RESUMEN
Oncogenic activation of RAS genes via point mutations occurs in 20%-30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that ribosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.
Asunto(s)
Glicina/análogos & derivados , Proteínas de Unión al ARN/química , Transducción de Señal/efectos de los fármacos , Sulfonas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Neoplásica/efectos de los fármacos , Cristalografía por Rayos X , Dimerización , Glicina/administración & dosificación , Glicina/química , Glicina/farmacología , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Neoplasias Pancreáticas/tratamiento farmacológico , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Sulfonas/administración & dosificación , Sulfonas/química , Proteínas ras/metabolismo , Quinasa Tipo Polo 1RESUMEN
Oligosaccharides have myriad functions throughout biological processes1,2. Chemical synthesis of these structurally complex molecules facilitates investigation of their functions. With a dense concentration of stereocentres and hydroxyl groups, oligosaccharide assembly through O-glycosylation requires simultaneous control of site, stereo- and chemoselectivities3,4. Chemists have traditionally relied on protecting group manipulations for this purpose5-8, adding considerable synthetic work. Here we report a glycosylation platform that enables selective coupling between unprotected or minimally protected donor and acceptor sugars, producing 1,2-cis-O-glycosides in a catalyst-controlled, site-selective manner. Radical-based activation9 of allyl glycosyl sulfones forms glycosyl bromides. A designed aminoboronic acid catalyst brings this reactive intermediate close to an acceptor through a network of non-covalent hydrogen bonding and reversible covalent B-O bonding interactions, allowing precise glycosyl transfer. The site of glycosylation can be switched with different aminoboronic acid catalysts by affecting their interaction modes with substrates. The method accommodates a wide range of sugar types, amenable to the preparation of naturally occurring sugar chains and pentasaccharides containing 11 free hydroxyls. Experimental and computational studies provide insights into the origin of selectivity outcomes.
Asunto(s)
Glicósidos , Oligosacáridos , Ácidos Borónicos/química , Bromuros/química , Catálisis , Glicósidos/química , Glicósidos/síntesis química , Glicosilación , Enlace de Hidrógeno , Oligosacáridos/química , Oligosacáridos/síntesis química , Sulfonas/químicaRESUMEN
Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular , Glicina/análogos & derivados , Neoplasias Pulmonares/patología , Sulfonas/farmacología , Tubulina (Proteína)/metabolismo , Contaminación de Medicamentos , Resistencia a Antineoplásicos , Glicina/farmacología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Mutación , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Células Tumorales CultivadasRESUMEN
We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Neoplasias/patología , Preparaciones Farmacéuticas/metabolismo , Sulfonas/farmacología , Tubulina (Proteína)/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Contaminación de Medicamentos , Glicina/farmacología , Humanos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Preparaciones Farmacéuticas/química , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine1-3. Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments4. Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.
Asunto(s)
Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Proteínas de Transporte de Glicina en la Membrana Plasmática/química , Glicina/metabolismo , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Cristalografía , Humanos , Modelos Moleculares , Piperazinas/química , Piperazinas/farmacología , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único , Sulfonas/química , Sulfonas/farmacología , SincrotronesRESUMEN
Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.
Asunto(s)
Mitosis/efectos de los fármacos , Mitosis/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Centro Organizador de los Microtúbulos/efectos de los fármacos , Centro Organizador de los Microtúbulos/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estabilidad Proteica , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Sulfonas/farmacología , Sulfonas/uso terapéutico , Ubiquitina/metabolismo , Ubiquitinación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.
Asunto(s)
Aminoquinolinas/farmacología , Inflamación/prevención & control , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Sulfonas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Células Cultivadas , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidoresRESUMEN
NLRP3 inflammasome activation has emerged as a critical initiator of inflammatory response in ischemic retinopathy. Here, we identified the effect of a potent, selective NLRP3 inhibitor, MCC950, on autophagy and apoptosis under hypoxia. Neonatal mice were exposed to hyperoxia for 5 days to establish oxygen-induced retinopathy (OIR) model. Intravitreal injection of MCC950 was given, and then autophagy and apoptosis markers were assessed. Retinal autophagy, apoptosis, and related pathways were evaluated by western blot, immunofluorescent labeling, transmission electron microscopy, and TUNEL assay. Autophagic activity in Müller glia after NLRP3 inflammasome inhibition, together with its influence on photoreceptor death, was studied using western blot, immunofluorescence staining, mRFP-GFP-LC3 adenovirus transfection, cell viability, proliferation, and apoptosis assays. Results showed that activation of NLRP3 inflammasome in Müller glia was detected in OIR model. MCC950 could improve impaired retinal autophagic flux and attenuate retinal apoptosis while it regulated the retinal AMPK/mTOR/ULK-1 pathway. Suppressed autophagy and depressed proliferation capacity resulting from hypoxia was promoted after MCC950 treatment in Müller glia. Inhibition of AMPK and ULK-1 pathway significantly interfered with the MCC950-induced autophagy activity, indicating MCC950 positively modulated autophagy through AMPK/mTOR/ULK-1 pathway in Müller cells. Furthermore, blockage of autophagy in Müller glia significantly induced apoptosis in the cocultured 661W photoreceptor cells, whereas MCC950 markedly preserved the density of photoreceptor cells. These findings substantiated the therapeutic potential of MCC950 against impaired autophagy and subsequent apoptosis under hypoxia. Such protective effect might involve the modulation of AMPK/mTOR/ULK-1 pathway. Targeting NLRP3 inflammasome in Müller glia could be beneficial for photoreceptor survival under hypoxic conditions.
Asunto(s)
Apoptosis , Autofagia , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Células Fotorreceptoras de Vertebrados , Sulfonamidas , Animales , Ratones , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Óxidos S-Cíclicos/farmacología , Células Ependimogliales/metabolismo , Células Ependimogliales/efectos de los fármacos , Furanos/farmacología , Hipoxia/metabolismo , Indenos/farmacología , Inflamasomas/metabolismo , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonas/farmacologíaRESUMEN
Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.
Asunto(s)
Antineoplásicos/farmacología , Regulación Neoplásica de la Expresión Génica , Pruebas Genéticas/métodos , Glicina/análogos & derivados , Microtúbulos/efectos de los fármacos , Sulfonas/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/genética , Antineoplásicos/química , Sistemas CRISPR-Cas , Colchicina/farmacología , Resistencia a Antineoplásicos , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glicina/química , Glicina/farmacología , Células HeLa , Humanos , Células K562 , Cinesinas/genética , Cinesinas/metabolismo , Lentivirus/genética , Lentivirus/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Mutación , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Síndromes Mielodisplásicos/patología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Vinblastina/farmacologíaRESUMEN
Allosteric modulators of G-protein-coupled receptors (GPCRs) enhance signaling by binding to GPCRs concurrently with their orthosteric ligands, offering a novel approach to overcome the efficacy limitations of conventional orthosteric ligands. However, the structural mechanism by which allosteric modulators mediate GPCR signaling remains largely unknown. Here, to elucidate the mechanism of µ-opioid receptor (MOR) activation by allosteric modulators, we conducted solution NMR analyses of MOR by monitoring the signals from methionine methyl groups. We found that the intracellular side of MOR exists in an equilibrium between three conformations with different activities. Interestingly, the populations in the equilibrium determine the apparent signaling activity of MOR. Our analyses also revealed that the equilibrium is not fully shifted to the conformation with the highest activity even in the full agonist-bound state, where the intracellular half of TM6 is outward-shifted. Surprisingly, an allosteric modulator for MOR, BMS-986122, shifted the equilibrium toward the conformation with the highest activity, leading to the increased activity of MOR in the full agonist-bound state. We also determined that BMS-986122 binds to a cleft in the transmembrane region around T162 on TM3. Together, these results suggest that BMS-986122 binding to TM3 increases the activity of MOR by rearranging the direct interactions of TM3 and TM6, thus stabilizing TM6 in the outward-shifted position which is favorable for G-protein binding. These findings shed light on the rational developments of novel allosteric modulators that activate GPCRs further than orthosteric ligands alone and pave the way for next-generation GPCR-targeting therapeutics.
Asunto(s)
Receptores Opioides mu , Sulfonas , Regulación Alostérica , Sitio Alostérico , Sitios de Unión , Ligandos , Conformación Proteica/efectos de los fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/química , Transducción de Señal , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfonederived acylenzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acylenzyme complexes.
Asunto(s)
Compuestos de Azabiciclo , Inhibidores de beta-Lactamasas , Penicilinas , Sulfonas , Triazoles , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/químicaRESUMEN
Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), which is the most common form of kidney cancer. The VHL tumor suppressor protein marks hypoxia-inducible factor 1 (HIF1) and HIF2 for proteasomal degradation when oxygen is present. The inappropriate accumulation of HIF2 drives tumor formation by VHL tumor suppressor protein (pVHL)defective ccRCC. Belzutifan, a first-in-class allosteric HIF2 inhibitor, has advanced to phase 3 testing for advanced ccRCC and is approved for ccRCCs arising in patients with VHL disease, which is caused by germline VHL mutations. HIF2 can suppress p53 function in some settings and preliminary data suggested that an intact p53 pathway, as measured by activation in response to DNA damage, was necessary for HIF2 dependence. Here, we correlated HIF2 dependence and p53 status across a broader collection of ccRCC cell lines. We also genetically manipulated p53 function in ccRCC lines that were or were not previously HIF2-dependent and then assessed their subsequent sensitivity to HIF2 ablation using CRISPR-Cas9 or the HIF2 inhibitor PT2399, which is closely related to belzutifan. From these studies, we conclude that p53 status does not dictate HIF2 dependence, at least in preclinical models, and thus is unlikely to be a useful biomarker for predicting which ccRCC patients will respond to HIF2 inhibitors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinoma de Células Renales , Indanos , Neoplasias Renales , Sulfonas , Proteína p53 Supresora de Tumor , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Indanos/farmacología , Indanos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Sulfonas/farmacología , Sulfonas/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
SignificanceBisphenol A (BPA), found in many plastic products, has weak estrogenic effects that can be harmful to human health. Thus, structurally related replacements-bisphenol S (BPS) and bisphenol F (BPF)-are coming into wider use with very few data about their biological activities. Here, we compared the effects of BPA, BPS, and BPF on human mammary organoids established from normal breast tissue. BPS disrupted organoid architecture and induced supernumerary branching. At a proteomic level, the bisphenols altered the abundance of common targets and those that were unique to each compound. The latter included proteins linked to tumor-promoting processes. These data highlighted the importance of testing the human health effects of replacements that are structurally related to chemicals of concern.
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Compuestos de Bencidrilo , Carcinogénesis , Estrógenos , Glándulas Mamarias Humanas , Fenoles , Proteoma , Sulfonas , Compuestos de Bencidrilo/toxicidad , Carcinogénesis/inducido químicamente , Estrógenos/toxicidad , Humanos , Glándulas Mamarias Humanas/efectos de los fármacos , Glándulas Mamarias Humanas/patología , Organoides/efectos de los fármacos , Organoides/patología , Fenoles/toxicidad , Proteoma/efectos de los fármacos , Proteómica , Sulfonas/toxicidadRESUMEN
Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.
Asunto(s)
Guanidinas , Lipopolisacáridos , Miometrio , Canales de Potasio de Dominio Poro en Tándem , Intercambiador 1 de Sodio-Hidrógeno , Sulfonas , Animales , Femenino , Ratones , Embarazo , Escherichia coli , Lipopolisacáridos/toxicidad , Miometrio/metabolismo , ARN Interferente Pequeño/metabolismo , Contracción Uterina/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Intercambiador 1 de Sodio-Hidrógeno/metabolismoRESUMEN
BACKGROUND: Bisphenol S (BPS) is a substitute for bisphenol A in plastic manufacturing and, as a potential endocrine disruptor, may alter the physiology of the oviduct, in which fertilization and early embryo development take place in mammals. The objective of this study was to assess the effect of a daily dietary exposure to BPS combined with a contrasted diet on the oviduct fluid proteome using an ovine model. RESULTS: Eighty adult cyclic ewes were allotted to four groups (20/group): overfed (OF) consuming 50 µg/kg/day of BPS in their diet, underfed (UF) consuming 50 µg/kg/day of BPS, and non-exposed controls in each diet group. After three months, the mean body condition score, plasma levels of glucose and non-esterified fatty acids were significantly higher in OF than in UF females. The proteins in collected OF samples (50 µg) were analyzed by nanoliquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS). Overall, 1563 proteins were identified, among which 848 were quantified. Principal component analysis of the data revealed a clear discrimination of samples according to the diet and a segregation between BPS-exposed and non-exposed females in overfed ewes. Hierarchical clustering of differentially abundant proteins (DAPs) identified two clusters of 101 and 78 DAPs according to the diet. Pairwise comparisons between groups revealed a stronger effect of BPS in OF than in UF females (70 vs. 24 DAPs) and a stronger effect of the diet in BPS-exposed than non-exposed females (56 vs. 36 DAPs). Functional analysis of DAPs showed an enrichment in metabolic processes, immune system, cell response to stress, and reproductive processes. CONCLUSIONS: This work highlights for the first time the important impact of BPS on the oviduct proteome, with larger effects seen in OF than UF females. These results, together with previous ones, raise health concerns for everyone and call for a greater regulation of BPS in the food industry.
Asunto(s)
Oviductos , Fenoles , Proteoma , Sulfonas , Animales , Femenino , Ovinos , Fenoles/toxicidad , Proteoma/metabolismo , Oviductos/metabolismo , Oviductos/efectos de los fármacos , Sulfuros/administración & dosificación , Proteómica , Administración Oral , DietaRESUMEN
Macrocyclic peptides are promising scaffolds for the covalent ligand discovery. However, platforms enabling the direct identification of covalent macrocyclic ligands in a high-throughput manner are limited. In this study, we present an mRNA display platform allowing selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS). Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date. This study outlines the workflow for integrating chemical functionalizationâinstallation of a covalent warheadâwith mRNA display and showcases its application in targeted covalent ligand discovery.
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ARN Mensajero , ARN Mensajero/antagonistas & inhibidores , Ciclización , Sulfuros/química , Sulfuros/farmacología , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/síntesis química , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Compuestos Macrocíclicos/síntesis química , Sulfonas/química , Sulfonas/farmacología , Descubrimiento de Drogas , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Inhibidores de Proteasas/síntesis química , Estructura MolecularRESUMEN
Sphingosine kinase 1 (SPHK1) has been shown to play a key role in the pathogenesis of asthma where SPHK1-generated sphingosine-1-phosphate (S1P) is known to mediate innate and adaptive immunity while promoting mast cell degranulation. Goblet cell metaplasia (GCM) contributes to airway obstruction in asthma and has been demonstrated in animal models. We investigated the role of PF543, a SPHK1-specific inhibitor, in preventing the pathogenesis of GCM using a murine (C57BL/6) model of allergen-induced acute asthma. Treatment with PF543 before triple allergen exposure (DRA: House dust mite, Ragweed pollen, and Aspergillus) reduced inflammation, eosinophilic response, and GCM followed by reduced airway hyperreactivity to intravenous methacholine. Furthermore, DRA exposure was associated with increased expression of SPHK1 in the airway epithelium which was reduced by PF543. DRA-induced reduction of acetylated α-tubulin in airway epithelium was associated with an increased expression of NOTCH2 and SPDEF which was prevented by PF543. In vitro studies using human primary airway epithelial cells showed that inhibition of SPHK1 using PF543 prevented an allergen-induced increase of both NOTCH2 and SPDEF. siRNA silencing of SPHK1 prevented the allergen-induced increase of both NOTCH2 and SPDEF. NOTCH2 silencing was associated with a reduction of SPDEF but not that of SPHK1 upon allergen exposure. Our studies demonstrate that inhibition of SPHK1 protected allergen-challenged airways by preventing GCM and airway hyperreactivity, associated with downregulation of the NOTCH2-SPDEF signaling pathway. This suggests a potential novel link between SPHK1, GCM, and airway remodeling in asthma.NEW & NOTEWORTHY The role of SPHK1-specific inhibitor, PF543, in preventing goblet cell metaplasia (GCM) and airway hyperreactivity (AHR) is established in an allergen-induced mouse model. This protection was associated with the downregulation of NOTCH2-SPDEF signaling pathway, suggesting a novel link between SPHK1, GCM, and AHR.
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Asma , Células Caliciformes , Lisofosfolípidos , Fosfotransferasas (Aceptor de Grupo Alcohol) , Pirrolidinas , Esfingosina/análogos & derivados , Sulfonas , Animales , Humanos , Ratones , Células Caliciformes/metabolismo , Ratones Endogámicos C57BL , Asma/patología , Epitelio/metabolismo , Factores de Transcripción/metabolismo , Metaplasia/metabolismo , Metaplasia/patología , Alérgenos , MetanolRESUMEN
INTRODUCTION: The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting. METHODS: In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose-response curves was the variable used to quantify the cellular response towards TKIs. RESULTS: Spheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = - 7.33), lorlatinib (AOC fold change = - 6.17), ceritinib (AOC fold change = - 2.8) and entrectinib (AOC fold change = - 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = - 2.35), entrectinib (AOC fold change = - 2.44) and ceritinib (AOC fold change = - 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = - 2.19) and entrectinib (spheroid AOC fold change = - 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = - 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells. CONCLUSION: In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.
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Aminopiridinas , Benzamidas , Carcinoma de Pulmón de Células no Pequeñas , Indazoles , Lactamas , Neoplasias Pulmonares , Pirazoles , Pirimidinas , Sulfonas , Humanos , Proteínas Tirosina Quinasas/genética , Crizotinib , Sistemas CRISPR-Cas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas , Resistencia a MedicamentosRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) remains a leading life-threatening health challenge worldwide, with pressing needs for novel therapeutic strategies. Sphingosine kinase 1 (SphK1), a well-established pro-cancer enzyme, is aberrantly overexpressed in a multitude of malignancies, including HCC. Our previous research has shown that genetic ablation of Sphk1 mitigates HCC progression in mice. Therefore, the development of PF-543, a highly selective SphK1 inhibitor, opens a new avenue for HCC treatment. However, the anti-cancer efficacy of PF-543 has not yet been investigated in primary cancer models in vivo, thereby limiting its further translation. METHODS: Building upon the identification of the active form of SphK1 as a viable therapeutic target in human HCC specimens, we assessed the capacity of PF-543 in suppressing tumor progression using a diethylnitrosamine-induced mouse model of primary HCC. We further delineated its underlying mechanisms in both HCC and endothelial cells. Key findings were validated in Sphk1 knockout mice and lentiviral-mediated SphK1 knockdown cells. RESULTS: SphK1 activity was found to be elevated in human HCC tissues. Administration of PF-543 effectively abrogated hepatic SphK1 activity and significantly suppressed HCC progression in diethylnitrosamine-treated mice. The primary mechanism of action was through the inhibition of tumor neovascularization, as PF-543 disrupted endothelial cell angiogenesis even in a pro-angiogenic milieu. Mechanistically, PF-543 induced proteasomal degradation of the critical glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, thus restricting the energy supply essential for tumor angiogenesis. These effects of PF-543 could be reversed upon S1P supplementation in an S1P receptor-dependent manner. CONCLUSIONS: This study provides the first in vivo evidence supporting the potential of PF-543 as an effective anti-HCC agent. It also uncovers previously undescribed links between the pro-cancer, pro-angiogenic and pro-glycolytic roles of the SphK1/S1P/S1P receptor axis. Importantly, unlike conventional anti-HCC drugs that target individual pro-angiogenic drivers, PF-543 impairs the PFKFB3-dictated glycolytic energy engine that fuels tumor angiogenesis, representing a novel and potentially safer therapeutic strategy for HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Fosfotransferasas (Aceptor de Grupo Alcohol) , Pirrolidinas , Sulfonas , Animales , Humanos , Ratones , Angiogénesis , Carcinoma Hepatocelular/genética , Dietilnitrosamina , Células Endoteliales , Neoplasias Hepáticas/genética , Metanol , Neovascularización Patológica , Fosfofructoquinasa-2 , Receptores de Esfingosina-1-FosfatoRESUMEN
BACKGROUND: Oncogenic mutations in the RAS gene are associated with uncontrolled cell growth, a hallmark feature contributing to tumorigenesis. While diverse therapeutic strategies have been diligently applied to treat RAS-mutant cancers, successful targeting of the RAS gene remains a persistent challenge in the field of cancer therapy. In our study, we discover a promising avenue for addressing this challenge. METHODS: In this study, we tested the viability of several cell lines carrying oncogenic NRAS, KRAS, and HRAS mutations upon treatment with IkappaBalpha (IκBα) inhibitor BAY 11-7082. We performed both cell culture-based viability assay and in vivo subcutaneous xenograft-based assay to confirm the growth inhibitory effect of BAY 11-7082. We also performed large RNA sequencing analysis to identify differentially regulated genes and pathways in the context of oncogenic NRAS, KRAS, and HRAS mutations upon treatment with BAY 11-7082. RESULTS: We demonstrate that oncogenic NRAS, KRAS, and HRAS activate the expression of IκBα kinase. BAY 11-7082, an inhibitor of IκBα kinase, attenuates the growth of NRAS, KRAS, and HRAS mutant cancer cells in cell culture and in mouse model. Mechanistically, BAY 11-7082 inhibitor treatment leads to suppression of the PI3K-AKT signaling pathway and activation of apoptosis in all RAS mutant cell lines. Additionally, we find that BAY 11-7082 treatment results in the downregulation of different biological pathways depending upon the type of RAS protein that may also contribute to tumor growth inhibition. CONCLUSION: Our study identifies BAY 11-7082 to be an efficacious inhibitor for treating RAS oncogene (HRAS, KRAS, and NRAS) mutant cancer cells. This finding provides new therapeutic opportunity for effective treatment of RAS-mutant cancers.