RESUMEN
Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Diseño de Fármacos , Janus Quinasa 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , Animales , Artritis Experimental/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Janus Quinasa 1/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Ratas , Ratas Endogámicas Lew , Relación Estructura-Actividad , TYK2 Quinasa/metabolismoRESUMEN
Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).
Asunto(s)
Antituberculosos/farmacología , Artritis Experimental/prevención & control , Janus Quinasa 1/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirimidinas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , Tuberculosis/complicaciones , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/microbiología , Femenino , Estructura Molecular , Ratas , Ratas Endogámicas Lew , Tuberculosis/microbiologíaRESUMEN
Biochemical screening is a major source of lead generation for novel targets. However, during the process of small molecule lead optimization, compounds with excellent biochemical activity may show poor cellular potency, making structure-activity relationships difficult to decipher. This may be due to low membrane permeability of the molecule, resulting in insufficient intracellular drug concentration. The Cell Squeeze platform increases permeability regardless of compound structure by mechanically disrupting the membrane, which can overcome permeability limitations and bridge the gap between biochemical and cellular studies. In this study, we show that poorly permeable Janus kinase (JAK) inhibitors are delivered into primary cells using Cell Squeeze, inhibiting up to 90% of the JAK pathway, while incubation of JAK inhibitors with or without electroporation had no significant effect. We believe this robust intracellular delivery approach could enable more effective lead optimization and deepen our understanding of target engagement by small molecules and functional probes.
Asunto(s)
Inhibidores de las Cinasas Janus/farmacología , Quinasas Janus/metabolismo , Dispositivos Laboratorio en un Chip , Leucocitos Mononucleares/efectos de los fármacos , Membrana Celular , Células Cultivadas , Humanos , Inhibidores de las Cinasas Janus/química , Leucocitos Mononucleares/fisiología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2', 3' -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2'-5' and 3'-5' phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Nucleotidiltransferasas/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/farmacología , Anticuerpos/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Ensayo de Inmunoadsorción Enzimática , Polarización de Fluorescencia , Humanos , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Nucleótidos Cíclicos/inmunología , Nucleotidiltransferasas/metabolismo , Unión Proteica , Pirazoles/síntesis química , Pirimidinas/síntesis químicaRESUMEN
Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.
Asunto(s)
Descubrimiento de Drogas , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Isoquinolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Relación Dosis-Respuesta a Droga , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Isoquinolinas/administración & dosificación , Isoquinolinas/química , Lactamas , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Histone deacetylases (HDACs) regulate diverse cellular processes, and are promising targets for a number of diseases. Here we describe the design and utilization of a largazole-based chemical probe to quantitatively measure the intracellular occupancy of HDAC1 and HDAC2 by dacinostat. Surprisingly, the probe was unable to enrich HDAC3 despite its nanomolar potency in a biochemical assay, further proving the necessity of cell-based target occupancy assays to understand compound potency in physiologically-relevant settings. This occupancy assay has the potential to aid the development of novel HDAC1/2 inhibitors in drug discovery.
RESUMEN
Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.
Asunto(s)
Portadores de Fármacos/química , Liberación de Fármacos , Inmunoconjugados/química , Animales , Catepsina B/metabolismo , Línea Celular Tumoral , Portadores de Fármacos/metabolismo , Humanos , Hígado/citología , Lisosomas/enzimología , Espectrometría de Masas , Ratones , RatasRESUMEN
Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies, the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case, the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/química , Quinazolinas/farmacología , Temperatura , Química Clic , Relación Dosis-Respuesta a Droga , Endorribonucleasas/metabolismo , Inhibidores Enzimáticos/química , Células HEK293 , Humanos , Estructura Molecular , Quinazolinas/química , Ácidos Sulfínicos/química , Tirosina/químicaRESUMEN
Friedreich's Ataxia is a genetic disease caused by expansion of an intronic trinucleotide repeat in the frataxin (FXN) gene yielding diminished FXN expression and consequently disease. Since increasing FXN protein levels is desirable to ameliorate pathology, we explored the role of major cellular proteostasis pathways and mitochondrial proteases in FXN processing and turnover. We targeted p97/VCP, the ubiquitin proteasome pathway (UPP), and autophagy with chemical inhibitors in cell lines and patient-derived cells. p97 inhibition by DBeQ increased precursor FXN levels, while UPP and autophagic flux modulators had variable effects predominantly on intermediate FXN. Our data suggest that these pathways cannot be modulated to influence mature functional FXN levels. We also targeted known mitochondrial proteases by RNA interference and discovered a novel protease PITRM1 that regulates intermediate FXN levels. Treatment with the aforementioned chemical and genetic modulators did not have a differential effect in patient cells containing lower amounts of FXN. Interestingly, a number of treatments caused a change in total amount of FXN protein, without an effect on mature FXN. Our results imply that regulation of FXN protein levels is complex and that total amounts can be modulated chemically and genetically without altering the absolute amount of mature FXN protein.
Asunto(s)
Proteínas de Unión a Hierro/metabolismo , Mapas de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Transducción de Señal , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Humanos , Proteínas de Unión a Hierro/genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteolisis , Quinazolinas/farmacología , Expansión de Repetición de Trinucleótido , Ubiquitina/metabolismo , Proteína que Contiene Valosina , FrataxinaRESUMEN
A nontoxic and inexpensive photocatalytic initiation of anti-Markovnikov hydrothiolation of olefins using visible light is reported. This method is characterized by low catalyst loading, thereby enabling a mild and selective method for radical initiation in thiol-ene reactions between a wide scope of olefins and thiols.
RESUMEN
This paper describes the design and synthesis of a novel series of dual inhibitors of acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2). Key findings include the discovery of an initial lead that was modestly potent and subsequent medicinal chemistry optimization with a focus on lipophilic efficiency (LipE) to balance overall druglike properties. Free-Wilson methodology provided a clear breakdown of the contributions of specific structural elements to the overall LipE, a rationale for prioritization of virtual compounds for synthesis, and a highly successful prediction of the LipE of the resulting analogues. Further preclinical assays, including in vivo malonyl-CoA reduction in both rat liver (ACC1) and rat muscle (ACC2), identified an advanced analogue that progressed to regulatory toxicity studies.
Asunto(s)
Acetil-CoA Carboxilasa/antagonistas & inhibidores , Bencimidazoles/síntesis química , Hipoglucemiantes/síntesis química , Indazoles/síntesis química , Indoles/síntesis química , Pirazoles/síntesis química , Compuestos de Espiro/síntesis química , Animales , Bencimidazoles/química , Diseño de Fármacos , Humanos , Hipoglucemiantes/química , Indazoles/química , Indoles/química , Isoenzimas/antagonistas & inhibidores , Hígado/enzimología , Músculo Esquelético/enzimología , Pirazoles/química , Relación Estructura-Actividad Cuantitativa , Ratas , Compuestos de Espiro/químicaRESUMEN
Focused SAR studies were carried out around 5-heteroaryl and 1-amide portions of the 2-chlorobenzamide scaffold, resulting in the discovery of a potent, metabolically stable and centrally penetrable antagonist against P2X(7) receptor.
Asunto(s)
Antiinflamatorios/química , Benzamidas/química , Antagonistas del Receptor Purinérgico P2 , Pirimidinas/química , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Benzamidas/síntesis química , Benzamidas/farmacocinética , Sistema Nervioso Central/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Masculino , Microsomas Hepáticos/metabolismo , Pirimidinas/síntesis química , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7 , Relación Estructura-ActividadRESUMEN
Activation of the p38 kinase pathway in immune cells leads to the transcriptional and translational regulation of proinflammatory cytokines. Mitogen-activated protein kinase-activated protein kinase 2 (MK2), a direct downstream substrate of p38 kinase, regulates lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6) production through modulating the stability and translation of these mRNAs. Developing small-molecule inhibitors of MK2 may yield anti-inflammatory efficacy with a different safety profile relative to p38 kinase inhibitors. This article describes the pharmacologic properties of a benzothiophene MK2 inhibitor, PF-3644022 [(10R)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8H-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one]. PF-3644022 is a potent freely reversible ATP-competitive compound that inhibits MK2 activity (K(i) = 3 nM) with good selectivity when profiled against 200 human kinases. In the human U937 monocytic cell line or peripheral blood mononuclear cells, PF-3644022 potently inhibits TNFalpha production with similar activity (IC(50) = 160 nM). PF-3644022 blocks TNFalpha and IL-6 production in LPS-stimulated human whole blood with IC(50) values of 1.6 and 10.3 microM, respectively. Inhibition of TNFalpha in U937 cells and blood correlates closely with inhibition of phospho-heat shock protein 27, a target biomarker of MK2 activity. PF-3644022 displays good pharmacokinetic parameters in rats and is orally efficacious in both the rat acute LPS-induced TNFalpha model and the chronic streptococcal cell wall-induced arthritis model. Dose-dependent inhibition of TNFalpha production in the acute model and inhibition of paw swelling in the chronic model is observed with ED(50) values of 6.9 and 20 mg/kg, respectively. PF-3644022 efficacy in the chronic inflammation model is strongly correlated with maintaining a C(min) higher than the EC(50) measured in the rat LPS-induced TNFalpha model.
Asunto(s)
Antiinflamatorios , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inflamación/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Enfermedad Aguda , Adenosina Trifosfato/metabolismo , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Unión Competitiva/efectos de los fármacos , Pared Celular/química , Enfermedad Crónica , Relación Dosis-Respuesta a Droga , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Humanos , Inflamación/inducido químicamente , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Masculino , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Ratas Endogámicas Lew , Ratas Sprague-Dawley , Streptococcus , Células U937 , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The work described herein demonstrates the utility of structure-based drug design (SBDD) in shifting the binding mode of an HTS hit from a DFG-in to a DFG-out binding mode resulting in a class of novel potent CSF-1R kinase inhibitors suitable for lead development.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento , Enlace de Hidrógeno , Conformación Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismoRESUMEN
Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.
Asunto(s)
MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Tiofenos/química , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Diseño de Fármacos , Humanos , MAP Quinasa Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.
Asunto(s)
MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Tiofenos/química , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , MAP Quinasa Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
Abnormal proliferation mediated by disruption of the normal cell cycle mechanisms is a hallmark of virtually all cancer cells. Compounds targeting complexes between cyclin-dependent kinases (CDK) and cyclins, such as CDK2/cyclin A and CDK2/cyclin E, and inhibiting their kinase activity are regarded as promising antitumor agents to complement the existing therapies. From a high-throughput screening effort, we identified a new class of CDK2/cyclin A/E inhibitors. The hit-to-lead expansion of this class is described. X-ray crystallographic data of early compounds in this series, as well as in vitro testing funneled for rapidly achieving in vivo efficacy, led to a nanomolar inhibitor of CDK2/cyclin A (N-(5-cyclopropyl-1H-pyrazol-3-yl)-2-(2-naphthyl)acetamide (41), PNU-292137, IC50 = 37 nM) with in vivo antitumor activity (TGI > 50%) in a mouse xenograft model at a dose devoid of toxic effects.