RESUMEN
Nucleic acid-sensing pathways play critical roles in innate immune activation through the production of type I interferon (IFN-I) and proinflammatory cytokines. These factors are required for effective antitumor immune responses. Pharmacological modulators of the pre-mRNA spliceosome splicing factor 3b subunit 1 (SF3B1) are under clinical investigation as cancer cytotoxic agents. However, potential roles of these agents in aberrant RNA generation and subsequent RNA-sensing pathway activation have not been studied. In this study, we observed that SF3B1 pharmacological modulation using pladienolide B (Plad B) induces production of aberrant RNA species and robust IFN-I responses via engagement of the dsRNA sensor retinoic acid-inducible gene I (RIG-I) and downstream interferon regulatory factor 3. We found that Plad B synergized with canonical RIG-I agonism to induce the IFN-I response. In addition, Plad B induced NF-κB responses and secretion of proinflammatory cytokines and chemokines. Finally, we showed that cancer cells bearing the hotspot SF3B1K700E mutation, which leads to global aberrant splicing, had enhanced IFN-I response to canonical RIG-I agonism. Together, these results demonstrate that pharmacological modulation of SF3B1 in cancer cells can induce an enhanced IFN-I response dependent on RIG-I expression. The study suggests that spliceosome modulation may not only induce direct cancer cell cytotoxicity but also initiate an innate immune response via activation of RNA-sensing pathways.
Asunto(s)
Proteína 58 DEAD Box/metabolismo , Interferón Tipo I/metabolismo , Fosfoproteínas/metabolismo , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , Receptores Inmunológicos/metabolismo , Empalmosomas/metabolismo , Células A549 , Sustitución de Aminoácidos , Animales , Proteína 58 DEAD Box/genética , Humanos , Interferón Tipo I/genética , Ratones , Mutación Missense , Fosfoproteínas/genética , Precursores del ARN/genética , Factores de Empalme de ARN/genética , Receptores Inmunológicos/genética , Empalmosomas/genética , Células THP-1RESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.2001882.].
RESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.
Asunto(s)
Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/efectos de los fármacos , Animales , Línea Celular , Sistema Libre de Células , Colesterol/sangre , Escherichia coli/genética , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Masculino , Espectrometría de Masas , Terapia Molecular Dirigida , Proproteína Convertasa 9/sangre , Proproteína Convertasa 9/genética , Biosíntesis de Proteínas/fisiología , Conejos , Ratas , Ratas Sprague-Dawley , Ribosomas/metabolismo , Ribosomas/fisiologíaRESUMEN
A series of N-(piperidin-3-yl)-N-(pyridin-2-yl)piperidine/piperazine-1-carboxamides were identified as small molecule PCSK9 mRNA translation inhibitors. Analogues from this new chemical series, such as 4d and 4g, exhibited improved PCSK9 potency, ADME properties, and in vitro safety profiles when compared to earlier lead structures.
Asunto(s)
Amidas/química , Inhibidores de PCSK9 , Piperidinas/química , Inhibidores de Proteasas/química , Amidas/metabolismo , Amidas/farmacología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Humanos , Concentración 50 Inhibidora , Células de Riñón Canino Madin Darby , Conformación Molecular , Proproteína Convertasa 9/metabolismo , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.
Asunto(s)
Conotoxinas/química , Péptido 1 Similar al Glucagón/química , Péptidos/química , Proteínas Recombinantes de Fusión/química , Ponzoñas/química , Animales , Células CHO , Conotoxinas/genética , Cricetinae , Cricetulus , Exenatida , Péptido 1 Similar al Glucagón/genética , Humanos , Péptidos/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/genética , Ponzoñas/genéticaRESUMEN
Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesteraseâ 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free inâ vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. Inâ vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.
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Hígado/efectos de los fármacos , Inhibidores de PCSK9 , Proproteína Convertasa 9/biosíntesis , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Dosis-Respuesta a Droga , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/enzimología , Hígado/metabolismo , Estructura Molecular , Proproteína Convertasa 9/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Prostaglandin E receptor subtype 3 (EP3) antagonism may treat a variety of symptoms from inflammation to cardiovascular and metabolic diseases. Previously, most EP3 antagonists were large acidic ligands that mimic the substrate, prostaglandin E2 (PGE2). This manuscript describes the optimization of a neutral small molecule amide series with improved lipophilic efficiency (LipE) also known as lipophilic ligand efficiency (LLE) ((a) Nat. Rev. Drug Disc.2007, 6, 881; (b) Annu. Rep. Med. Chem.2010, 45, 380).
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Amidas/farmacología , Subtipo EP3 de Receptores de Prostaglandina E/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Hit-to-lead medicinal chemistry efforts are described starting from a screening hit 1, leading to a new class of aryl sulfonamide-based MR antagonist, exemplified by 17, that possesses favourable MR binding affinity, selectivity profile against closely related NHRs, physicochemical properties and metabolic stability.
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Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Humanos , Antagonistas de Receptores de Mineralocorticoides/síntesis química , Modelos Moleculares , Relación Estructura-Actividad , Sulfonamidas/síntesis químicaRESUMEN
A series of small-molecule YEATS4 binders have been discovered as part of an ongoing research effort to generate high-quality probe molecules for emerging and/or challenging epigenetic targets. Analogues such as 4d and 4e demonstrate excellent potency and selectivity for YEATS4 binding versus YEATS1,2,3 and exhibit good physical properties and in vitro safety profiles. A new X-ray crystal structure confirms direct binding of this chemical series to YEATS4 at the lysine acetylation recognition site of the YEATS domain. Multiple analogues engage YEATS4 with nanomolar potency in a whole-cell nanoluciferase bioluminescent resonance energy transfer assay. Rodent pharmacokinetic studies demonstrate the competency of several analogues as in vivo-capable binders.
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Regulación de la Expresión Génica , Procesamiento Proteico-Postraduccional , Dominios Proteicos , Acetilación , Epigénesis GenéticaRESUMEN
Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0-4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.
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Glucosa-6-Fosfato , Glucógeno Sintasa , Arginina/metabolismo , Glucosa-6-Fosfato/metabolismo , Glucógeno Sintasa/química , Glucógeno Sintasa/metabolismo , Humanos , Fosforilación , Uridina Difosfato/metabolismoRESUMEN
Discovery efforts leading to the identification of ervogastat (PF-06865571), a systemically acting diacylglycerol acyltransferase (DGAT2) inhibitor that has advanced into clinical trials for the treatment of non-alcoholic steatohepatitis (NASH) with liver fibrosis, are described herein. Ervogastat is a first-in-class DGAT2 inhibitor that addressed potential development risks of the prototype liver-targeted DGAT2 inhibitor PF-06427878. Key design elements that culminated in the discovery of ervogastat are (1) replacement of the metabolically labile motif with a 3,5-disubstituted pyridine system, which addressed potential safety risks arising from a cytochrome P450-mediated O-dearylation of PF-06427878 to a reactive quinone metabolite precursor, and (2) modifications of the amide group to a 3-THF group, guided by metabolite identification studies coupled with property-based drug design.
Asunto(s)
Diacilglicerol O-Acetiltransferasa , Enfermedad del Hígado Graso no Alcohólico , Humanos , Diseño de Fármacos , Cirrosis Hepática , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológicoRESUMEN
Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.
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Receptor del Péptido 1 Similar al Glucagón , Hipoglucemiantes , Animales , Receptor del Péptido 1 Similar al Glucagón/agonistas , Humanos , Hipoglucemiantes/farmacología , Péptidos/químicaRESUMEN
Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle wasting and weakness, leading to premature death from respiratory and/or cardiac failure. A clinically relevant question is whether myostatin inhibition can improve function of the diaphragm, which exhibits a severe and progressive pathology comparable with that in DMD. We hypothesized that antibody-directed myostatin inhibition would improve the pathophysiology of diaphragm muscle strips from young mdx mice (when the pathology is mild) and adult mdx mice (when the pathology is quite marked). Five weeks treatment with a mouse chimera of anti-human myostatin antibody (PF-354, 10 mg/kg/week) increased muscle mass (P < 0.05) and increased diaphragm median fiber cross-sectional area (CSA, P < 0.05) in young C57BL/10 and mdx mice, compared with saline-treated controls. PF-354 had no effect on specific force (sPo, maximum force normalized to muscle CSA) of diaphragm muscle strips from young C57BL/10 mice, but increased sPo by 84% (P < 0.05) in young mdx mice. In contrast, 8 weeks of PF-354 treatment did not improve muscle mass, median fiber CSA, collagen infiltration, or sPo of diaphragm muscle strips from adult mdx mice. PF-354 antibody-directed myostatin inhibition completely restored the functional capacity of diaphragm strips to control levels when treatment was initiated early, but not in the later stages of disease progression, suggesting that such therapies may only have a limited window of efficacy for DMD and related conditions.
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Envejecimiento , Diafragma/patología , Distrofia Muscular Animal/metabolismo , Miostatina/química , Animales , Concentración 50 Inhibidora , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular , Fibras Musculares Esqueléticas/patología , Distrofia Muscular Animal/patología , Miostatina/antagonistas & inhibidores , Miostatina/metabolismo , Factores de TiempoRESUMEN
The atypical chemokine receptor CXCR7 has been studied in various disease settings including immunological diseases and heart disease. Efforts to elucidate the role of CXCR7 have been limited by the lack of suitable chemical tools with a range of pharmacological profiles. A high-throughput screen was conducted to discover novel chemical matter with the potential to modulate CXCR7 receptor activity. This led to the identification of a series of diphenylacetamides confirmed in a CXCL12 competition assay indicating receptor binding. Further evaluation of this series revealed a lack of activity in the functional assay measuring ß-arrestin recruitment. The most potent representative, compound 10 (K i = 597 nM), was determined to be an antagonist in the ß-arrestin assay (IC50 = 622 nM). To our knowledge, this is the first reported small molecule ß-arrestin antagonist for CXCR7, useful as an in vitro chemical tool to elucidate the effects of CXCL12 displacement with ß-arrestin antagonism in models for diseases such as cardiac injury and suitable as starting point for hit optimization directed toward an in vivo tool compound for studying CXCR7 receptor pharmacology.
RESUMEN
The promise of phenotypic screening resides in its track record of novel biology and first-in-class therapies. However, challenges stemming from major differences between target-based and phenotypic screening do exist. These challenges prompted us to rethink the critical stage of hit triage and validation on the road to clinical candidates and novel drug targets. Whereas this process is usually straightforward for target screening hits, phenotypic screening hits act through a variety of mostly unknown mechanisms within a large and poorly understood biological space. Our analysis suggests successful hit triage and validation is enabled by three types of biological knowledge-known mechanisms, disease biology, and safety-while structure-based hit triage may be counterproductive.
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Triaje , Descubrimiento de Drogas , Humanos , FenotipoRESUMEN
The estrogen receptor (ER) subtypes, ERalpha and ERbeta, modulate numerous signaling cascades in the brain to result in a variety of cell fates including neuronal differentiation. We report here that 17beta-estradiol (E2) rapidly stimulates the autophosphorylation of alpha-Ca(2+)/calmodulin-dependent kinase II (alphaCaMKII) in immortalized NLT GnRH neurons, primary hippocampal neurons, and Cos7 cells co-transfected with ERalpha and alphaCaMKII. The E2-induced alphaCaMKII autophosphorylation is ERalpha- and Ca(2+)/calmodulin (CaM)-dependent. Interestingly, the hormone-dependent association of ERalpha with alphaCaMKII attenuates the positive effect of E2 on alphaCaMKII autophosphorylation, suggesting that ERalpha plays a complex role in modulating alphaCaMKII activity and may function to fine-tune alphaCaMKII-triggered signaling events. However, it appears as though the activating signal of E2 dominates the negative effect of ER since there is a clear, positive downstream response to E2-activated alphaCaMKII; pharmacological inhibitors and RNAi technology show that targets of ERalpha-mediated alphaCaMKII signaling include extracellular signal-regulated kinase 1/2 (ERK1/2), cAMP response element-binding protein (CREB), and microtubule associated protein 2 (MAP2). These findings suggest a novel model for the modulation of alphaCaMKII signaling by ERalpha, which provides a molecular link as to how E2 might influence brain function.
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Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/metabolismo , Receptor alfa de Estrógeno/metabolismo , Transducción de Señal/fisiología , Animales , Autorradiografía , Calcio/metabolismo , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina/genética , Células Cultivadas , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Estradiol/farmacología , Receptor alfa de Estrógeno/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Ratones , Mutación/fisiología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Transfección/métodosRESUMEN
Sodium-phosphate cotransporter 2a, or NaPi2a (SLC34A1), is a solute-carrier (SLC) transporter located in the kidney proximal tubule that reabsorbs glomerular-filtered phosphate. Inhibition of NaPi2a may enhance urinary phosphate excretion and correct maladaptive mineral and hormonal derangements associated with increased cardiovascular risk in chronic kidney disease-mineral and bone disorder (CKD-MBD). To date, only nonselective NaPi inhibitors have been described. Herein, we detail the discovery of the first series of selective NaPi2a inhibitors, resulting from optimization of a high-throughput screening hit. The oral PK profile of inhibitor PF-06869206 (6f) in rodents allows for the exploration of the pharmacology of selective NaPi2a inhibition.
RESUMEN
A novel series of morpholine-based nonsteroidal mineralocorticoid receptor antagonists is reported. Starting from a pyrrolidine HTS hit 9 that possessed modest potency but excellect selectivity versus related nuclear hormone receptors, a series of libraries led to identification of morpholine lead 10. After further optimization, cis disubstituted morpholine 22 was discovered, which showed a 45-fold boost in binding affinity and corresponding functional potency compared to 13. While 22 had high clearance in rat, it provided sufficient exposure at high doses to favorably assess in vivo efficacy (increased urinary Na+/K+ ratio) and safety. In contrast to rat, the dog and human MetID and PK profiles of 22 were adequate, suggesting that it could be suitable as a potential clinical asset.
Asunto(s)
Antagonistas de Receptores de Mineralocorticoides/química , Antagonistas de Receptores de Mineralocorticoides/farmacología , Morfolinos/química , Morfolinos/farmacología , Oxazinas/química , Receptores de Mineralocorticoides/metabolismo , Animales , Ensayos Clínicos Fase I como Asunto , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Proteica , Ratas , Ratas Wistar , Receptores de Mineralocorticoides/química , Relación Estructura-ActividadRESUMEN
While there is evidence that distinct protein isoforms resulting from alternative pre-mRNA splicing play critical roles in neuronal development and function, little is known about molecules regulating alternative splicing in the nervous system. Using Caenorhabditis elegans as a model for studying neuron/target communication, we report that unc-75 mutant animals display neuroanatomical and behavioral defects indicative of a role in modulating GABAergic and cholinergic neurotransmission but not neuronal development. We show that unc-75 encodes an RRM domain-containing RNA binding protein that is exclusively expressed in the nervous system and neurosecretory gland cells. UNC-75 protein, as well as a subset of related C. elegans RRM proteins, localizes to dynamic nuclear speckles; this localization pattern supports a role for the protein in pre-mRNA splicing. We found that human orthologs of UNC-75, whose splicing activity has recently been documented in vitro, are expressed nearly exclusively in brain and when expressed in C. elegans, rescue unc-75 mutant phenotypes and localize to subnuclear puncta. Furthermore, we report that the subnuclear-localized EXC-7 protein, the C. elegans ortholog of the neuron-restricted Drosophila ELAV splicing factor, acts in parallel to UNC-75 to also affect cholinergic synaptic transmission. In conclusion, we identified a new neuronal, putative pre-mRNA splicing factor, UNC-75, and show that UNC-75, as well as the C. elegans homolog of ELAV, is required for the fine tuning of synaptic transmission. These findings thus provide a novel molecular link between pre-mRNA splicing and presynaptic function.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Mapeo Cromosómico , Perfilación de la Expresión Génica , Datos de Secuencia Molecular , Neuronas Motoras/citología , Filogenia , Transmisión Sináptica/genéticaRESUMEN
Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a ß-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.