RESUMEN
Sickle cell disease (SCD) is caused by a point mutation in the ß-globin gene that creates hemoglobin S (HbS). Upon deoxygenation, HbS forms long polymers that distort the shape of red blood cells, causing hemolysis and vaso-occlusion. Voxelotor inhibits HbS polymerization, the root cause of SCD complications. To view this Bench to Bedside, open or download the PDF.
Asunto(s)
Anemia de Células Falciformes/genética , Benzaldehídos/uso terapéutico , Hemoglobina Falciforme/antagonistas & inhibidores , Pirazinas/uso terapéutico , Pirazoles/uso terapéutico , Globinas beta/genética , Anemia de Células Falciformes/epidemiología , Hemoglobina Falciforme/genética , Humanos , Mutación Puntual/genética , Polimerizacion/efectos de los fármacosRESUMEN
Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.
Asunto(s)
Huesos/metabolismo , Colon/metabolismo , Motilidad Gastrointestinal/genética , Canales Iónicos/metabolismo , ARN/metabolismo , Serotonina/biosíntesis , Serotonina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Huesos/citología , Calcio/metabolismo , Colitis/genética , Colitis/metabolismo , Colitis/prevención & control , Colon/fisiología , Heces/química , Femenino , Motilidad Gastrointestinal/fisiología , Células HEK293 , Humanos , Inmunohistoquímica , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Canales Iónicos/genética , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microbiota/efectos de los fármacos , Factor 88 de Diferenciación Mieloide/metabolismo , Osteoclastos/metabolismo , Pirazinas/farmacología , ARN/farmacología , Ribonucleasa Pancreática/administración & dosificación , Serotonina/sangre , Serotonina/deficiencia , Tiadiazoles/farmacologíaRESUMEN
The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.
Asunto(s)
Bacterias/metabolismo , Microbioma Gastrointestinal , Microbiota , Péptido Sintasas/metabolismo , Pirazinas/metabolismo , Animales , Bacillus subtilis/genética , Bacterias/clasificación , Bacterias/genética , Escherichia coli/genética , Heces/microbiología , Humanos , Péptido Sintasas/genética , FilogeniaRESUMEN
Pyridines and related N-heteroarenes are commonly found in pharmaceuticals, agrochemicals and other biologically active compounds1,2. Site-selective C-H functionalization would provide a direct way of making these medicinally active products3-5. For example, nicotinic acid derivatives could be made by C-H carboxylation, but this remains an elusive transformation6-8. Here we describe the development of an electrochemical strategy for the direct carboxylation of pyridines using CO2. The choice of the electrolysis setup gives rise to divergent site selectivity: a divided electrochemical cell leads to C5 carboxylation, whereas an undivided cell promotes C4 carboxylation. The undivided-cell reaction is proposed to operate through a paired-electrolysis mechanism9,10, in which both cathodic and anodic events play critical roles in altering the site selectivity. Specifically, anodically generated iodine preferentially reacts with a key radical anion intermediate in the C4-carboxylation pathway through hydrogen-atom transfer, thus diverting the reaction selectivity by means of the Curtin-Hammett principle11. The scope of the transformation was expanded to a wide range of N-heteroarenes, including bipyridines and terpyridines, pyrimidines, pyrazines and quinolines.
Asunto(s)
Dióxido de Carbono , Electroquímica , Pirazinas , Piridinas , Pirimidinas , Quinolinas , Hidrógeno/química , Pirazinas/química , Piridinas/química , Pirimidinas/química , Electroquímica/métodos , Dióxido de Carbono/química , Quinolinas/química , Preparaciones Farmacéuticas/síntesis química , Preparaciones Farmacéuticas/químicaRESUMEN
Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
Asunto(s)
Pirazinas/química , Virus ARN/genética , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Recombinación Genética , Ribonucleótidos/química , Animales , Antivirales , Catálisis , Células Cultivadas , Técnicas Genéticas , Genoma , Genoma Viral , Recombinación Homóloga , Humanos , Cinética , Ratones , Ratones Transgénicos , Simulación de Dinámica Molecular , Mutagénesis , Nucleótidos/genética , Conformación Proteica , ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , RNA-Seq , Transgenes , VirulenciaRESUMEN
Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1-3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5-7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.
Asunto(s)
Aciltransferasas , Proteínas de la Membrana , Vía de Señalización Wnt , Acilación/efectos de los fármacos , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Antineoplásicos , Sitios de Unión , Coenzima A/metabolismo , Microscopía por Crioelectrón , Histidina , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Palmitoil Coenzima A , Pirazinas/farmacología , Piridinas/farmacología , Serina , Especificidad por Sustrato , Vía de Señalización Wnt/efectos de los fármacos , Proteína Wnt3ARESUMEN
While effective anti-cancer drugs targeting the CHK1 kinase are advancing in the clinic, drug resistance is rapidly emerging. Here, we demonstrate that CRISPR-mediated knockout of the little-known gene FAM122A/PABIR1 confers cellular resistance to CHK1 inhibitors (CHK1is) and cross-resistance to ATR inhibitors. Knockout of FAM122A results in activation of PP2A-B55α, a phosphatase that dephosphorylates the WEE1 protein and rescues WEE1 from ubiquitin-mediated degradation. The resulting increase in WEE1 protein expression reduces replication stress, activates the G2/M checkpoint, and confers cellular resistance to CHK1is. Interestingly, in tumor cells with oncogene-driven replication stress, CHK1 can directly phosphorylate FAM122A, leading to activation of the PP2A-B55α phosphatase and increased WEE1 expression. A combination of a CHK1i plus a WEE1 inhibitor can overcome CHK1i resistance of these tumor cells, thereby enhancing anti-cancer activity. The FAM122A expression level in a tumor cell can serve as a useful biomarker for predicting CHK1i sensitivity or resistance.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Pirazinas/farmacología , Pirazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Nucleares/metabolismo , Fosfoproteínas/fisiología , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/genética , Pirazinas/metabolismo , Pirazoles/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The coronavirus disease 2019 (COVID-19) that is wreaking havoc on worldwide public health and economies has heightened awareness about the lack of effective antiviral treatments for human coronaviruses (CoVs). Many current antivirals, notably nucleoside analogs (NAs), exert their effect by incorporation into viral genomes and subsequent disruption of viral replication and fidelity. The development of anti-CoV drugs has long been hindered by the capacity of CoVs to proofread and remove mismatched nucleotides during genome replication and transcription. Here, we review the molecular basis of the CoV proofreading complex and evaluate its potential as a drug target. We also consider existing nucleoside analogs and novel genomic techniques as potential anti-CoV therapeutics that could be used individually or in combination to target the proofreading mechanism.
Asunto(s)
Antivirales/uso terapéutico , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Infecciones por Coronavirus/epidemiología , Genoma Viral , Pandemias , Neumonía Viral/tratamiento farmacológico , Neumonía Viral/epidemiología , ARN Viral/genética , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/química , Adenosina Monofosfato/uso terapéutico , Alanina/análogos & derivados , Alanina/química , Alanina/uso terapéutico , Amidas/química , Amidas/uso terapéutico , Antivirales/química , Betacoronavirus/genética , Betacoronavirus/patogenicidad , COVID-19 , Infecciones por Coronavirus/virología , Citidina/análogos & derivados , Humanos , Hidroxilaminas , Terapia Molecular Dirigida/métodos , Mutación , Neumonía Viral/virología , Pirazinas/química , Pirazinas/uso terapéutico , ARN Viral/antagonistas & inhibidores , ARN Viral/metabolismo , Ribonucleósidos/química , Ribonucleósidos/uso terapéutico , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Transcripción Genética , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Piezo1 and Piezo2 are recently reported mechanosensory ion channels that transduce mechanical stimuli from the environment into intracellular biochemical signals in various tissues and organ systems. Here, we show that Piezo1 and Piezo2 display a robust expression during jawbone development. Deletion of Piezo1 in neural crest cells causes jawbone malformations in a small but significant number of mice. We further demonstrate that disruption of Piezo1 and Piezo2 in neural crest cells causes more striking defects in jawbone development than any single knockout, suggesting essential but partially redundant roles of Piezo1 and Piezo2. In addition, we observe defects in other neural crest derivatives such as malformation of the vascular smooth muscle in double knockout mice. Moreover, TUNEL examinations reveal excessive cell death in osteogenic cells of the maxillary and mandibular arches of the double knockout mice, suggesting that Piezo1 and Piezo2 together regulate cell survival during jawbone development. We further demonstrate that Yoda1, a Piezo1 agonist, promotes mineralization in the mandibular arches. Altogether, these data firmly establish that Piezo channels play important roles in regulating jawbone formation and maintenance.
Asunto(s)
Canales Iónicos , Maxilares , Cresta Neural , Animales , Ratones , Regulación del Desarrollo de la Expresión Génica , Canales Iónicos/metabolismo , Canales Iónicos/genética , Maxilares/embriología , Maxilares/metabolismo , Mandíbula/embriología , Mandíbula/metabolismo , Ratones Noqueados , Cresta Neural/metabolismo , Osteogénesis/genética , Pirazinas , TiadiazolesRESUMEN
The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.
Asunto(s)
Dipeptidil Peptidasa 4/inmunología , Inmunoterapia/métodos , Linfocitos/inmunología , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiocinas/inmunología , Quimiocinas/metabolismo , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Femenino , Citometría de Flujo , Linfocitos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/genética , Pirazinas/farmacología , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Fosfato de Sitagliptina , Triazoles/farmacologíaRESUMEN
Regulation of transcription is a fundamental process that allows bacteria to respond to external stimuli with appropriate timing and magnitude of response. In the soil bacterium Bacillus subtilis, transcriptional regulation is at the core of developmental processes needed for cell survival. Gene expression in cells transitioning from exponential phase to stationary phase is under the control of a group of transcription factors called transition state regulators (TSRs). TSRs influence numerous developmental processes including the decision between biofilm formation and motility, genetic competence, and sporulation, but the extent to which TSRs influence bacterial physiology remains to be fully elucidated. Here, we demonstrate two TSRs, ScoC and AbrB, along with the MarR-family transcription factor PchR negatively regulate production of the iron chelator pulcherrimin in B. subtilis. Genetic analysis of the relationship between the three transcription factors indicate that all are necessary to limit pulcherrimin production during exponential phase and influence the rate and total amount of pulcherrimin produced. Similarly, expression of the pulcherrimin biosynthesis gene yvmC was found to be under control of ScoC, AbrB, and PchR and correlated with the amount of pulcherrimin produced by each background. Lastly, our in vitro data indicate a weak direct role for ScoC in controlling pulcherrimin production along with AbrB and PchR. The layered regulation by two distinct regulatory systems underscores the important role for pulcherrimin in B. subtilis physiology.
Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Transcripción Genética , Biopelículas/crecimiento & desarrollo , PirazinasRESUMEN
ABSTRACT: Voxelotor is an inhibitor of sickle hemoglobin polymerization that is used to treat sickle cell disease. Although voxelotor has been shown to improve anemia, the clinical benefit on the brain remains to be determined. This study quantified the cerebral hemodynamic effects of voxelotor in children with sickle cell anemia (SCA) using noninvasive diffuse optical spectroscopies. Specifically, frequency-domain near-infrared spectroscopy combined with diffuse correlation spectroscopy were used to noninvasively assess regional oxygen extraction fraction (OEF), cerebral blood volume, and an index of cerebral blood flow (CBFi). Estimates of CBFi were first validated against arterial spin-labeled magnetic resonance imaging (ASL-MRI) in 8 children with SCA aged 8 to 18 years. CBFi was significantly positively correlated with ASL-MRI-measured blood flow (R2 = 0.651; P = .015). Next, a single-center, open-label pilot study was completed in 8 children with SCA aged 4 to 17 years on voxelotor, monitored before treatment initiation and at 4, 8, and 12 weeks (NCT05018728). By 4 weeks, both OEF and CBFi significantly decreased, and these decreases persisted to 12 weeks (both P < .05). Decreases in CBFi were significantly correlated with increases in blood hemoglobin (Hb) concentration (P = .025), whereas the correlation between decreases in OEF and increases in Hb trended toward significance (P = .12). Given that previous work has shown that oxygen extraction and blood flow are elevated in pediatric SCA compared with controls, these results suggest that voxelotor may reduce cerebral hemodynamic impairments. This trial was registered at www.ClinicalTrials.gov as #NCT05018728.
Asunto(s)
Anemia de Células Falciformes , Circulación Cerebrovascular , Oxígeno , Humanos , Anemia de Células Falciformes/sangre , Niño , Adolescente , Masculino , Femenino , Oxígeno/sangre , Oxígeno/metabolismo , Preescolar , Imagen por Resonancia Magnética/métodos , Pirazinas/uso terapéutico , Pirazinas/administración & dosificación , Proyectos Piloto , Benzaldehídos/uso terapéutico , Benzaldehídos/farmacología , Benzaldehídos/administración & dosificación , Espectroscopía Infrarroja Corta/métodos , PirazolesRESUMEN
ABSTRACT: The phase 2 CLL2-BAAG trial tested the measurable residual disease (MRD)-guided triple combination of acalabrutinib, venetoclax, and obinutuzumab after optional bendamustine debulking in 45 patients with relapsed/refractory chronic lymphocytic leukemia (CLL). MRD was measured by flow cytometry (FCM; undetectable MRD <10-4) in peripheral blood (PB) and circulating tumor DNA (ctDNA) using digital droplet polymerase chain reaction of variable-diversity-joining (VDJ) rearrangements and CLL-related mutations in plasma. The median number of previous treatments was 1 (range, 1-4); 18 patients (40%) had received a Bruton tyrosine kinase inhibitor (BTKi) and/or venetoclax before inclusion, 14 of 44 (31.8%) had TP53 aberrations, and 34 (75.6%) had unmutated immunoglobulin heavy-chain variable region genes. With a median observation time of 36.3 months and all patients off-treatment for a median of 21.9 months, uMRD <10-4 in PB was achieved in 42 of the 45 patients (93.3%) at any time point, including 17 of 18 (94.4%) previously exposed to venetoclax/BTKi and 13 of 14 (92.9%) with TP53 aberrations. The estimated 3-year progression-free and overall survival rates were 85.0% and 93.8%, respectively. Overall, 585 paired FCM/ctDNA samples were analyzed and 18 MRD recurrences (5 with and 13 without clinical progression) occurred after the end of treatment. Twelve samples were first detected by ctDNA, 3 by FCM, and 3 synchronously. In conclusion, time-limited MRD-guided acalabrutinib, venetoclax, and obinutuzumab achieved deep remissions in almost all patients with relapsed/refractory CLL. The addition of ctDNA-based analyses to FCM MRD assessment seems to improve early detection of relapses. This trial was registered at www.clinicaltrials.gov as #NCT03787264.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas , Compuestos Bicíclicos Heterocíclicos con Puentes , ADN Tumoral Circulante , Leucemia Linfocítica Crónica de Células B , Neoplasia Residual , Pirazinas , Sulfonamidas , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/mortalidad , Sulfonamidas/administración & dosificación , Sulfonamidas/uso terapéutico , Anciano , Persona de Mediana Edad , Femenino , Masculino , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , ADN Tumoral Circulante/genética , ADN Tumoral Circulante/sangre , Pirazinas/administración & dosificación , Pirazinas/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Anciano de 80 o más Años , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/uso terapéutico , Benzamidas/administración & dosificación , Benzamidas/uso terapéutico , Adulto , RecurrenciaRESUMEN
SLFN11 sensitizes cancer cells to a broad range of DNA-targeted therapies. Here we show that, in response to replication stress induced by camptothecin, SLFN11 tightly binds chromatin at stressed replication foci via RPA1 together with the replication helicase subunit MCM3. Unlike ATR, SLFN11 neither interferes with the loading of CDC45 and PCNA nor inhibits the initiation of DNA replication but selectively blocks fork progression while inducing chromatin opening across replication initiation sites. The ATPase domain of SLFN11 is required for chromatin opening, replication block, and cell death but not for the tight binding of SLFN11 to chromatin. Replication stress by the CHK1 inhibitor Prexasertib also recruits SLFN11 to nascent replicating DNA together with CDC45 and PCNA. We conclude that SLFN11 is recruited to stressed replication forks carrying extended RPA filaments where it blocks replication by changing chromatin structure across replication sites.
Asunto(s)
Proteínas Nucleares/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Camptotecina , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Daño del ADN , ADN Helicasas/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Humanos , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas Nucleares/metabolismo , Pirazinas , Pirazoles , Proteína de Replicación A/metabolismoRESUMEN
The renal collecting duct is continuously exposed to a wide spectrum of fluid flow rates and osmotic gradients. Expression of a mechanoactivated Piezo1 channel is the most prominent in the collecting duct. However, the status and regulation of Piezo1 in functionally distinct principal and intercalated cells (PCs and ICs) of the collecting duct remain to be determined. We used pharmacological Piezo1 activation to quantify Piezo1-mediated [Ca2+]i influx and single-channel activity separately in PCs and ICs of freshly isolated collecting ducts with fluorescence imaging and electrophysiological tools. We also employed a variety of systemic treatments to examine their consequences on Piezo1 function in PCs and ICs. Piezo1 selective agonists, Yoda-1 or Jedi-2, induced a significantly greater Ca2+ influx in PCs than in ICs. Using patch clamp analysis, we recorded a Yoda-1-activated nonselective channel with 18.6 ± 0.7 pS conductance on both apical and basolateral membranes. Piezo1 activity in PCs but not ICs was stimulated by short-term diuresis (injections of furosemide) and reduced by antidiuresis (water restriction for 24 h). However, prolonged stimulation of flow by high K+ diet decreased Yoda-1-dependent Ca2+ influx without changes in Piezo1 levels. Water supplementation with NH4Cl to induce metabolic acidosis stimulated Piezo1 activity in ICs but not in PCs. Overall, our results demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1-mediated [Ca2+]i influx in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli.
Asunto(s)
Calcio , Canales Iónicos , Túbulos Renales Colectores , Membrana Celular , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/metabolismo , Pirazinas/farmacología , Tiadiazoles/farmacología , Agua/metabolismo , Canales Iónicos/agonistas , Canales Iónicos/metabolismo , Animales , Ratones , Calcio/metabolismoRESUMEN
The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.
Asunto(s)
Apoptosis , Aterosclerosis , Células Espumosas , Canales Iónicos , Macrófagos , Fagocitosis , Animales , Canales Iónicos/metabolismo , Canales Iónicos/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Aterosclerosis/genética , Ratones , Células Espumosas/metabolismo , Células Espumosas/patología , Humanos , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Tiofenos/farmacología , Masculino , Especies Reactivas de Oxígeno/metabolismo , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/genética , Mitocondrias/metabolismo , Pirazinas , TiadiazolesRESUMEN
Piezo1 channels are essential mechanically activated ion channels in vertebrates. Their selective activation by the synthetic chemical activator Yoda1 opened new avenues to probe their gating mechanisms and develop novel pharmaceuticals. Yet, the nature and extent of Piezo1 functions modulated by this small molecule remain unclear. Here we close this gap by conducting a comprehensive biophysical investigation of the effects of Yoda1 on mouse Piezo1 in mammalian cells. Using calcium imaging, we first show that cysteine bridges known to inhibit mechanically evoked Piezo1 currents also inhibit activation by Yoda1, suggesting Yoda1 acts by energetically modulating mechanosensory domains. The presence of Yoda1 alters single-channel dwell times and macroscopic kinetics consistent with a dual and reciprocal energetic modulation of open and shut states. Critically, we further discovered that the electrophysiological effects of Yoda1 depend on membrane potential and temperature, two other Piezo1 modulators. This work illuminates a complex interplay between physical and chemical modulators of Piezo1 channels.
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Canales Iónicos , Mecanotransducción Celular , Pirazinas , Tiadiazoles , Animales , Canales Iónicos/agonistas , Canales Iónicos/metabolismo , Mecanotransducción Celular/fisiología , Potenciales de la Membrana , Ratones , Pirazinas/farmacología , Temperatura , Tiadiazoles/farmacologíaRESUMEN
BACKGROUND: Acute infusion reactions to oxaliplatin, a chemotherapeutic used to treat gastrointestinal cancers, are observed in about 20% of patients. Rapid drug desensitization (RDD) protocols often allow the continuation of oxaliplatin in patients with no alternative options. Breakthrough symptoms, including anaphylaxis, can still occur during RDD. OBJECTIVE: Our aim was to evaluate whether pretreatment with acalabrutinib, a Bruton tyrosine kinase inhibitor, can prevent anaphylaxis during RDD in a patient sensitized to oxaliplatin. METHODS: A 52-year-old male with locally advanced gastric carcinoma developed anaphylaxis during his fifth cycle of oxaliplatin. As he required 6 additional cycles to complete his curative-intent treatment regimen, he underwent RDD to oxaliplatin but still developed severe acute reactions. The risks and benefits of adding acalabrutinib before and during RDD were reviewed, and the patient elected to proceed. RESULTS: With acalabrutinib taken before and during the RDD, the patient was able to tolerate oxaliplatin RDD without complication. Consistent with its mechanism of action, acalabrutinib completely blocked the patient's positive skin prick response to oxaliplatin. Acalabrutinib did not alter the percentage of circulating basophils (1.24% vs 0.98%) before the RDD but did protect against basopenia (0.74% vs 0.09%) after the RDD. Acalabrutinib was associated with a drastic reduction in the ability of basophils to upregulate CD63 in vitro following incubation with oxaliplatin (0.11% vs 2.38%) or polyclonal anti-human IgE antibody (0.08% vs 44.2%). CONCLUSIONS: Five doses of acalabrutinib, 100 mg, orally twice daily starting during the evening 2 days before and continuing through RDD allowed a sensitized patient to receive oxaliplatin successfully and safely.
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Agammaglobulinemia Tirosina Quinasa , Antineoplásicos , Benzamidas , Desensibilización Inmunológica , Hipersensibilidad a las Drogas , Oxaliplatino , Pirazinas , Humanos , Oxaliplatino/efectos adversos , Persona de Mediana Edad , Masculino , Hipersensibilidad a las Drogas/inmunología , Hipersensibilidad a las Drogas/prevención & control , Desensibilización Inmunológica/métodos , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Pirazinas/efectos adversos , Pirazinas/administración & dosificación , Pirazinas/uso terapéutico , Benzamidas/uso terapéutico , Benzamidas/administración & dosificación , Antineoplásicos/efectos adversos , Anafilaxia/prevención & control , Anafilaxia/inducido químicamente , Anafilaxia/inmunología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/inmunologíaRESUMEN
INTRODUCTION: Thoracic aortic aneurysm (TAA) is a severe vascular disease that threatens human life, characterized by focal dilatation of the entire aortic wall, with a diameter 1.5 times larger than normal. PIEZO1, a mechanosensitive cationic channel, monitors mechanical stimulations in the environment, transduces mechanical signals into electrical signals, and converts them into biological signals to activate intracellular signaling pathways. However, the role of PIEZO1 in TAA is still unclear. METHODS: We analyzed a single-cell database to investigate the expression level of PIEZO1 in TAA. We constructed a conditional knockout mouse model of Piezo1 and used the PIEZO1 agonist Yoda1 to intervene in the TAA model mice established by co-administration of BAPN and ANG-II. Finally, we explored the effect of Yoda1 on TAA in vitro. RESULTS AND DISCUSSION: We observed decreased PIEZO1 expression in TAA at both RNA and protein levels. Single-cell sequencing identified a specific reduction in Piezo1 expression in endothelial cells. Administration of PIEZO1 agonist Yoda1 prevented the formation of TAA. In PIEZO1 endothelial cell conditional knockout mice, Yoda1 inhibited TAA formation by interfering with PIEZO1. In vivo and in vitro experiments demonstrated that the effect of Yoda1 on endothelial cells involved macrophage infiltration, extracellular matrix degradation, and neovascularization. This study highlights the role of PIEZO1 in TAA and its potential as a therapeutic target, providing opportunities for clinical translation.
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Aneurisma de la Aorta Torácica , Modelos Animales de Enfermedad , Células Endoteliales , Canales Iónicos , Ratones Noqueados , Análisis de la Célula Individual , Animales , Aneurisma de la Aorta Torácica/metabolismo , Aneurisma de la Aorta Torácica/genética , Aneurisma de la Aorta Torácica/patología , Canales Iónicos/metabolismo , Canales Iónicos/genética , Ratones , Células Endoteliales/metabolismo , Humanos , Masculino , Pirazinas , TiadiazolesRESUMEN
Bioluminescence is a fascinating natural phenomenon, wherein organisms produce light through specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc) derived from the sea pansy Renilla reniformis is notable for its blue light emission and has potential applications in bioluminescent tagging. Our study focuses on RLuc8, a variant of RLuc with eight amino acid substitutions. Recent studies have shown that the luminescent emitter coelenteramide can adopt multiple protonation states, which may be influenced by nearby residues at the enzyme's active site, demonstrating a complex interplay between protein structure and bioluminescence. Herein, using the quantum mechanical consistent force field method and the semimacroscopic protein dipole-Langevin dipole method with linear response approximation, we show that the phenolate state of coelenteramide in RLuc8 is the primary light-emitting species in agreement with experimental results. Our calculations also suggest that the proton transfer (PT) from neutral coelenteramide to Asp162 plays a crucial role in the bioluminescence process. Additionally, we reproduced the observed emission maximum for the amide anion in RLuc8-D120A and the pyrazine anion in the presence of a Na+ counterion in RLuc8-D162A, suggesting that these are the primary emitters. Furthermore, our calculations on the neutral emitter in the engineered AncFT-D160A enzyme, structurally akin to RLuc8-D162A but with a considerably blue-shifted emission peak, aligned with the observed data, possibly explaining the variance in emission peaks. Overall, this study demonstrates an effective approach to investigate chromophores' bimolecular states while incorporating the PT process in emission spectra calculations, contributing valuable insights for future studies of PT in photoproteins.