RESUMO
Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.
Assuntos
Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such, could potentially serve as a target for the development of therapeutics as a treatment for those diseases. Previous work has described GW4064 as an FXR agonist with an interesting activity profile. This manuscript will describe the synthesis of novel analogs of GW4064 and the activity profile of those analogs.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Oxazolidinonas/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazolidinonas/química , Relação Estrutura-AtividadeRESUMO
We have applied hydrogen-deuterium exchange mass spectrometry, in conjunction with differential scanning calorimetry and protein stability analysis, to examine solution dynamics of the integrin α1 I domain induced by the binding of divalent cations, full-length type IV collagen, or a function-blocking monoclonal antibody. These studies revealed features of integrin activation and α1I-ligand complexes that were not detected by static crystallographic data. Mg(2+) and Mn(2+) stabilized α1I but differed in their effects on exchange rates in the αC helix. Ca(2+) impacted α1I conformational dynamics without altering its gross thermal stability. Interaction with collagen affected the exchange rates in just one of three metal ion-dependent adhesion site (MIDAS) loops, suggesting that MIDAS loop 2 plays a primary role in mediating ligand binding. Collagen also induced changes consistent with increased unfolding in both the αC and allosteric C-terminal helices of α1I. The antibody AQC2, which binds to α1I in a ligand-mimetic manner, also reduced exchange in MIDAS loop 2 and increased exchange in αC, but it did not impact the C-terminal region. This is the first study to directly demonstrate the conformational changes induced upon binding of an integrin I domain to a full-length collagen ligand, and it demonstrates the utility of the deuterium exchange mass spectrometry method to study the solution dynamics of integrin/ligand and integrin/metal ion interactions. Based on the ligand and metal ion binding data, we propose a model for collagen-binding integrin activation that explains the differing abilities of Mg(2+), Mn(2+), and Ca(2+) to activate I domain-containing integrins.
Assuntos
Colágeno Tipo IV/metabolismo , Integrina alfa1/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Animais , Colágeno Tipo IV/química , Humanos , Integrina alfa1/química , Integrina alfa1/genética , Magnésio/química , Manganês/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , RatosRESUMO
The ability to generate and design antibodies recognizing specific targets has revolutionized the pharmaceutical industry and medical imaging. Engineering antibody therapeutics in some cases requires modifying their constant domains to enable new and altered interactions. Engineering novel specificities into antibody constant domains has proved challenging due to the complexity of inter-domain interactions. Covarying networks of residues that tend to cluster on the protein surface and near binding sites have been identified in some proteins. However, the underlying role these networks play in the protein resulting in their conservation remains unclear in most cases. Resolving their role is crucial, because residues in these networks are not viable design targets if their role is to maintain the fold of the protein. Conversely, these networks of residues are ideal candidates for manipulating specificity if they are primarily involved in binding, such as the myriad interdomain interactions maintained within antibodies. Here, we identify networks of evolutionarily-related residues in C-class antibody domains by evaluating covariation, a measure of propensity with which residue pairs vary dependently during evolution. We computationally test whether mutation of residues in these networks affects stability of the folded antibody domain, determining their viability as design candidates. We find that members of covarying networks cluster at domain-domain interfaces, and that mutations to these residues are diverse and frequent during evolution, precluding their importance to domain stability. These results indicate that networks of covarying residues exist in antibody domains for functional reasons unrelated to thermodynamic stability, making them ideal targets for antibody design.
Assuntos
Anticorpos/química , Regiões Constantes de Imunoglobulina/química , Fragmentos Fab das Imunoglobulinas/química , Sequência de Aminoácidos , Animais , Anticorpos/genética , Sítios de Ligação , Evolução Molecular , Regiões Constantes de Imunoglobulina/genética , Fragmentos Fab das Imunoglobulinas/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.
Assuntos
Isoxazóis/química , Isoxazóis/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Estilbenos/química , Estilbenos/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Conformação Molecular , Dados de Sequência Molecular , Receptores Citoplasmáticos e Nucleares/metabolismoRESUMO
To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.
Assuntos
Isoxazóis/química , Naftalenos/química , Quinolinas/química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Sítios de Ligação , Glicemia/metabolismo , Cristalografia por Raios X , Diabetes Mellitus Experimental/metabolismo , Cães , Transferência Ressonante de Energia de Fluorescência , Humanos , Isoxazóis/síntese química , Isoxazóis/farmacocinética , Ligantes , Camundongos , Conformação Molecular , Estrutura Terciária de Proteína , Quinolinas/síntese química , Quinolinas/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Aumento de Peso/efeitos dos fármacosRESUMO
When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.
Assuntos
Ribossomos/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Anticorpos/metabolismo , Biocatálise , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estabilidade Proteica , Reprodutibilidade dos Testes , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterase/antagonistas & inibidoresRESUMO
Inhibition of mutant IDH1 is being evaluated clinically as a treatment option for oncology. Here we describe the structure-based design and optimization of quinoline lead compounds to identify FT-2102, a potent, orally bioavailable, brain penetrant, and selective mIDH1 inhibitor. FT-2102 has excellent ADME/PK properties and reduces 2-hydroxyglutarate levels in an mIDH1 xenograft tumor model. This compound has been selected as a candidate for clinical development in hematologic malignancies, solid tumors, and gliomas with mIDH1.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Isocitrato Desidrogenase/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Piridinas/uso terapêutico , Quinolinas/uso terapêutico , Quinolonas/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Feminino , Humanos , Isocitrato Desidrogenase/metabolismo , Camundongos Endogâmicos BALB C , Estrutura Molecular , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Quinolinas/síntese química , Quinolinas/metabolismo , Quinolonas/síntese química , Quinolonas/metabolismo , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of alternately 3,5-substituted isoxazoles was prepared. Several of these analogs were potent full FXR agonists. A subset of this series, with a tether between the isoxazole ring and the 3-position aryl substituent, were equipotent FXR agonists to GW 4064 1a, with the 2,6-dimethyl phenol analog 1t having greater FRET FXR potency than GW 4064 1a.
Assuntos
Isoxazóis/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Animais , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Isoxazóis/química , Isoxazóis/farmacologia , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-AtividadeRESUMO
Two series of conformationally constrained analogs of the FXR agonist GW 4064 1 were prepared. Replacement of the metabolically labile stilbene with either benzothiophene or naphthalene rings led to the identification of potent full agonists 2a and 2g.
Assuntos
Isoxazóis/química , Naftalenos/química , Receptores Citoplasmáticos e Nucleares/agonistas , Tiofenos/química , Animais , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Isoxazóis/farmacologia , Naftalenos/farmacocinética , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade , Tiofenos/farmacocinéticaRESUMO
Mutations at the arginine residue (R132) in isocitrate dehydrogenase 1 (IDH1) are frequently identified in various human cancers. Inhibition of mutant IDH1 (mIDH1) with small molecules has been clinically validated as a promising therapeutic treatment for acute myeloid leukemia and multiple solid tumors. Herein, we report the discovery and optimization of a series of quinolinones to provide potent and orally bioavailable mIDH1 inhibitors with selectivity over wild-type IDH1. The X-ray structure of an early lead 24 in complex with mIDH1-R132H shows that the inhibitor unexpectedly binds to an allosteric site. Efforts to improve the in vitro and in vivo absorption, distribution, metabolism, and excretion (ADME) properties of 24 yielded a preclinical candidate 63. The detailed preclinical ADME and pharmacology studies of 63 support further development of quinolinone-based mIDH1 inhibitors as therapeutic agents in human trials.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Isocitrato Desidrogenase/antagonistas & inibidores , Quinolonas/química , Quinolonas/farmacologia , Sítio Alostérico/efeitos dos fármacos , Animais , Disponibilidade Biológica , Linhagem Celular Tumoral , Cristalografia por Raios X , Cães , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/genética , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutação Puntual , Quinolonas/farmacocinéticaRESUMO
Starting from the known FXR agonist GW 4064 1a, a series of stilbene replacements were prepared. The 6-substituted 1-naphthoic acid 1b was an equipotent FXR agonist with improved developability parameters relative to 1a. Analog 1b also reduced the severity of cholestasis in the ANIT acute cholestatic rat model.
Assuntos
Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Colestase/tratamento farmacológico , Proteínas de Ligação a DNA/agonistas , Isoxazóis/síntese química , Isoxazóis/farmacologia , Naftalenos/síntese química , Naftalenos/farmacologia , Receptores Citoplasmáticos e Nucleares/agonistas , Fatores de Transcrição/agonistas , Administração Oral , Animais , Ácidos Carboxílicos/química , Colestase/metabolismo , Colestase/patologia , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Cães , Mucosa Gástrica/metabolismo , Haplorrinos , Isoxazóis/química , Camundongos , Conformação Molecular , Estrutura Molecular , Naftalenos/química , Ratos , Receptores Citoplasmáticos e Nucleares/química , Estômago/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Transcrição/químicaRESUMO
Defining complete sets of gene family members from diverse species provides the foundation for comparative studies. Using a bioinformatic approach, we have defined the entire nuclear receptor complement within the first available complete sequence of a non-human vertebrate (the teleost fish Fugu rubripes). In contrast to the human set (48 total nuclear receptors), we found 68 nuclear receptors in the Fugu genome. All 68 Fugu receptors had a clear human homolog, thus defining no new nuclear receptor subgroups. A reciprocal analysis showed that each human receptor had one or more Fugu orthologs, excepting CAR (NR1I3) and LXRbeta (NR1H2). These 68 receptors add striking diversity to the known nuclear receptor superfamily and provide important comparators to human nuclear receptors. We have compared several pharmacologically relevant human nuclear receptors (FXR, LXRalpha/beta, CAR, PXR, VDR and PPARalpha/gamma/delta) to their Fugu orthologs. This comparison included expression analysis across five Fugu tissue types. All of the Fugu receptors that were analyzed by PCR in this study were expressed, indicating that the majority of the additional Fugu receptors are likely to be functional.
Assuntos
Receptores Citoplasmáticos e Nucleares/genética , Takifugu/genética , Sequência de Aminoácidos , Animais , Receptor Constitutivo de Androstano , Feminino , Expressão Gênica , Variação Genética , Genoma , Humanos , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.
Assuntos
Compostos de Anilina/síntese química , Proteínas de Ligação a DNA/agonistas , Maleimidas/síntese química , Receptores Citoplasmáticos e Nucleares/agonistas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Transportadores de Cassetes de Ligação de ATP/genética , Compostos de Anilina/química , Compostos de Anilina/farmacologia , Sítios de Ligação , Linhagem Celular , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Genes Reporter , Histona Acetiltransferases , Humanos , Ligantes , Receptores X do Fígado , Luciferases/genética , Maleimidas/química , Maleimidas/farmacologia , Modelos Moleculares , Estrutura Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Coativador 1 de Receptor Nuclear , Receptores Nucleares Órfãos , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/química , Relação Estrutura-Atividade , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Engineered antibodies are emerging as a promising class of therapeutic biomolecules, as well as having applications in medical research. Knowledge on conserved functional and structural regions within antibody domains is imperative in order to rationally design stable and specific antibodies. Of particular interest for the design of therapeutics are antibody variable and constant domains, which are responsible for antigen binding and immune response. These antibody domains are part of the larger immunoglobulin (Ig) V-class and C-class families, respectively. We find that, although both classes belong to the Ig-fold superfamily, the sets of conserved residue positions and identities differ between these classes. We exploit these evolutionary differences to derive a metric based on sequence positional entropy that distinguishes C-class from V-class sequences utilizing only sequence information. By distinguishing different domain families using sequence information alone, we enable the application of domain-specific design strategies without the need for secondary or tertiary structural information.
Assuntos
Biologia Computacional/métodos , Imunoglobulinas/química , Imunoglobulinas/genética , Algoritmos , Imunoglobulinas/metabolismo , Modelos Moleculares , Domínios e Motivos de Interação entre ProteínasRESUMO
Monoclonal antibodies capable of recognizing antigens with high affinity and specificity represent a wellestablished class of biological agents. Since the development of hybridoma technology in 1975, advances in recombinant DNA technologies and computational and biophysical methods have allowed us to develop a better understanding of the relationships between antibody sequence, structure, and function. These advances enable us to manipulate antibody sequences with the goal of improving upon, or creating new biological or biophysical properties. In this review we will focus on recent successes in using structure-guided computational methods to design antibodies and antibody-like molecules with optimized affinity and specificity to antigen and for enhancing protein stability.
Assuntos
Anticorpos Monoclonais/química , Desenho Assistido por Computador , Desenho de Fármacos , Estrutura Molecular , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Estabilidade de Medicamentos , Engenharia de Proteínas/métodosRESUMO
Monoclonal antibodies capable of recognizing antigens with high affinity and specificity represent a well established class of biological agents. Since the development of hybridoma technology in 1975, advances in recombinant DNA technologies and computational and biophysical methods have allowed us to develop a better understanding of the relationships between antibody sequence, structure, and function. These advances enable us to manipulate antibody sequences with the goal of improving upon, or creating new biological or biophysical properties. In this review we will focus on recent successes in using structure-guided computational methods to design antibodies and antibody-like molecules with optimized affinity and specificity to antigen and for enhancing protein stability.
RESUMO
A cocrystal structure of T1317 (3) bound to hLXRbeta was utilized in the design of a series of substituted N-phenyl tertiary amines. Profiling in binding and functional assays led to the identification of LXR modulator GSK9772 ( 20) as a high-affinity LXRbeta ligand (IC 50 = 30 nM) that shows separation of anti-inflammatory and lipogenic activities in human macrophage and liver cell lines, respectively. A cocrystal structure of the structurally related analog 19 bound to LXRbeta reveals regions within the receptor that can affect receptor modulation through ligand modification. Mechanistic studies demonstrate that 20 is greater than 10-fold selective for LXR-mediated transrepression of proinflammatory gene expression versus transactivation of lipogenic signaling pathways, thus providing an opportunity for the identification of LXR modulators with improved therapeutic indexes.
Assuntos
Aminas/química , Aminas/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Desenho de Fármacos , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Cristalografia por Raios X , Receptores X do Fígado , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Receptores Nucleares Órfãos , Relação Estrutura-AtividadeRESUMO
Ecdysteroid pulses trigger the major developmental transitions during the Drosophila life cycle. These hormonal responses are thought to be mediated by the ecdysteroid receptor (EcR) and its heterodimeric partner Ultraspiracle (USP). We provide evidence for a second ecdysteroid signaling pathway mediated by DHR38, the Drosophila ortholog of the mammalian NGFI-B subfamily of orphan nuclear receptors. DHR38 also heterodimerizes with USP, and this complex responds to a distinct class of ecdysteroids in a manner that is independent of EcR. This response is unusual in that it does not involve direct binding of ecdysteroids to either DHR38 or USP. X-ray crystallographic analysis of DHR38 reveals the absence of both a classic ligand binding pocket and coactivator binding site, features that seem to be common to all NGFI-B subfamily members. Taken together, these data reveal the existence of a separate structural class of nuclear receptors that is conserved from fly to humans.