmacologia , Organofosfonatos/farmacologia , Fosfopiruvato Hidratase/antagonistas & inibidores , Fosfopiruvato Hidratase/química , Pirrolidinonas/farmacologia , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Organofosfonatos/química , Ligação Proteica , Pirrolidinonas/química , Análise Espectral , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions--autophosphorylation of tyrosine-397--requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.
Assuntos
Quinase 1 de Adesão Focal/química , Motivos de Aminoácidos , Animais , Cristalografia por Raios X , Dimerização , Ativação Enzimática , Quinase 1 de Adesão Focal/fisiologia , Adesões Focais , Células HEK293 , Humanos , Modelos Moleculares , Fosforilação , Fosfotirosina/fisiologia , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Espalhamento de RadiaçãoRESUMO
Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.
Assuntos
Inibidores Enzimáticos/farmacologia , Organofosfonatos/farmacologia , Fosfopiruvato Hidratase/antagonistas & inibidores , Pirrolidinonas/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Organofosfonatos/química , Fosfopiruvato Hidratase/metabolismo , Pirrolidinonas/química , Relação Estrutura-AtividadeRESUMO
LiaR is a 'master regulator' of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR(D191N) increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Daptomicina/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sequência Consenso , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Bacteriana , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Modelos Moleculares , Mutação , Óperon , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/química , Dobramento de Proteína , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Agregação Patológica de Proteínas/patologia , Domínios Proteicos , Multimerização Proteica , TermodinâmicaRESUMO
Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Ligação a DNA/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Histonas/química , Isoxazóis/química , Fragmentos de Peptídeos/química , Processamento de Proteína Pós-Traducional , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Biotinilação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Histonas/antagonistas & inibidores , Histonas/metabolismo , Humanos , Isoxazóis/síntese química , Isoxazóis/farmacologia , Cinética , Ligantes , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Maleabilidade , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sulfonamidas/síntese química , Sulfonamidas/química , Sulfonamidas/farmacologia , meta-Aminobenzoatos/síntese química , meta-Aminobenzoatos/química , meta-Aminobenzoatos/farmacologiaRESUMO
Staphylococcus aureus VraR, a vancomycin-resistance-associated response regulator, activates a cell-wall-stress stimulon in response to antibiotics that inhibit cell wall formation. X-ray crystal structures of VraR in both unphosphorylated and beryllofluoride-activated states have been determined, revealing a mechanism of phosphorylation-induced dimerization that features a deep hydrophobic pocket at the center of the receiver domain interface. Unphosphorylated VraR exists in a closed conformation that inhibits dimer formation. Phosphorylation at the active site promotes conformational changes that are propagated throughout the receiver domain, promoting the opening of a hydrophobic pocket that is essential for homodimer formation and enhanced DNA-binding activity. This prominent feature in the VraR dimer can potentially be exploited for the development of novel therapeutics to counteract antibiotic resistance in this important pathogen.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Multimerização Proteica , Staphylococcus aureus/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Bacteriana/fisiologia , Fosforilação/fisiologia , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismoRESUMO
Interferon-induced ISG15 conjugation plays an important antiviral role against several viruses, including influenza viruses. The NS1 protein of influenza B virus (NS1B) specifically binds only human and nonhuman primate ISG15s and inhibits their conjugation. To elucidate the structural basis for the sequence-specific recognition of human ISG15, we determined the crystal structure of the complex formed between human ISG15 and the N-terminal region of NS1B (NS1B-NTR). The NS1B-NTR homodimer interacts with two ISG15 molecules in the crystal and also in solution. The two ISG15-binding sites on the NS1B-NTR dimer are composed of residues from both chains, namely residues in the RNA-binding domain (RBD) from one chain, and residues in the linker between the RBD and the effector domain from the other chain. The primary contact region of NS1B-NTR on ISG15 is composed of residues at the junction of the N-terminal ubiquitin-like (Ubl) domain and the short linker region between the two Ubl domains, explaining why the sequence of the short linker in human and nonhuman primate ISG15s is essential for the species-specific binding of these ISG15s. In addition, the crystal structure identifies NS1B-NTR binding sites in the N-terminal Ubl domain of ISG15, and shows that there are essentially no contacts with the C-terminal Ubl domain of ISG15. Consequently, NS1B-NTR binding to ISG15 would not occlude access of the C-terminal Ubl domain of ISG15 to its conjugating enzymes. Nonetheless, transfection assays show that NS1B-NTR binding of ISG15 is responsible for the inhibition of interferon-induced ISG15 conjugation in cells.
Assuntos
Citocinas/metabolismo , Vírus da Influenza B/química , Ubiquitinas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Humanos , Interferons/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
CDK2AP1 (cyclin-dependent kinase 2-associated protein 1), corresponding to the gene doc-1 (deleted in oral cancer 1), is a tumor suppressor protein. The doc-1 gene is absent or down-regulated in hamster oral cancer cells and in many other cancer cell types. The ubiquitously expressed CDK2AP1 protein is the only known specific inhibitor of CDK2, making it an important component of cell cycle regulation during G(1)-to-S phase transition. Here, we report the solution structure of CDK2AP1 by combined methods of solution state NMR and amide hydrogen/deuterium exchange measurements with mass spectrometry. The homodimeric structure of CDK2AP1 includes an intrinsically disordered 60-residue N-terminal region and a four-helix bundle dimeric structure with reduced Cys-105 in the C-terminal region. The Cys-105 residues are, however, poised for disulfide bond formation. CDK2AP1 is phosphorylated at a conserved Ser-46 site in the N-terminal "intrinsically disordered" region by IκB kinase ε.
Assuntos
Multimerização Proteica , Proteínas Supressoras de Tumor/química , Animais , Linhagem Celular Tumoral , Cricetinae , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Fase G1/fisiologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Fase S/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Sequência de Aminoácidos , Biopolímeros/química , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
Metabolic networks are interconnected and influence diverse cellular processes. The protein-metabolite interactions that mediate these networks are frequently low affinity and challenging to systematically discover. We developed mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS) to identify such interactions. Analysis of 33 enzymes from human carbohydrate metabolism identified 830 protein-metabolite interactions, including known regulators, substrates, and products as well as previously unreported interactions. We functionally validated a subset of interactions, including the isoform-specific inhibition of lactate dehydrogenase by long-chain acyl-coenzyme A. Cell treatment with fatty acids caused a loss of pyruvate-lactate interconversion dependent on lactate dehydrogenase isoform expression. These protein-metabolite interactions may contribute to the dynamic, tissue-specific metabolic flexibility that enables growth and survival in an ever-changing nutrient environment.
Assuntos
Metabolismo dos Carboidratos , L-Lactato Desidrogenase , Metaboloma , Humanos , Ácidos Graxos/metabolismo , L-Lactato Desidrogenase/metabolismo , Especificidade de Órgãos , Espectrometria de Massas/métodos , Regulação AlostéricaRESUMO
The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA binding activity. Using nuclear magnetic resonance, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Peptídeos Cíclicos/química , Proteínas de Bactérias/antagonistas & inibidores , Cristalização , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Biblioteca de Peptídeos , Peptídeos Cíclicos/antagonistas & inibidores , Estrutura Terciária de Proteína/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Bromodomain-containing protein 4 (BRD4) is an emerging epigenetic drug target for intractable inflammatory disorders. The lack of highly selective inhibitors among BRD4 family members has stalled the collective understanding of this critical system and the progress toward clinical development of effective therapeutics. Here we report the discovery of a potent BRD4 bromodomain 1 (BD1)-selective inhibitor ZL0590 (52) targeting a unique, previously unreported binding site, while exhibiting significant anti-inflammatory activities in vitro and in vivo. The X-ray crystal structural analysis of ZL0590 in complex with human BRD4 BD1 and the associated mutagenesis study illustrate a first-in-class nonacetylated lysine (KAc) binding site located at the helix αB and αC interface that contains important BRD4 residues (e.g., Glu151) not commonly shared among other family members and is spatially distinct from the classic KAc recognition pocket. This new finding facilitates further elucidation of the complex biology underpinning bromodomain specificity among BRD4 and its protein-protein interaction partners.
Assuntos
Anti-Inflamatórios/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Compostos de Fenilureia/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacocinética , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cristalografia por Raios X , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Compostos de Fenilureia/síntese química , Compostos de Fenilureia/metabolismo , Compostos de Fenilureia/farmacocinética , Ligação Proteica , Domínios Proteicos , Ratos , Fatores de Transcrição/metabolismoRESUMO
DNA double-strand break (DSB) repair is initiated by MRE11 nuclease for both homology-directed repair (HDR) and alternative end joining (Alt-EJ). Here, we found that GRB2, crucial to timely proliferative RAS/MAPK pathway activation, unexpectedly forms a biophysically validated GRB2-MRE11 (GM) complex for efficient HDR initiation. GRB2-SH2 domain targets the GM complex to phosphorylated H2AX at DSBs. GRB2 K109 ubiquitination by E3 ubiquitin ligase RBBP6 releases MRE11 promoting HDR. RBBP6 depletion results in prolonged GM complex and HDR defects. GRB2 knockout increased MRE11-XRCC1 complex and Alt-EJ. Reconstitution with separation-of-function GRB2 mutant caused HDR deficiency and synthetic lethality with PARP inhibitor. Cell and cancer genome analyses suggest biomarkers of low GRB2 for noncanonical HDR deficiency and high MRE11 and GRB2 expression for worse survival in HDR-proficient patients. These findings establish GRB2's role in binding, targeting, and releasing MRE11 to promote efficient HDR over Alt-EJ DSB repair, with implications for genome stability and cancer biology.
RESUMO
Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2) plays a role in receptor tyrosine kinase (RTK), neurofibromin-1 (NF-1), and Kirsten rat sarcoma virus (KRAS) mutant-driven cancers, as well as in RTK-mediated resistance, making the identification of small-molecule therapeutics that interfere with its function of high interest. Our quest to identify potent, orally bioavailable, and safe SHP2 inhibitors led to the discovery of a promising series of pyrazolopyrimidinones that displayed excellent potency but had a suboptimal in vivo pharmacokinetic (PK) profile. Hypothesis-driven scaffold optimization led us to a series of pyrazolopyrazines with excellent PK properties across species but a narrow human Ether-à-go-go-Related Gene (hERG) window. Subsequent optimization of properties led to the discovery of the pyrimidinone series, in which multiple members possessed excellent potency, optimal in vivo PK across species, and no off-target activities including no hERG liability up to 100 µM. Importantly, compound 30 (IACS-15414) potently suppressed the mitogen-activated protein kinase (MAPK) pathway signaling and tumor growth in RTK-activated and KRASmut xenograft models in vivo.
Assuntos
Antineoplásicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Humanos , Camundongos , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Relação Estrutura-AtividadeRESUMO
Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-containing enzyme that mediates the rate-limiting step in the metabolism of l-tryptophan to kynurenine, has been widely explored as a potential immunotherapeutic target in oncology. We developed a class of inhibitors with a conformationally constrained bicyclo[3.1.0]hexane core. These potently inhibited IDO1 in a cellular context by binding to the apoenzyme, as elucidated by biochemical characterization and X-ray crystallography. A SKOV3 tumor model was instrumental in differentiating compounds, leading to the identification of IACS-9779 (62) and IACS-70465 (71). IACS-70465 has excellent cellular potency, a robust pharmacodynamic response, and in a human whole blood assay was more potent than linrodostat (BMS-986205). IACS-9779 with a predicted human efficacious once daily dose below 1 mg/kg to sustain >90% inhibition of IDO1 displayed an acceptable safety margin in rodent toxicology and dog cardiovascular studies to support advancement into preclinical safety evaluation for human development.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The nucleoid-associated protein H-NS and its paralogue StpA are global regulators of gene expression and form an integral part of the protein scaffold responsible for DNA condensation in Escherichia coli and Salmonella typhimurium. Although protein oligomerization is a requirement for this function, it is not entirely understood how this is accomplished. We address this by reporting on the self-association of H-NS and its hetero-association with StpA. We identify residues 1-77 of H-NS as being necessary and sufficient for high-order association. A multi-technique-based approach was used to measure the effects of salt concentration on the size distribution of H-NS and the thermal stability of H-NS and StpA dimers. The thermal stability of the StpA homodimer is significantly greater than that of H-NS(1-74). Investigation of the hetero-association of H-NS and StpA proteins suggested that the association of H-NS with StpA is more stable than the self-association of either H-NS or StpA with themselves. This provides a clear understanding of the method of oligomerization of these important proteins in effecting DNA condensation and reveals that the different associative properties of H-NS and StpA allow them to perform distinct, yet complementary roles in the bacterial nucleoid.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Bactérias/genética , Cromatografia em Gel , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Multimerização Proteica , Estabilidade Proteica , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismoRESUMO
The histone-like nucleoid-structuring protein (H-NS) plays an important role in both DNA packaging and global gene regulation in enterobacteria. Self-association of the N-terminal domain results in polydisperse oligomers that are critical to the function of the protein. This heterogeneity in oligomer size has so far prevented structure determination of the complete oligomerization domain by NMR or X-ray crystallography. In the absence of inorganic salt, the H-NS oligomerization domain is predominantly restricted to an equilibrium between a homodimer and homotetramer, allowing a protein solution to be prepared that is sufficiently homogeneous for successful crystallization. Crystallization was achieved by tailoring the conditions screened to those identified as minimizing the potential disruption of protein-solution homogeneity. This finding provides a significant step towards resolving the structure of this important prokaryotic protein.
Assuntos
Proteínas de Bactérias/química , Histonas/química , Multimerização Proteica , Salmonella typhimurium/química , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Histonas/metabolismo , Concentração Osmolar , Salmonella typhimurium/metabolismoRESUMO
Bromodomain-containing protein 4 (BRD4) represents a promising drug target for anti-inflammatory therapeutics. Herein, we report the design, synthesis, and pharmacological evaluation of novel chromone derivatives via scaffold hopping to discover a new class of orally bioavailable BRD4-selective inhibitors. Two potent BRD4 bromodomain 1 (BD1)-selective inhibitors 44 (ZL0513) and 45 (ZL0516) have been discovered with high binding affinity (IC50 values of 67-84 nM) and good selectivity over other BRD family proteins and distant BD-containing proteins. Both compounds significantly inhibited the expression of Toll-like receptor-induced inflammatory genes in vitro and airway inflammation in murine models. The cocrystal structure of 45 in complex with human BRD4 BD1 at a high resolution of 2.0 Å has been solved, offering a solid structural basis for its binding validation and further structure-based optimization. These BRD4 BD1 inhibitors demonstrated impressive in vivo efficacy and overall promising pharmacokinetic properties, indicating their therapeutic potential for the treatment of inflammatory diseases.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Cromonas/administração & dosagem , Cromonas/química , Descoberta de Drogas/métodos , Fatores de Transcrição/antagonistas & inibidores , Administração Oral , Animais , Disponibilidade Biológica , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Cromonas/farmacologia , Cristalização/métodos , Cristalização/tendências , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismoRESUMO
Tumor-associated macrophages (TAMs) have a significant presence in the tumor stroma across multiple human malignancies and are believed to be beneficial to tumor growth. Targeting CSF1R has been proposed as a potential therapy to reduce TAMs, especially the protumor, immune-suppressive M2 TAMs. Additionally, the high expression of CSF1R on tumor cells has been associated with poor survival in certain cancers, suggesting tumor dependency and therefore a potential therapeutic target. The CSF1-CSF1R signaling pathway modulates the production, differentiation, and function of TAMs; however, the discovery of selective CSF1R inhibitors devoid of type III kinase activity has proven to be challenging. We discovered a potent, highly selective, and orally bioavailable CSF1R inhibitor, IACS-9439 (1). Treatment with 1 led to a dose-dependent reduction in macrophages, promoted macrophage polarization toward the M1 phenotype, and led to tumor growth inhibition in MC38 and PANC02 syngeneic tumor models.