RESUMO
The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rho-associated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention.
Assuntos
Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Glucocorticoides/metabolismo , Treonina/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Humanos , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Receptores de Glucocorticoides/genética , Treonina/genética , Ativação TranscricionalRESUMO
Contrary to expectation N-aryl pyrrolidinones (and isosteric imidazolinones and oxazolinones) are more lipophilic and less soluble than the corresponding piperidinones (tetrahydropyrimidinones and oxazinones). Exploration of the basis for these results uncovered a subtle interplay of steric and electronic effects that result in different conformations for the two classes of compounds which drive the observed effects.
Assuntos
Pirrolidinonas , Conformação MolecularRESUMO
We have discovered a class of PI3Kγ inhibitors exhibiting over 1,000-fold selectivity over PI3Kα and PI3Kß. On the basis of X-ray crystallography, hydrogen-deuterium exchange-mass spectrometry and surface plasmon resonance experiments we propose that the cyclopropylethyl moiety displaces the DFG motif of the enzyme away from the adenosine tri-phosphate binding site, inducing a large conformational change in both the kinase- and helical domains of PI3Kγ. Site directed mutagenesis explained how the conformational changes occur. Our results suggest that these cyclopropylethyl substituted compounds selectively inhibit the active state of PI3Kγ, which is unique to these compounds and to the PI3Kγ isoform, explaining their excellent potency and unmatched isoform selectivity that were confirmed in cellular systems. This is the first example of a Class I PI3K inhibitor achieving its selectivity by affecting the DFG motif in a manner that bears similarity to DFG in/out for type II protein kinase inhibitors.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Adenosina Trifosfatases , Sítios de Ligação , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ftalimidas , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/fisiologia , Inibidores de Proteínas Quinases , Especificidade por SubstratoRESUMO
Regulation of inflammation is a central part of the maintenance of homeostasis by the immune system. One important class of regulatory protein that has been shown to have effects on the inflammatory process are the 14-3-3 proteins. Herein we describe the roles that have been identified for 14-3-3 in regulation of the inflammatory response. These roles encompass regulation of the response that affect inflammation at the genetic, molecular and cellular levels. At a genetic level 14-3-3 is involved in the regulation of multiple transcription factors and affects the transcription of key effectors of the immune response. At a molecular level many of the constituent parts of the inflammatory process, such as pattern recognition receptors, protease activated receptors and cytokines are regulated through phosphorylation and recognition by 14-3-3 whilst disruption of the recognition processes has been observed to result in clinical syndromes. 14-3-3 is also involved in the regulation of cell proliferation and differentiation, this has been shown to affect the immune system, particularly T- and B-cells. Finally, we discuss how abnormal levels of 14-3-3 contribute to undesirable immune responses and chronic inflammatory conditions.
Assuntos
Proteínas 14-3-3/metabolismo , Inflamação/metabolismo , Proteínas 14-3-3/genética , Animais , Humanos , Inflamação/genética , Ligação ProteicaRESUMO
Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 non-peptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption.
Assuntos
Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacocinética , Células CACO-2 , Humanos , Estrutura Molecular , Permeabilidade , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
A series of dual CCR3/H(1) antagonists based on a bispiperidine scaffold were discovered. Introduction of an acidic group overcame hERG liability. Bioavailability was optimised by modulation of physico-chemical properties and physical form to deliver a compound suitable for clinical evaluation.
Assuntos
Descoberta de Drogas , Antagonistas dos Receptores Histamínicos H1/química , Receptores CCR3/antagonistas & inibidores , Animais , Interações Medicamentosas , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Antagonistas dos Receptores Histamínicos H1/farmacocinética , Estrutura Molecular , Piperidinas/química , Ratos , Fatores de RiscoRESUMO
The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.
Assuntos
Descoberta de Drogas , Piperidinas/química , Piperidinas/farmacocinética , Receptores CCR3/antagonistas & inibidores , Animais , Humanos , Concentração Inibidora 50 , Estrutura Molecular , RatosRESUMO
The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.
Assuntos
Glucocorticoides , Receptores de Glucocorticoides , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica , Sítios de Ligação , Esteroides , DexametasonaRESUMO
Overactive phosphoinositide 3-kinase (PI3K) in cancer and immune dysregulation has spurred extensive efforts to develop therapeutic PI3K inhibitors. Although progress has been hampered by issues such as poor drug tolerance and drug resistance, several PI3K inhibitors have now received regulatory approval - the PI3Kα isoform-selective inhibitor alpelisib for the treatment of breast cancer and inhibitors mainly aimed at the leukocyte-enriched PI3Kδ in B cell malignancies. In addition to targeting cancer cell-intrinsic PI3K activity, emerging evidence highlights the potential of PI3K inhibitors in cancer immunotherapy. This Review summarizes key discoveries that aid the clinical translation of PI3Kα and PI3Kδ inhibitors, highlighting lessons learnt and future opportunities.
Assuntos
Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Animais , Humanos , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/genética , Imunoterapia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.
Assuntos
Asma/tratamento farmacológico , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Sulfonamidas/uso terapêutico , Tiazóis/uso terapêutico , Animais , Asma/induzido quimicamente , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Cristalografia por Raios X , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Estrutura Molecular , Ovalbumina , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Ratos Endogâmicos BN , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Tiazóis/síntese química , Tiazóis/metabolismo , Tiazóis/farmacocinéticaRESUMO
PROteolysis TArgeting Chimeras (PROTACs) targeting the degradation of MEK have been designed based on allosteric MEK inhibitors. Inhibition of the phosphorylation of ERK1/2 was less effective with the PROTACs than a small-molecule inhibitor; the best PROTACs, however, were more effective in inhibiting proliferation of A375 cells than an inhibitor.
Assuntos
MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Interleucina-6/metabolismo , Inibidores de Proteínas Quinases/síntese química , Sulfonamidas/síntese química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.
Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Exorribonucleases/antagonistas & inibidores , Organofosfatos/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Núcleo Celular/metabolismo , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Exorribonucleases/química , Exorribonucleases/metabolismo , Humanos , Inosina Monofosfato/metabolismo , Inosina Monofosfato/farmacologia , Inosina Monofosfato/toxicidade , Células K562 , Organofosfatos/metabolismo , Organofosfatos/toxicidade , Proteínas Proto-Oncogênicas c-abl/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Fosfato de Piridoxal/toxicidade , Alinhamento de Sequência , Bibliotecas de Moléculas Pequenas/toxicidadeRESUMO
Phosphoinositol 3-kinases (PI3Ks) γ and δ are key enzymes in hematopoietic cells and have been seen as high-value targets for the treatment of diseases with inflammatory and immunomodulatory components since their discovery and the identification of their roles. In this Perspective we review progress in the application of inhibitors of PI3Kγ and δ to inflammatory and immunological conditions over the past 6 years. We consider progress in the understanding of the roles of PI3Kγ and PI3Kδ in immunology and inflammation, the experience from clinical trials where inhibitors have been tested, and what has been learned about the safety of their use. The extensive medicinal chemistry efforts to discover both isoform selective and dual PI3Kγδ inhibitors are analyzed and detailed. Developments in understanding the structural chemistry of the PI3K enzymes and the factors that govern isoform selectivity are discussed. The effects observed with the known inhibitor compounds in animal models are described.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Inflamação/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Animais , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios Clínicos como Assunto , Humanos , Estrutura Molecular , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Ligação ProteicaRESUMO
In this paper, we describe the discovery and optimization of a new chemotype of isoform selective PI3Kγ inhibitors. Starting from an HTS hit, potency and physicochemical properties could be improved to give compounds such as 15, which is a potent and remarkably selective PI3Kγ inhibitor with ADME properties suitable for oral administration. Compound 15 was advanced into in vivo studies showing dose-dependent inhibition of LPS-induced airway neutrophilia in rats when administered orally.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Trifosfato de Adenosina/metabolismo , Administração Oral , Animais , Sítios de Ligação , Disponibilidade Biológica , Cristalografia por Raios X , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Isoenzimas , Transtornos Leucocíticos/induzido quimicamente , Transtornos Leucocíticos/tratamento farmacológico , Lipopolissacarídeos/toxicidade , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Ftalimidas/química , Ratos , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: PPIs are involved in every disease and specific modulation of these PPIs with small molecules would significantly improve our prospects of developing therapeutic agents. Both industry and academia have engaged in the identification and use of PPI inhibitors. However in comparison, the opposite strategy of employing small-molecule stabilizers of PPIs is underrepresented in drug discovery. Areas covered: PPI stabilization has not been exploited in a systematic manner. Rather, this concept validated by a number of therapeutically used natural products like rapamycin and paclitaxel has been shown retrospectively to be the basis of the activity of synthetic molecules originating from drug discovery projects among them lenalidomide and tafamidis. Here, the authors cover the growing number of synthetic small-molecule PPI stabilizers to advocate for a stronger consideration of this as a drug discovery approach. Expert opinion: Both the natural products and the growing number of synthetic molecules show that PPI stabilization is a viable strategy for drug discovery. There is certainly a significant challenge to adapt compound libraries, screening techniques and downstream methodologies to identify, characterize and optimize PPI stabilizers, but the examples of molecules reviewed here in our opinion justify these efforts.
Assuntos
Desenho de Fármacos , Descoberta de Drogas/métodos , Proteínas/metabolismo , Produtos Biológicos/farmacologia , Humanos , Preparações Farmacêuticas/síntese química , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Ligação Proteica , Estabilidade Proteica , Bibliotecas de Moléculas PequenasRESUMO
PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 > 9), and by careful manipulation of the pKa and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Relação Estrutura-Atividade , Administração por Inalação , Animais , Disponibilidade Biológica , Técnicas de Química Sintética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/administração & dosagem , Meia-Vida , Isoenzimas/antagonistas & inibidores , Camundongos Transgênicos , Permeabilidade , Ratos , Solubilidade , Tiazóis/químicaRESUMO
One pragmatic way to improve compound quality, while enhancing and accelerating drug discovery projects, is the ability to access a high quality, novel, diverse building block collection. Here, we outline general principles that should be applied to ensure that a building block collection has the greatest impact on drug discovery projects, by discussing design principles for novel reagents and what types of reagents are popular with medicinal chemists in general. We initiated a program in 2009 to address this, which has already delivered three candidate drugs, and the success of that program provides evidence that focussing on building block design is a useful strategy for drug discovery.