RESUMEN
The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.
Asunto(s)
Neoplasias Hepáticas , Ribonucleósidos , Arginina/metabolismo , Creatina/análogos & derivados , Creatina/orina , Humanos , Ribonucleósidos/orinaRESUMEN
Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacología , Ensayos Analíticos de Alto Rendimiento , Mycobacterium tuberculosis/genética , Relación Estructura-ActividadRESUMEN
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) inhibit reverse transcription and block the replication of HIV-1. Currently, NNRTIs are usually used as part of a three-drug combination given to patients as antiretroviral therapy. These combinations involve other classes of anti-HIV-1 drugs, commonly nucleoside reverse transcriptase inhibitors (NRTIs). However, attempts are being made to develop two-drug maintenance therapies, some of which involve an NNRTI and an integrase strand transfer inhibitor. This has led to a renewed interest in developing novel NNRTIs, with a major emphasis on designing compounds that can effectively inhibit the known NNRTI-resistant mutants. We have generated and tested novel rilpivirine (RPV) analogs. The new compounds were designed to exploit a small opening in the upper right periphery of the NNRTI-binding pocket. The best of the new compounds, 12, was a more potent inhibitor of the NNRTI-resistant mutants we tested than either doravirine or efavirenz but was inferior to RPV. We describe the limitations on the modifications that can be appended to the "upper right side" of the RPV core and the effects of substituting other cores for the central pyrimidine core of RPV and make suggestions about how this information can be used in NNRTI design.
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Fármacos Anti-VIH/química , Diseño de Fármacos , Farmacorresistencia Viral , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Piridonas , Triazoles , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Sitios de Unión , Farmacorresistencia Viral/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , Humanos , Simulación de Dinámica Molecular , Mutación , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinas/química , Rilpivirina/análogos & derivados , Triazoles/farmacología , Triazoles/uso terapéuticoRESUMEN
Creatine riboside (CR) is a novel metabolite of cancer metabolism. It is a urinary diagnostic biomarker of lung and liver cancer risk and prognosis. The level of CR is highly positive correlated in tumor and urine indicating that it is derived from human lung and liver cancers. A precise and sensitive ultra-pressure liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS) method was developed and validated for simultaneous quantification of the noninvasive biomarker CR, along with creatinine riboside (CNR), and their precursors creatine and creatinine, utilizing the labeled internal standard creatine riboside-13C,15N2 (CR-13C,15N2). Chromatography was carried out on a hydrophilic interaction chromatography column under a gradient mobile phase condition. MRM transitions were monitored for CR (264.1 > 132.1, m/z), CNR (246.1 > 113.9, m/z), creatine (132.0 > 72.0, m/z), creatinine (114.0 > 85.8, m/z) and CR-13C,15N2 (267.1 > 134.9, m/z) with a 11.0 min run time in the positive mode ionization. The calibration plot of the method was linear over the concentration range of 4.50-10,000 nM. Method validation was performed according to regulatory guidelines established for sensitivity, selectivity, calibration curve, stability at different storage conditions, reinjection reproducibility, ruggedness with acceptable accuracy, and precision. This assay was applied for the quantification of CR along with CNR, creatine and creatinine in a subset of urine and serum samples from the National Cancer Institute - Maryland (NCI-MD) cohort population controls and lung cancer cases. It can be standardized and used in multiple laboratories for cancer diagnosis and determining the efficacy of cancer therapy and monitoring cancer recurrence.
Asunto(s)
Neoplasias Pulmonares , Espectrometría de Masas en Tándem , Biomarcadores de Tumor , Cromatografía Líquida de Alta Presión , Creatina/análogos & derivados , Creatinina , Humanos , Neoplasias Pulmonares/diagnóstico , Maryland , National Cancer Institute (U.S.) , Recurrencia Local de Neoplasia , Regulación de la Población , Reproducibilidad de los Resultados , Ribonucleósidos , Estados UnidosRESUMEN
Introduction: Transporters comprising the blood-brain barrier complicate delivery of many therapeutics to the central nervous system. The present study ascertained whether the natural product botryllamide G is viable for in vivo inhibition of ABCG2 using lapatinib as a probe for ABCB1 and ABCG2-mediated efflux from the brain. Methods: Wild-type and Mdr1a/Mdr1b (-/-) mice were treated with botryllamide G and lapatinib ("doublet therapy"), and while a separate cohort of wild-type mice was treated with botryllamide, tariquidar and lapatinib ("triplet therapy"). Results: Botryllamide G demonstrates biphasic elimination with a rapid distribution, decreasing below the in vitro IC50 of 6.9 µM within minutes, yet with a relatively slower terminal half-life (4.6 h). In Mdr1a/Mdr1b (-/-) mice, doublet therapy resulted in a significant increase in brain lapatinib AUC at 8 h (2058 h*ng/mL vs 4007 h*ng/mL; P = .031), but not plasma exposure (P = .15). No significant differences were observed after 24 h. Lapatinib brain exposure was greater through 1 h when wild-type mice were administered triplet therapy (298 h*pg/mg vs 120 h*pg/mg; P < .001), but the triplet decreased brain AUC through 24 h vs. mice administered lapatinib alone (2878 h*pg/mg vs 4461hr*ng/mL; P < .001) and did not alter the brain:plasma ratio. Conclusions: In summary, the ABCG2 inhibitor, botryllamide G, increases brain exposure to lapatinib in mice lacking Abcb1, although the combination of botryllamide G and tariquidar increases brain exposure in wild-type mice only briefly (1 h). Additional research is needed to find analogs of this compound that have better pharmacokinetics and pharmacodynamic effects on ABCG2 inhibition.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Acrilamidas/farmacología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Lapatinib/farmacocinética , Fenoles/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Lapatinib/administración & dosificación , Lapatinib/metabolismo , Masculino , Ratones , Ratones Noqueados , Distribución Tisular , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Brain metastases are devastating complications of cancer. The blood-brain barrier (BBB), which protects the normal brain, morphs into an inadequately characterized blood-tumor barrier (BTB) when brain metastases form, and is surrounded by a neuroinflammatory response. These structures contribute to poor therapeutic efficacy by limiting drug uptake. Here, we report that experimental breast cancer brain metastases of low- and high permeability to a dextran dye exhibit distinct microenvironmental gene expression patterns. Astrocytic sphingosine-1 phosphate receptor 3 (S1P3) is upregulated in the neuroinflammatory response of the highly permeable lesions, and is expressed in patients' brain metastases. S1P3 inhibition functionally tightens the BTB in vitro and in vivo. S1P3 mediates its effects on BTB permeability through astrocytic secretion of IL-6 and CCL2, which relaxes endothelial cell adhesion. Tumor cell overexpression of S1P3 mimics this pathway, enhancing IL-6 and CCL-2 production and elevating BTB permeability. In conclusion, neuroinflammatory astrocytic S1P3 modulates BTB permeability.
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Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Receptores de Lisoesfingolípidos/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Antibióticos Antineoplásicos/farmacocinética , Antibióticos Antineoplásicos/farmacología , Astrocitos/patología , Barrera Hematoencefálica/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Doxorrubicina/farmacocinética , Doxorrubicina/farmacología , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Humanos , Inyecciones Intramusculares , Interleucina-6/genética , Interleucina-6/metabolismo , Ratones Desnudos , Miocardio , Permeabilidad , Receptores de Lisoesfingolípidos/genética , Transducción de Señal , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Xantenos/química , Xantenos/metabolismoRESUMEN
Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Current chemotherapy is quite toxic in growing children and more directed therapeutics are being sought. The IL-7R pathway is a major driver of ALL and here we evaluate two drugs directed to that pathway using a model of T cell ALL. Mutant gain-of-function IL-7Rα was transduced into an IL-7-dependent murine thymocyte line conferring ligand-independent survival and growth. JAK1 is associated with IL-7Rα and mediates signaling from the mutant receptor. In vitro, treating the transformed cell line with the JAK1/2 inhibitor ruxolitinib inhibited ligand-independent signaling and induced cell death. Transfer of the transformed cell line into mice resulted in aggressive leukemia and untreated mice succumbed in about three weeks. Treatment with ruxolitinib incorporated into chow showed a potent therapeutic benefit with reduction in leukemic burden and extension of survival. BCL-2 is an anti-apoptotic downstream mediator of the IL-7R survival mechanism. Venetoclax, an inhibitor of BCL-2, showed activity against the transformed cell line in vitro and could be combined with ruxolitinib in vivo. These findings support the therapeutic potential of treating T-ALL by targeting the IL-7R pathway.
RESUMEN
Progressive multifocal leukoencephalopathy (PML) is a lethal brain disease caused by uncontrolled replication of JC polyomavirus (JCV). JCV strains recovered from the brains of PML patients carry mutations that prevent the engagement of sialylated glycans, which are thought to serve as receptors for the infectious entry of wild-type JCV. In this report, we show that non-sialylated glycosaminoglycans (GAGs) can serve as alternative attachment receptors for the infectious entry of both wild-type and PML mutant JCV strains. After GAG-mediated attachment, PML mutant strains engage non-sialylated non-GAG co-receptor glycans, such as asialo-GM1. JCV-neutralizing monoclonal antibodies isolated from patients who recovered from PML appear to block infection by preventing the docking of post-attachment co-receptor glycans in an apical pocket of the JCV major capsid protein. Identification of the GAG-dependent/sialylated glycan-independent alternative entry pathway should facilitate the development of infection inhibitors, including recombinant neutralizing antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Virus JC/fisiología , Internalización del Virus , Anticuerpos Neutralizantes/farmacología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Línea Celular Tumoral , Gangliósidos/farmacología , Genotipo , Glicosaminoglicanos/metabolismo , Hemaglutinación/efectos de los fármacos , Humanos , Virus JC/genética , Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/metabolismo , Leucoencefalopatía Multifocal Progresiva/patología , Leucoencefalopatía Multifocal Progresiva/virología , Mutación , Neuraminidasa/metabolismo , Proteínas de Transporte de Nucleótidos/antagonistas & inhibidores , Proteínas de Transporte de Nucleótidos/genética , Proteínas de Transporte de Nucleótidos/metabolismo , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ácidos Siálicos/farmacología , Internalización del Virus/efectos de los fármacosRESUMEN
We previously demonstrated activation of the mitogen-activated protein kinase (MAPK) pathway in a series of romidepsin-selected T-cell lymphoma cell lines as a mechanism of resistance to the histone deacetylase inhibitor (HDI), romidepsin. As Ras mutation leads to activation of both the MAPK and the phosphoinositide 3-kinase (PI3K) pathway, we examined whether combining romidepsin with small molecule pathway inhibitors would lead to increased apoptosis in cancers harboring Ras mutations. We treated 18 Ras mutant or wild-type cell lines with romidepsin in the presence of a MEK inhibitor (PD-0325901) and/or an AKT inhibitor (MK-2206) and examined apoptosis by flow cytometry. A short-term treatment schedule of romidepsin (25 ng/ml for 6 h) was used to more closely model clinical administration. Romidepsin in combination with a MEK and an AKT inhibitor induced apoptosis preferentially in cells harboring mutant versus wild-type Ras (69.1% vs. 21.1%, p < 0.0001). Similar results were found in a subset of cell lines when belinostat was combined with the MEK and AKT inhibitors and when romidepsin was combined with the dual extracellular signaling-related kinase (ERK)/PI3K inhibitor, D-87503, which inhibited both the MAPK and PI3K pathways at 5-10 µM. The observed apoptosis was caspase-dependent and required Bak and Bax expression. Cells with wild-type or mutant Ras treated with romidepsin alone or in combination with the MEK inhibitor displayed increased expression of proapoptotic Bim. We thus conclude that cancers bearing Ras mutations, such as pancreatic cancer, can be targeted by the combination of an HDI and a dual inhibitor of the MAPK and PI3K pathways.
Asunto(s)
Apoptosis/efectos de los fármacos , Depsipéptidos/farmacología , Genes ras , Inhibidores de Histona Desacetilasas/farmacología , Mutación , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidoresRESUMEN
Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes.
Asunto(s)
Antineoplásicos/farmacología , Proteínas del Citoesqueleto/antagonistas & inhibidores , Estrés Fisiológico , Transcriptoma , Adamantano/análogos & derivados , Adamantano/farmacocinética , Adamantano/farmacología , Animales , Antineoplásicos/farmacocinética , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Línea Celular Tumoral , Perros , Femenino , Semivida , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/secundario , Fenoles/farmacocinética , Fenoles/farmacología , Quinolinas/farmacocinética , Quinolinas/farmacología , Quinolonas/farmacocinética , Quinolonas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Rilpivirine (RPV) is the latest non-nucleoside reverse transcriptase inhibitor (NNRTI) to be approved by Food and Drug Administration to combat HIV-1 infections. NNRTIs inhibit the chemical step in viral DNA synthesis by binding to an allosteric site located about 10 Å from the polymerase active site of reverse transcriptase (RT). Although NNRTIs potently inhibit the replication of wild-type HIV-1, the binding site is not conserved, and mutations arise in the binding pocket. Doravirine (DOR) is a new NNRTI in phase III clinical trials. METHODS: Using a single round HIV-1 infection assay, we tested RPV and DOR against a broad panel of NNRTI-resistant mutants to determine their respective activities. We also used molecular modeling to determine if the susceptibility profile of each compound was related to how they bind RT. RESULTS: Several mutants displayed decreased susceptibility to DOR. However, with the exception of E138K, our data suggest that the mutations that reduce the potency of DOR and RPV are non-overlapping. Thus, these 2 NNRTIs have the potential to be used together in combination therapy. We also show that the location at which DOR and RPV bind with the NNRTI binding pocket of RT correlates with the differences in their respective susceptibility to the panel of NNRTI-resistance mutations. CONCLUSIONS: This shows that (1) DOR is susceptible to a number of well-known NNRTI resistance mutations and (2) an understanding of the mutational susceptibilities and binding interactions of NNRTIs with RT could be used to develop pairs of compounds with non-overlapping mutational susceptibilities.
Asunto(s)
Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Mutación , Piridonas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Rilpivirina/farmacología , Triazoles/farmacología , Relación Dosis-Respuesta a Droga , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a class of antiretroviral compounds that bind in an allosteric binding pocket in HIV-1 RT, located about 10 Å from the polymerase active site. Binding of an NNRTI causes structural changes that perturb the alignment of the primer terminus and polymerase active site, preventing viral DNA synthesis. Rilpivirine (RPV) is the most recent NNRTI approved by the FDA, but like all other HIV-1 drugs, suboptimal treatment can lead to the development of resistance. To generate better compounds that could be added to the current HIV-1 drug armamentarium, we have developed several RPV analogs to combat viral variants that are resistant to the available NNRTIs. RESULTS: Using a single-round infection assay, we identified several RPV analogs that potently inhibited a broad panel of NNRTI resistant mutants. Additionally, we determined that several resistant mutants selected by either RPV or Doravirine (DOR) caused only a small increase in susceptibility to the most promising RPV analogs. CONCLUSIONS: The antiviral data suggested that there are RPV analogs that could be candidates for further development as NNRTIs, and one of the most promising compounds was modeled in the NNRTI binding pocket. This model can be used to explain why this compound is broadly effective against the panel of NNRTI resistance mutants.
Asunto(s)
Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Mutación , Rilpivirina/análogos & derivados , Rilpivirina/farmacología , Humanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Succinatos/farmacología , Triterpenos/farmacología , Virión/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Alquilación , Aminación , Secuencia de Aminoácidos , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Cápside/efectos de los fármacos , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Farmacorresistencia Viral/efectos de los fármacos , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , VIH-1/metabolismo , Células HeLa , Humanos , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Polimorfismo Genético , Relación Estructura-Actividad , Succinatos/síntesis química , Succinatos/química , Linfocitos T/efectos de los fármacos , Linfocitos T/virología , Triterpenos/síntesis química , Triterpenos/química , Virión/genética , Virión/metabolismo , Replicación Viral/efectos de los fármacos , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
BACKGROUND: The small molecule NSC676914A was previously identified as an NF-κB inhibitor in TPA-stimulated HEK293 cells (Mol Can Ther 8:571-581, 2009). We hypothesized that this effect would also be seen in ovarian cancer cells, and serve as its mechanism of cytotoxicity. OVCAR3 and HEK293 cell lines stably containing a NF-κB luciferase reporter gene were generated. METHODS: Levels of NF-κB activity were assessed by luciferase reporter assays, after stimulation with LPA, LPS, TPA, and TNFα, in the presence or absence of a known NF-κB inhibitor or NSC676914A, and cytotoxicity was measured. RESULTS: NSC676914A was toxic to both OVCAR3 and HEK293 cells. We also investigated the cytotoxicity of NSC676914A on a panel of lymphoma cell lines with well characterized mutations previously shown to determine sensitivity or resistance to NF-κB inhibition. The compound did not show predicted patterns of effects on NF-κB activity in either lymphoma, ovarian or HEK293 cell lines. In HEK293 cells, the small molecule inhibited NF-κB when cells were stimulated, while in OVCAR3 cells it only partially inhibited NF-κB. Interestingly, we observed rescue of cell death with ROS inhibition. CONCLUSIONS: The current study suggests that the effect of NSC676914A on NF-κB depends on cell type and the manner in which the pathway is stimulated. Furthermore, as it is similarly toxic to lymphoma, OVCAR3 and HEK293 cells, NSC676914A shows promising NF-κB-independent anti-cancer activity in ovarian tumor cells.
RESUMEN
Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.
Asunto(s)
VIH-2/genética , ARN Viral/química , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismo , Regiones no Traducidas 5' , Secuencia de Bases , Ácido Edético/análogos & derivados , Ácido Edético/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Pliegue del ARNRESUMEN
BACKGROUND: The recently approved anti-AIDS drug rilpivirine (TMC278, Edurant) is a nonnucleoside inhibitor (NNRTI) that binds to reverse transcriptase (RT) and allosterically blocks the chemical step of DNA synthesis. In contrast to earlier NNRTIs, rilpivirine retains potency against well-characterized, clinically relevant RT mutants. Many structural analogues of rilpivirine are described in the patent literature, but detailed analyses of their antiviral activities have not been published. This work addresses the ability of several of these analogues to inhibit the replication of wild-type (WT) and drug-resistant HIV-1. RESULTS: We used a combination of structure activity relationships and X-ray crystallography to examine NNRTIs that are structurally related to rilpivirine to determine their ability to inhibit WT RT and several clinically relevant RT mutants. Several analogues showed broad activity with only modest losses of potency when challenged with drug-resistant viruses. Structural analyses (crystallography or modeling) of several analogues whose potencies were reduced by RT mutations provide insight into why these compounds were less effective. CONCLUSIONS: Subtle variations between compounds can lead to profound differences in their activities and resistance profiles. Compounds with larger substitutions replacing the pyrimidine and benzonitrile groups of rilpivirine, which reorient pocket residues, tend to lose more activity against the mutants we tested. These results provide a deeper understanding of how rilpivirine and related compounds interact with the NNRTI binding pocket and should facilitate development of novel inhibitors.
Asunto(s)
Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/enzimología , Nitrilos/farmacología , Pirimidinas/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Línea Celular , Cristalografía , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/fisiología , Humanos , Modelos Moleculares , Estructura Molecular , Mutación , Nitrilos/síntesis química , Nitrilos/química , Pirimidinas/síntesis química , Pirimidinas/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , RilpivirinaRESUMEN
O(6)-Benzylguanine is an irreversible inactivator of O(6)-alkylguanine-DNA alkyltransferase currently in clinical trials to overcome alkyltransferase-mediated resistance to certain cancer chemotherapeutic alkylating agents. In order to produce more soluble alkyltransferase inhibitors, we have synthesized three aminomethyl-substituted O(6)-benzylguanines and the three methyl analogs and found that the substitution of aminomethyl at the meta-position greatly enhances inactivation of alkyltransferase, whereas para-substitution has little effect and ortho-substitution virtually eliminates activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R(2)) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED(50)) values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanina/análogos & derivados , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Simulación por Computador , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Guanina/síntesis química , Guanina/química , Guanina/farmacología , Humanos , Enlace de Hidrógeno , Ligandos , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) is an important source of resistance to some therapeutic alkylating agents and attempts to circumvent this resistance by the use of hAGT inhibitors have reached clinical trials. Several human polymorphisms in the MGMT gene that encodes hAGT have been described including L84F and the linked double alteration I143V/K178R. We have investigated the inactivation of these variants and the much rarer variant W65C by O(6)-benzylguanine, which is currently in clinical trials, and a number of other second generation hAGT inhibitors that contain folate derivatives (O(4)-benzylfolic acid, the 3' and 5' folate esters of O(6)-benzyl-2'-deoxyguanosine and the folic acid gamma ester of O(6)-(p-hydroxymethyl)benzylguanine). The I143V/K178R variant was resistant to all of these compounds. The resistance was due solely to the I143V change. These results suggest that the frequency of the I143V/K178R variant among patients in the clinical trials with hAGT inhibitors and the correlation with response should be considered.
Asunto(s)
Inhibidores Enzimáticos/farmacología , O(6)-Metilguanina-ADN Metiltransferasa/antagonistas & inhibidores , Ensayos Clínicos como Asunto , Humanos , O(6)-Metilguanina-ADN Metiltransferasa/genética , Polimorfismo GenéticoRESUMEN
O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine residues at different rates depending on the identity of the alkyl group as well as the sequence context. To elucidate the mechanism(s) underlying the differences in rates, we examined the repair of five alkyl groups in three different sequence contexts. The kinact and Km values were determined by measuring the rates of repair of oligodeoxynucleotide duplexes containing the O6-alkylguanine residues with various concentrations of AGT in excess. The time course of the reactions all followed pseudo-first-order kinetics except for one of the O6-ethylguanine substrates, which could be analyzed in a two-phase exponential equation. The differences in rates of repair between the different alkyl groups and the different sequence contexts are dependent on rates of alkyl transfer and not substrate recognition. The relative rates of reaction are in general benzyl>methyl>ethyl>2-hydroxyethyl>4-(3-pyridyl)-4-oxobutyl, but the absolute rates are dependent on sequence. The kinact values between benzyl and 4-(3-pyridyl)-4-oxobutyl range from 2300 to 350000 depending on sequence. The sequence-dependent variation in kinact varied the most for O6-[4-(3-pyridyl)-4-oxobutyl]guanine, which ranged from 0.022 to 0.000016 s(-1). The results are consistent with a mechanism in which the O6-alkylguanine can bind to AGT in either a reactive or an unreactive orientation, the proportion of which depends on the sequence context.
Asunto(s)
Reparación del ADN , Guanina/análogos & derivados , Guanina/química , O(6)-Metilguanina-ADN Metiltransferasa/química , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , Oligonucleótidos/química , Especificidad por SustratoRESUMEN
O6-Alkylguanine-DNA alkyltransferase (alkyltransferase) provides an important source of resistance to some cancer chemotherapeutic alkylating agents. Folate ester derivatives of O6-benzyl-2'-deoxyguanosine and of O6-[4-(hydroxymethyl)benzyl]guanine were synthesized and tested for their ability to inactivate human alkyltransferase. Inactivation of alkyltransferase by the gamma-folate ester of O6-[4-(hydroxymethyl)benzyl]guanine was similar to that of the parent base. The gamma-folate esters of O6-benzyl-2'-deoxyguanosine were more potent alkyltransferase inactivators than the parent nucleoside. The 3'-ester was considerably more potent than the 5'-ester and was more than an order of magnitude more active than O6-benzylguanine, which is currently in clinical trials to enhance therapy with alkylating agents. They were also able to sensitize human tumor cells to killing by 1,3-bis(2-chloroethyl)-1-nitrosourea, with O6-benzyl-3'-O-(gamma-folyl)-2'-deoxyguanosine being most active. These compounds provide a new class of highly water-soluble alkyltransferase inactivators and form the basis to construct more tumor-specific and potent compounds targeting this DNA repair protein.