RESUMEN
Overexpression of γ-glutamyl transpeptidase (GGT1) has been implicated in an array of human diseases including asthma, reperfusion injury, and cancer. Inhibitors are needed for therapy, but development of potent, specific inhibitors of GGT1 has been hampered by a lack of structural information regarding substrate binding and cleavage. To enhance our understanding of the molecular mechanism of substrate cleavage, we have solved the crystal structures of human GGT1 (hGGT1) with glutathione (a substrate) and a phosphate-glutathione analog (an irreversible inhibitor) bound in the active site. These are the first structures of any eukaryotic GGT with the cysteinylglycine region of the substrate-binding site occupied. These structures and the structure of apo-hGGT reveal movement of amino acid residues within the active site as the substrate binds. Asn-401 and Thr-381 each form hydrogen bonds with two atoms of GSH spanning the γ-glutamyl bond. Three different atoms of hGGT1 interact with the carboxyl oxygen of the cysteine of GSH. Interactions between the enzyme and substrate change as the substrate moves deeper into the active site cleft. The substrate reorients and a new hydrogen bond is formed between the substrate and the oxyanion hole. Thr-381 is locked into a single conformation as an acyl bond forms between the substrate and the enzyme. These data provide insight on a molecular level into the substrate specificity of hGGT1 and provide an explanation for seemingly disparate observations regarding the enzymatic activity of hGGT1 mutants. This knowledge will aid in the design of clinically useful hGGT1 inhibitors.
Asunto(s)
Dipéptidos/metabolismo , Inhibidores Enzimáticos/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dipéptidos/química , Humanos , Modelos Moleculares , Conformación Proteica , gamma-Glutamiltransferasa/química , gamma-Glutamiltransferasa/metabolismoRESUMEN
Existing single cell mass spectrometry (SCMS) sampling platforms are largely designed to work only with immobilized cells and not the suspended cells isolated from patient samples. Here, we present a novel method that integrates a commercially available cell manipulation system commonly used for in vitro fertilization with the Single-probe SCMS sampling technology. The combined Single-probe SCMS/cell manipulating platform is capable of rapidly analyzing intracellular species in real time from a suspension leukemia cell line. A broad range of molecular species was detected, and species of interest were verified using tandem MS (MS/MS). Experimental results were analyzed utilizing statistical analyses such as principle component analysis (PCA) and t-tests. The developed SCMS/cell manipulation system is a versatile tool to provide rapid single cell analysis of broad types of patient cell samples.
Asunto(s)
Análisis de la Célula Individual/métodos , Espectrometría de Masas en Tándem/métodos , Humanos , Células K562 , Metaboloma/efectos de los fármacos , Metabolómica/métodos , Paclitaxel/farmacología , Análisis de Componente Principal , Análisis de la Célula Individual/instrumentaciónRESUMEN
Analyzing cellular constituents on the single-cell level through mass spectrometry (MS) allows for a wide range of compounds to be studied simultaneously. However, there is a need for quantitative single-cell mass spectrometry (qSCMS) methods to fully characterize drug efficacy from individual cells within cell populations. In this study, qSCMS experiments were carried out using the Single-probe MS technique. The method was successfully used to perform rapid absolute quantifications of the anticancer drug irinotecan in individual mammalian cancer cells under ambient conditions in real time. Traditional liquid chromatography/mass spectrometry (LC/MS) quantifications of irinotecan in cell lysate samples were used to compare the results from Single-probe qSCMS. This technique showcases heterogeneity of drug efficacy on the single-cell level.
Asunto(s)
Antineoplásicos/análisis , Irinotecán/análisis , Línea Celular Tumoral , Humanos , Espectrometría de Masas/métodos , Análisis de la Célula Individual/métodosAsunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Enzimas Desubicuitinizantes , Ubiquitina Tiolesterasa/genéticaRESUMEN
γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within the active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. These data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.
Asunto(s)
gamma-Glutamiltransferasa/química , Aminobutiratos/química , Aminobutiratos/farmacología , Apoenzimas/química , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Ácido Glutámico/metabolismo , Humanos , Modelos Moleculares , Organofosfonatos/química , Organofosfonatos/farmacología , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , gamma-Glutamiltransferasa/antagonistas & inhibidores , gamma-Glutamiltransferasa/genéticaRESUMEN
We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 µm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.
Asunto(s)
Antineoplásicos/análisis , Espectrometría de Masas/instrumentación , Metaboloma , Análisis de la Célula Individual/instrumentación , Adenosina Difosfato/análisis , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/análisis , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Colestenonas/análisis , Doxorrubicina/análisis , Células HeLa , Humanos , Espectrometría de Masas/métodos , Paclitaxel/análisis , Fosfatidilcolinas/análisis , Fosfatidilcolinas/metabolismo , Saponinas/análisis , Análisis de la Célula Individual/métodosRESUMEN
Cephalostatin 1, OSW-1, ritterazine B and schweinfurthin A are natural products that potently, and in some cases selectively, inhibit the growth of cultured human cancer cell lines. The cellular targets of these small molecules have yet to be identified. We have discovered that these molecules target oxysterol binding protein (OSBP) and its closest paralog, OSBP-related protein 4L (ORP4L)--proteins not known to be involved in cancer cell survival. OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal transduction, lipid transport and lipid metabolism. The functions of OSBP and the ORPs, however, remain largely enigmatic. Based on our findings, we have named the aforementioned natural products ORPphilins. Here we used ORPphilins to reveal new cellular activities of OSBP. The ORPphilins are powerful probes of OSBP and ORP4L that will be useful in uncovering their cellular functions and their roles in human diseases.
Asunto(s)
Productos Biológicos/farmacología , Colestenonas/farmacología , Neoplasias/metabolismo , Fenazinas/farmacología , Receptores de Esteroides/metabolismo , Saponinas/farmacología , Compuestos de Espiro/farmacología , Esteroides/farmacología , Productos Biológicos/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colestenonas/antagonistas & inhibidores , Humanos , Hidroxicolesteroles/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Fenazinas/antagonistas & inhibidores , Receptores de Esteroides/genética , Saponinas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Esfingomielinas/biosíntesis , Compuestos de Espiro/antagonistas & inhibidores , Esteroides/antagonistas & inhibidores , Estilbenos/antagonistas & inhibidores , Estilbenos/farmacologíaRESUMEN
Oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4) have emerged as potentially druggable targets in antiviral and precision cancer drug development. Multiple structurally diverse small molecules function through targeting the OSBP/ORP family of proteins, including the antiviral steroidal compounds OSW-1 and T-00127-HEV2. Here, the structure-activity relationships of oxysterols and related compound binding to human OSBP and ORP4 are characterized. Oxysterols with hydroxylation at various side chain positions (i.e., C-20, C-24, C-25, and C-27)âbut not C-22âconfer high affinity interactions with OSBP and ORP4. A library of 20(S)-hydroxycholesterol analogues with varying sterol side chains reveal that side chain length modifications are not well tolerated for OSBP and ORP4 interactions. This side chain requirement is contradicted by the high affinity binding of T-00127-HEV2, a steroidal compound lacking the side chain. The binding results, in combination with docking studies using homology models of OSBP and ORP4, suggest multiple modes of steroidal ligand binding to OSBP and ORP4.
Asunto(s)
Oxiesteroles , Receptores de Esteroides , Humanos , Antivirales/farmacología , Hidroxicolesteroles/metabolismo , Ligandos , Unión Proteica , Receptores de Esteroides/metabolismo , Relación Estructura-ActividadRESUMEN
The development of precision drugs for the selective treatment of ovarian cancer will require targeting proliferative factors selectively expressed in ovarian tumors or targeting unique physiological microenvironments specific for ovarian tumors. Here, we report that oxysterol-binding protein (OSBP)-related protein 4 (ORP4) is a potential druggable precision target in ovarian cancer cells. ORP4 has limited expression in normal tissues and was recently recognized to be a cancer-specific driver of cellular proliferation, including in patient-isolated leukemias. We demonstrate that ORP4 is strongly expressed in a panel of ovarian cancer cell lines. The antiproliferative natural product compound OSW-1 targets ORP4 and OSBP. Our results demonstrate that the OSW-1 compound has high antiproliferative potency in both monolayer and three-dimensional ovarian cancer spheroid models, especially compared to the standard-of-care agents cisplatin and paclitaxel. OSW-1 compound treatment induces a loss of ORP4 expression after 48 h, which is coincident with the cytotoxic effects of OSW-1. The absence of extracellular lipids markedly potentiated the cytotoxicity of OSW-1, which was reversed by addition of extracellular free cholesterol. OSBP, but not ORP4, is reported to transport cholesterol and other lipids between organelles. Our results indicate that the targeting of ORP4 is responsible for the antiproliferative activity of the OSW-1 compound, but that in the absence of exogenously supplied cholesterol, which might be similar to the in vivo ovarian cancer microenvironment, possible OSW-1 targeting of OSBP further potentiates the anticancer activity of the compound. Overall, ORP4 and potentially OSBP are revealed as potential druggable targets for the development of novel treatments for ovarian cancer.
RESUMEN
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.
Asunto(s)
Bronquios/inmunología , Bronquios/virología , Inmunidad Innata , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/virología , SARS-CoV-2/inmunología , Anciano , Envejecimiento/inmunología , Bronquios/citología , Bronquios/metabolismo , COVID-19/inmunología , Muerte Celular/genética , Células Cultivadas , Senescencia Celular/genética , Citocinas/biosíntesis , Citocinas/genética , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Femenino , Humanos , Inflamación , Interferones/biosíntesis , Interferones/genética , Masculino , RNA-Seq , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , SARS-CoV-2/fisiología , Transducción de Señal/genéticaRESUMEN
In clinical cancer medicine, the current inability to quantify intracellular chemotherapy drug concentrations in individual human cells limits the personalization and overall effectiveness of drug administration. New bioanalytical methods capable of real-time measurement of drug levels in live single cancer cells would allow for more adaptive and personalized administration of chemotherapy drugs, potentially leading to better clinical outcomes with fewer side effects. In this study, we report the development of a new quantitative single cell mass spectrometry (qSCMS) method capable of providing absolute drug amounts and concentrations in single cancer cells. Using this qSCMS system, quantitative analysis of the intracellular drug gemcitabine present in individual bladder cancer cells is reported, including in bladder cancer cells isolated from patients undergoing standard-of-care gemcitabine chemotherapy. The development of single cell pharmacology bioanalytical methods can potentially lead to more effective and safely administered drug medications in patients, especially in the treatment of cancer.
RESUMEN
Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.
Asunto(s)
Espectrometría de Masas/instrumentación , Análisis de la Célula Individual/instrumentación , Humanos , Células K562RESUMEN
Oxysterol-binding protein (OSBP) is a lipid transport and regulatory protein required for the replication of Enterovirus genus viruses, which includes many significant human pathogens. Short-term exposure (i.e., 1-6 h) to a low dose (i.e., 1 nM) of the natural product compound OSW-1 induces a reduction of cellular OSBP levels by â¼90% in multiple different cell lines with no measurable cytotoxicity, defect in cellular proliferation, or global proteome reduction. Interestingly, the reduction of OSBP levels persists multiple days after the low-dose, transient OSW-1 compound treatment is ended and the intracellular OSW-1 compound levels drop to undetectable levels. The reduction in OSBP levels is inherited in multiple generations of cells that are propagated after the OSW-1 compound treatment is stopped. The enduring multiday, multigenerational reduction of OSBP levels triggered by the OSW-1 compound is not due to proteasome degradation of OSBP or due to a reduction in OSBP mRNA levels. OSW-1 compound treatment induces transient autophagy in cells, but blocking autophagy does not rescue OSBP levels. Although the specific cellular mechanism of long-term OSBP repression is not yet identified, these results clearly show the existence of an OSBP specific cellular regulation process that is triggered upon treatment with an OSBP-binding compound. The stable reduction of OSBP levels upon short-term, transient OSW-1 compound treatment will be a powerful tool to understand OSBP regulation and cellular function. Additionally, the persistent reduction in OSBP levels triggered by the transient OSW-1 compound treatment substantially reduces viral replication in treated cells. Therefore, the long-term, compound-induced reduction of OSBP in cells presents a new route to broad spectrum anti- Enterovirus activity, including as a novel route to antiviral prophylactic treatment through small molecule targeting a human host protein.
Asunto(s)
Antivirales/farmacología , Enterovirus/efectos de los fármacos , Receptores de Esteroides/química , Replicación Viral/efectos de los fármacos , Antivirales/administración & dosificación , Relación Dosis-Respuesta a Droga , Enterovirus/metabolismo , Enterovirus/fisiología , Humanos , Proteínas Virales/metabolismoRESUMEN
Oxysterol-binding Protein (OSBP) is a human lipid-transport protein required for the cellular replication of many types of viruses, including several human pathogens. The structurally-diverse small molecule compounds OSW-1, itraconazole (ITZ), T-00127-HEV2 (THEV) and TTP-8307 (TTP) inhibit viral replication through interaction with the OSBP protein. The OSW-1 compound reduces intracellular OSBP, and the reduction of OSBP protein levels persists multiple days after the OSW-1-compound treatment is stopped. The OSW-1-induced reduction of OSBP levels inhibited Enterovirus replication prophylactically in cells. In this report, the OSBP-interacting compounds ITZ, THEV, and TTP are shown not to reduce OSBP levels in cells, unlike the OSW-1-compound, and the OSW-1 compound is determined to be the only compound capable of providing prophylactic antiviral activity in cells. Furthermore, OSW-1 and THEV inhibit the binding of 25-hydroxycholesterol (25-OHC) to OSBP indicating that these compounds bind at the conserved sterol ligand binding site. The ITZ and TTP compounds do not inhibit 25-hydroxycholesterol binding to OSBP, and therefore ITZ and TTP interact with OSBP through other, unidentified binding sites. Co-administration of the THEV compound partially blocks the cellular activity of OSW-1, including the reduction of cellular OSBP protein levels; co-administration of the ITZ and TTP compounds have minimal effect on OSW-1 cellular activity further supporting different modes of interaction with these compounds to OSBP. OSW-1, ITZ, THEV, and TTP treatment alter OSBP cellular localization and levels, but in four distinct ways. Co-administration of OSW-1 and ITZ induced OSBP cellular localization patterns with features similar to the effects of ITZ and OSW-1 treatment alone. Based on these results, OSBP is capable of interacting with multiple structural classes of antiviral small molecule compounds at different binding sites, and the different compounds have distinct effects on OSBP cellular activity.
Asunto(s)
Antivirales/farmacología , Enterovirus/efectos de los fármacos , Receptores de Esteroides/antagonistas & inhibidores , Receptores de Esteroides/metabolismo , Replicación Viral/efectos de los fármacos , Línea Celular , Células HEK293 , Células HeLa , Humanos , Hidroxicolesteroles/metabolismo , Unión ProteicaRESUMEN
One bond and a water molecule separate title compound 1 from the potently bioactive peptide metabolite diazonamide A. Compositionally similar, yet topographically distinct, diazonamide A and 1 are both toxic towards cultured human cancer cells although the mechanisms underlying their actions likely differ. The quest towards completely synthetic diazonamides continues.
Asunto(s)
Factores Biológicos/síntesis química , Compuestos Heterocíclicos de 4 o más Anillos/síntesis química , Oxazoles/síntesis química , Factores Biológicos/química , Compuestos Heterocíclicos de 4 o más Anillos/química , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Oxazoles/químicaRESUMEN
We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through a unique mechanism. A biotinylated form of diazonamide A affinity purifies ornithine delta-amino transferase (OAT), a mitochondrial enzyme, from HeLa cell and Xenopus egg extracts. In the latter system, the interaction between diazonamide A and OAT is regulated by RanGTP. We find that specific OAT knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death, the effects largely phenocopying diazonamide A treatment in these cell lines. Our experiments reveal an unanticipated, paradoxical role for OAT in mitotic cell division and identify the protein as a target for chemotherapeutic drug development.