RESUMEN
In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Lipoproteínas/metabolismo , ARN Pequeño no Traducido/metabolismo , Factor sigma/metabolismo , Estrés Fisiológico/fisiología , Proteínas de la Membrana Bacteriana Externa/genética , Péptidos y Proteínas de Señalización Intracelular , Lipoproteínas/genética , Fenotipo , Regiones Promotoras Genéticas/genética , Biosíntesis de Proteínas/fisiología , ARN Pequeño no Traducido/genética , Secuencias Reguladoras de Ácido Ribonucleico/genéticaRESUMEN
Regeneration of the adult intestinal epithelium is mediated by a pool of cycling stem cells, which are located at the base of the crypt, that express leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5). The Frizzled (FZD) 7 receptor (FZD7) is enriched in LGR5+ intestinal stem cells and plays a critical role in their self-renewal. Yet, drug discovery approaches and structural bases for targeting specific FZD isoforms remain poorly defined. FZD proteins interact with Wnt signaling proteins via, in part, a lipid-binding groove on the extracellular cysteine-rich domain (CRD) of the FZD receptor. Here we report the identification of a potent peptide that selectively binds to the FZD7 CRD at a previously uncharacterized site and alters the conformation of the CRD and the architecture of its lipid-binding groove. Treatment with the FZD7-binding peptide impaired Wnt signaling in cultured cells and stem cell function in intestinal organoids. Together, our data illustrate that targeting the lipid-binding groove holds promise as an approach for achieving isoform-selective FZD receptor inhibition.
Asunto(s)
Receptores Frizzled/antagonistas & inhibidores , Receptores Frizzled/metabolismo , Intestinos/efectos de los fármacos , Células Madre/efectos de los fármacos , Animales , Sitios de Unión , Células CHO , Membrana Celular/metabolismo , Cricetulus , Cristalografía por Rayos X , Descubrimiento de Drogas , Femenino , Citometría de Flujo , Células HEK293 , Humanos , Intestinos/citología , Lípidos/química , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Unión Proteica , Multimerización de Proteína , Regeneración , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos , Células Madre/patología , Resonancia por Plasmón de Superficie , Vía de Señalización WntRESUMEN
The version of this article originally published contained older versions of the Life Sciences Reporting Summary and the Supplementary Text and Figures. The error has been corrected in the HTML and PDF versions of the article.
RESUMEN
The Escherichia coli σ(E) envelope stress response monitors and repairs the outer membrane, a function central to the life of Gram-negative bacteria. The σ(E) stress response was characterized as a single-tier activation network comprised of ~100 genes, including the MicA and RybB noncoding sRNAs. These highly expressed sRNAs were thought to carry out the specialized function of halting de novo synthesis of several abundant porins when envelope homeostasis was perturbed. Using a systematic target profiling and validation approach we discovered that MicA and RybB are each global mRNA repressors of both distinct and shared targets, and that the two sRNAs constitute a posttranscriptional repression arm whose regulatory scope rivals that of the protein-based σ(E) activation arm. Intriguingly, porin mRNAs constitute only ~1/3 of all targets and new nonporin targets predict roles for MicA and RybB in crosstalk with other regulatory responses. This work also provides an example of evolutionarily unrelated sRNAs that are coinduced and bind the same targets, but at different sites. Our finding that expression of either MicA or RybB sRNA protects the cell from the loss of viability experienced when σ(E) activity is inadequate illustrates the importance of the posttranscriptional repression arm of the response. σ(E) is a paradigm of a single-tier stress response with a clear division of labor in which highly expressed noncoding RNAs (MicA, RybB) endow a transcriptional factor intrinsically restricted to gene activation (σ(E)) with the opposite repressor function.
Asunto(s)
Proteínas de Escherichia coli/genética , ARN Pequeño no Traducido/genética , Regulón/genética , Factor sigma/genética , Secuencia de Bases , Northern Blotting , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Porinas/genética , Porinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor sigma/metabolismo , Estrés FisiológicoRESUMEN
Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.
Asunto(s)
Neoplasias de la Mama , Fosfatidilinositol 3-Quinasas , Humanos , Femenino , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , MutaciónRESUMEN
NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.
Asunto(s)
Linfocitos B/efectos de los fármacos , Riñón/efectos de los fármacos , Lupus Eritematoso Sistémico/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Animales , Linfocitos B/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocina TWEAK/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Inflamación/genética , Subunidad p40 de la Interleucina-12/efectos de los fármacos , Subunidad p40 de la Interleucina-12/inmunología , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos NZB , Terapia Molecular Dirigida , Proteinuria/inmunología , Receptores OX40/metabolismo , Transducción de Señal , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/inmunología , Quinasa de Factor Nuclear kappa BRESUMEN
NF-κB-inducing kinase (NIK) is a protein kinase central to the noncanonical NF-κB pathway downstream from multiple TNF receptor family members, including BAFF, which has been associated with B cell survival and maturation, dendritic cell activation, secondary lymphoid organ development, and bone metabolism. We report herein the discovery of lead chemical series of NIK inhibitors that were identified through a scaffold-hopping strategy using structure-based design. Electronic and steric properties of lead compounds were modified to address glutathione conjugation and amide hydrolysis. These highly potent compounds exhibited selective inhibition of LTßR-dependent p52 translocation and transcription of NF-κB2 related genes. Compound 4f is shown to have a favorable pharmacokinetic profile across species and to inhibit BAFF-induced B cell survival in vitro and reduce splenic marginal zone B cells in vivo.
Asunto(s)
Descubrimiento de Drogas , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Quinasa de Factor Nuclear kappa BRESUMEN
BACKGROUND: Bordetella pertussis, the causative agent of whooping cough, is a highly clonal pathogen of the respiratory tract. Its lack of genetic diversity, relative to many bacterial pathogens, could limit its ability to adapt to a hostile and changing host environment. This limitation might be overcome by phase variation, as observed for other mucosal pathogens. One of the most common mechanisms of phase variation is reversible expansion or contraction of homopolymeric tracts (HPTs). RESULTS: The genomes of B. pertussis and the two closely related species, B. bronchiseptica and B. parapertussis, were screened for homopolymeric tracts longer than expected on the basis of chance, given their nucleotide compositions. Sixty-nine such HPTs were found in total among the three genomes, 74% of which were polymorphic among the three species. Nine HPTs were genotyped in a collection of 90 geographically and temporally diverse B. pertussis strains using the polymerase chain reaction/ligase detection reaction (PCR/LDR) assay. Six HPTs were polymorphic in this collection of B. pertussis strains. Of note, one of these polymorphic HPTs was found in the fimX promoter, where a single base insertion variant was present in seven strains, all of which were isolated prior to introduction of the pertussis vaccine. Transcript abundance of fimX was found to be 3.8-fold lower in strains carrying the longer allele. HPTs in three other genes, tcfA, bapC, and BP3651, varied widely in composition across the strain collection and displayed allelic polymorphism within single cultures. CONCLUSION: Allelic polymorphism at homopolymeric tracts is common within the B. pertussis genome. Phase variability may be an important mechanism in B. pertussis for evasion of the immune system and adaptation to different niches in the human host. High sensitivity and specificity make the PCR/LDR assay a powerful tool for investigating allelic variation at HPTs. Using this method, allelic diversity and phase variation were demonstrated at several B. pertussis loci.
Asunto(s)
Bordetella pertussis/genética , Evolución Molecular , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Alelos , Proteínas Bacterianas/genética , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/fisiología , Bordetella parapertussis/genética , Bordetella pertussis/aislamiento & purificación , Bordetella pertussis/fisiología , ADN Bacteriano , Proteínas Fimbrias/genética , Genoma Bacteriano , Humanos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras GenéticasRESUMEN
We report here structure-guided optimization of a novel series of NF-κB inducing kinase (NIK) inhibitors. Starting from a modestly potent, low molecular weight lead, activity was improved by designing a type 11/2 binding mode that accessed a back pocket past the methionine-471 gatekeeper. Divergent binding modes in NIK and PI3K were exploited to dampen PI3K inhibition while maintaining NIK inhibition within these series. Potent compounds were discovered that selectively inhibit the nuclear translocation of NF-κB2 (p52/REL-B) but not canonical NF-κB1 (REL-A/p50).
Asunto(s)
Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de Anillo en Puente/farmacología , Isoxazoles/farmacología , Oxazepinas/farmacología , Oxazoles/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Perros , Células HEK293 , Células HeLa , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/química , Compuestos Heterocíclicos de Anillo en Puente/síntesis química , Compuestos Heterocíclicos de Anillo en Puente/química , Humanos , Imidazoles/farmacología , Isoxazoles/síntesis química , Isoxazoles/química , Ratones , Subunidad p50 de NF-kappa B/metabolismo , Subunidad p52 de NF-kappa B/metabolismo , Oxazepinas/síntesis química , Oxazepinas/química , Oxazoles/síntesis química , Oxazoles/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Transducción de Señal/efectos de los fármacos , Quinasa de Factor Nuclear kappa BRESUMEN
The Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant in vitro and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474.