RESUMEN

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.

Asunto(s)

Glucocorticoides , Receptores de Glucocorticoides , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas 14-3-3/metabolismo , Regulación de la Expresión Génica , Sitios de Unión , Esteroides , Dexametasona

RESUMEN

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.

Asunto(s)

Pirrolidinonas , Conformación Molecular

RESUMEN

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.

Asunto(s)

Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Animales , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Enfermedades del Sistema Inmune/genética , Inmunoterapia , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Fosfatidilinositol 3-Quinasas/metabolismo

RESUMEN

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.

Asunto(s)

Asma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Sulfonamidas/uso terapéutico , Tiazoles/uso terapéutico , Animales , Asma/inducido químicamente , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Cristalografía por Rayos X , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Estructura Molecular , Ovalbúmina , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas Endogámicas BN , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinética , Tiazoles/síntesis química , Tiazoles/metabolismo , Tiazoles/farmacocinética

RESUMEN

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.

Asunto(s)

Proteínas 14-3-3/metabolismo , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Glucocorticoides/metabolismo , Treonina/metabolismo , Proteínas 14-3-3/genética , Células HEK293 , Humanos , Mutación , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Receptores de Glucocorticoides/genética , Treonina/genética , Activación Transcripcional

RESUMEN

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.

Asunto(s)

Proteínas 14-3-3/metabolismo , Inflamación/metabolismo , Proteínas 14-3-3/genética , Animales , Humanos , Inflamación/genética , Unión Proteica

RESUMEN

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.

Asunto(s)

Proteínas 14-3-3/antagonistas & inhibidores , Exorribonucleasas/antagonistas & inhibidores , Organofosfatos/farmacología , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/toxicidad , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Humanos , Inosina Monofosfato/metabolismo , Inosina Monofosfato/farmacología , Inosina Monofosfato/toxicidad , Células K562 , Organofosfatos/metabolismo , Organofosfatos/toxicidad , Proteínas Proto-Oncogénicas c-abl/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacología , Fosfato de Piridoxal/toxicidad , Alineación de Secuencia , Bibliotecas de Moléculas Pequeñas/toxicidad

RESUMEN

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.

Asunto(s)

MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteolisis/efectos de los fármacos , Sulfonamidas/farmacología , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Interleucina-6/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Sulfonamidas/síntesis química , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo

RESUMEN

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.

Asunto(s)

Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenosina Trifosfatasas , Sitios de Unión , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ftalimidas , Unión Proteica , Conformación Proteica , Isoformas de Proteínas/fisiología , Inhibidores de Proteínas Quinasas , Especificidad por Sustrato

RESUMEN

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.

Asunto(s)

Enfermedades Autoinmunes/tratamiento farmacológico , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Inflamación/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Animales , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Ensayos Clínicos como Asunto , Humanos , Estructura Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3/química , Inhibidores de las Quinasa Fosfoinosítidos-3/metabolismo , Unión Proteica

RESUMEN

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.

Asunto(s)

Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Adenosina Trifosfato/metabolismo , Administración Oral , Animales , Sitios de Unión , Disponibilidad Biológica , Cristalografía por Rayos X , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacocinética , Humanos , Isoenzimas , Trastornos Leucocíticos/inducido químicamente , Trastornos Leucocíticos/tratamiento farmacológico , Lipopolisacáridos/toxicidad , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Ftalimidas/química , Ratas , Relación Estructura-Actividad

RESUMEN

The sexes perform different reproductive roles and have evolved sometimes strikingly different phenotypes. One focal point of adaptive divergence occurs in the context of diet and metabolism, and males and females of a range of species have been shown to require different nutrients to maximize their fitness. Biochemical analyses in Drosophila melanogaster have confirmed that dimorphism in dietary requirements is associated with molecular sex differences in metabolite titres. In addition, they also showed significant within-sex genetic variation in the metabolome. To date however, it is unknown whether this metabolic variation translates into differences in reproductive fitness. The answer to this question is crucial to establish whether genetic variation is selectively neutral or indicative of constraints on sex-specific physiological adaptation and optimization. Here we assay genetic variation in consumption and metabolic fitness effects by screening male and female fitness of thirty D. melanogaster genotypes across four protein-to-carbohydrate ratios. In addition to confirming sexual dimorphism in consumption and fitness, we find significant genetic variation in male and female dietary requirements. Importantly, these differences are not explained by feeding responses and probably reflect metabolic variation that, in turn, suggests the presence of genetic constraints on metabolic dimorphism.

Asunto(s)

Drosophila melanogaster/fisiología , Aptitud Genética , Variación Genética , Animales , Drosophila melanogaster/genética , Conducta Alimentaria , Femenino , Genotipo , Masculino , Caracteres Sexuales , Factores Sexuales

RESUMEN

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.

Asunto(s)

Diseño de Fármacos , Descubrimiento de Drogas/métodos , Proteínas/metabolismo , Productos Biológicos/farmacología , Humanos , Preparaciones Farmacéuticas/síntesis química , Preparaciones Farmacéuticas/química , Preparaciones Farmacéuticas/metabolismo , Unión Proteica , Estabilidad Proteica , Bibliotecas de Moléculas Pequeñas

RESUMEN

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.

Asunto(s)

Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Relación Estructura-Actividad , Administración por Inhalación , Animales , Disponibilidad Biológica , Técnicas de Química Sintética , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/administración & dosificación , Semivida , Isoenzimas/antagonistas & inhibidores , Ratones Transgénicos , Permeabilidad , Ratas , Solubilidad , Tiazoles/química

RESUMEN

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.

Asunto(s)

Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacocinética , Células CACO-2 , Humanos , Estructura Molecular , Permeabilidad , Estereoisomerismo , Relación Estructura-Actividad

RESUMEN

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.

Asunto(s)

Diseño de Fármacos , Indicadores y Reactivos/química

RESUMEN

Profiling of eight stereoisomeric T. cruzi growth inhibitors revealed vastly different in vitro properties such as solubility, lipophilicity, pKa, and cell permeability for two sets of four stereoisomers. Using computational chemistry and NMR spectroscopy, we identified the formation of an intramolecular NH→NR3 hydrogen bond in the set of stereoisomers displaying lower solubility, higher lipophilicity, and higher cell permeability. The intramolecular hydrogen bond resulted in a significant pKa difference that accounts for the other structure-property relationships. Application of this knowledge could be of particular value to maintain the delicate balance of size, solubility, and lipophilicity required for cell penetration and oral administration for chemical probes or therapeutics with properties at, or beyond, Lipinski's rule of 5.

Asunto(s)

Permeabilidad de la Membrana Celular/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Algoritmos , Animales , Células CACO-2 , Biología Computacional , Diseño de Fármacos , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Lípidos/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Bibliotecas de Moléculas Pequeñas , Solubilidad , Estereoisomerismo , Relación Estructura-Actividad