RESUMEN
Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.
Asunto(s)
Adenosina Monofosfato/metabolismo , Dominio Catalítico , Procesamiento Proteico-Postraduccional , Selenoproteínas/metabolismo , Secuencia Conservada , Humanos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Selenoproteínas/químicaRESUMEN
Marine-derived fungi have emerged as a source for novel metabolites with a broad range of bioactivities. However, accessing the full potential of fungi under standard laboratory conditions remains challenging. LC-MS-based metabolomics in combination with varied culture conditions is a fast and powerful tool to detect new metabolites. Here, three developmental forms of the marine-derived fungus Aspergillus alliaceus were analyzed and 14 fungal metabolites, including new brominated polyketides (11-14) were isolated. Structure elucidation relied mainly on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry and DFT-based computations. We sequenced the A. alliaceus genome, identified the bianthrone-producing biosynthetic gene cluster, and conducted expression analysis on genes involved in sexual development and biosynthesis. The NCI-60 cell line panel revealed selective in vitro activity against triple-negative breast cancer (TNBC) for the halogenated allianthrones and their full anti-proliferative and cytotoxic effects were evaluated in five TNBC cell lines.
RESUMEN
Ovarian cancer often has a poor clinical prognosis because of late detection, frequently after metastatic progression, as well as acquired resistance to taxane-based therapy. Herein, we evaluate a novel class of covalent microtubule stabilizers, the C-22,23-epoxytaccalonolides, for their efficacy against taxane-resistant ovarian cancer models in vitro and in vivo. Taccalonolide AF, which covalently binds ß-tubulin through its C-22,23-epoxide moiety, demonstrates efficacy against taxane-resistant models and shows superior persistence in clonogenic assays after drug washout due to irreversible target engagement. In vivo, intraperitoneal administration of taccalonolide AF demonstrated efficacy against the taxane-resistant NCI/ADR-RES ovarian cancer model both as a flank xenograft, as well as in a disseminated orthotopic disease model representing localized metastasis. Taccalonolide-treated animals had a significant decrease in micrometastasis of NCI/ADR-RES cells to the spleen, as detected by quantitative RT-PCR, without any evidence of systemic toxicity. Together, these findings demonstrate that taccalonolide AF retains efficacy in taxane-resistant ovarian cancer models in vitro and in vivo and that its irreversible mechanism of microtubule stabilization has the unique potential for intraperitoneal treatment of locally disseminated taxane-resistant disease, which represents a significant unmet clinical need in the treatment of ovarian cancer patients.
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Hidrocarburos Aromáticos con Puentes/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neoplasias Ováricas/tratamiento farmacológico , Esteroides/farmacología , Taxoides/farmacología , Moduladores de Tubulina/farmacología , Animales , Apoptosis , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Micrometástasis de Neoplasia , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The taccalonolides are a class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance due to their unique mechanism of microtubule stabilization imparted by the covalent binding of the C-22-C-23 epoxide moiety to tubulin. A taccalonolide (8) with a fluorescein group attached with a linker at C-6 was generated, and biochemical and cell-based assays showed that it bound directly to tubulin and stabilized microtubules. This pharmacological probe has allowed, for the first time, a direct visualization of a taccalonolide binding to microtubules, verifying their cellular binding site. This C-6-modified taccalonolide showed potency comparable to the untagged compound in biochemical experiments; however, its potency was lower in cellular assays, presumably due to decreased cellular permeability. These studies provide a valuable tool to facilitate the further understanding of taccalonolide pharmacology and demonstrate that C-6 is a promising site for a linker to be added to this novel class of microtubule stabilizers for targeted drug delivery.
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Microtúbulos/efectos de los fármacos , Esteroides/química , Esteroides/farmacología , Moduladores de Tubulina/farmacología , Proliferación Celular/efectos de los fármacos , Células HeLa , Humanos , Estructura Molecular , Relación Estructura-Actividad , Moduladores de Tubulina/químicaRESUMEN
One unexploited family of cancer biomarkers comprise glycoproteins, carbohydrates, and glycolipids (the Tumor Glycocode).A class of glycolipid cancer biomarkers, the tumor-marker gangliosides (TMGs) are presented here as potential diagnostics for detecting cancer, especially at early stages, as the biological function of TMGs makes them etiological. We propose that a quantitative matrix of the Cancer Biomarker Glycocode and artificial intelligence-driven algorithms will expand the menu of validated cancer biomarkers as a step to resolve some of the challenges in cancer diagnosis, and yield a combination that can identify a specific cancer, in a tissue-agnostic manner especially at early stages, to enable early intervention. Diagnosis is critical to reducing cancer mortality but many cancers lack efficient and effective diagnostic tests, especially for early stage disease. Ideal diagnostic biomarkers are etiological, samples are preferably obtained via non-invasive methods (e.g. liquid biopsy of blood or urine), and are quantitated using assays that yield high diagnostic sensitivity and specificity for efficient diagnosis, prognosis, or predicting response to therapy. Validated biomarkers with these features are rare. While the advent of proteomics and genomics has led to the identification of a multitude of proteins and nucleic acid sequences as cancer biomarkers, relatively few have been approved for clinical use. The use of multiplex arrays and artificial intelligence-driven algorithms offer the option of combining data of known biomarkers; however, for most, the sensitivity and the specificity are below acceptable criteria, and clinical validation has proven difficult. One strategic solution to this problem is to expand the biomarker families beyond those currently exploited. One unexploited family of cancer biomarkers comprise glycoproteins, carbohydrates, and glycolipids (the Tumor Glycocode). Here, we focus on a family of glycolipid cancer biomarkers, the tumor-marker gangliosides (TMGs). We discuss the diagnostic potential of TMGs for detecting cancer, especially at early stages. We include prior studies from the literature to summarize findings for ganglioside quantification, expression, detection, and biological function and its role in various cancers. We highlight the examples of TMGs exhibiting ideal properties of cancer diagnostic biomarkers, and the application of GD2 and GD3 for diagnosis of early stage cancers with high sensitivity and specificity. We propose that a quantitative matrix of the Cancer Biomarker Glycocode and artificial intelligence-driven algorithms will expand the menu of validated cancer biomarkers as a step to resolve some of the challenges in cancer diagnosis, and yield a combination that can identify a specific cancer, in a tissue-agnostic manner especially at early stages, to enable early intervention.
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We report a water-soluble poly(phenylene ethynylene) (PPE-Pt(IV)) that is functionalized with oxidized oxaliplatin Pt(IV) units and its use for photoactivated chemotherapy. The photoactivation strategy is based on photoinduced electron transfer from the PPE backbone to oxaliplatin Pt(IV) as an electron acceptor; this process triggers the release of oxaliplatin, which is a clinically used anticancer drug. Mechanistic studies carried out using steady-state and time-resolved fluorescence spectroscopy coupled with picosecond-nanosecond transient absorption support the hypothesis that electron transfer triggers the drug release. Photoactivation is effective, producing oxaliplatin with a good chemical yield in less than 1 h of photolysis (400 nm, 5 mW cm-2). Photorelease of oxaliplatin from PPE-Pt(IV) can also be effected with two-photon excitation by using 100 fs pulsed light at 725 nm. Cytotoxicity studies using SK-OV-3 human ovarian cancer cells demonstrate that without photoactivation PPE-Pt(IV) is not cytotoxic at concentrations up to 10 µM in polymer repeating unit (PRU) concentration. However, following a short period of 460 nm irradiation, oxaliplatin is released from PPE-Pt(IV), resulting in cytotoxicity at concentrations as low as 2.5 µM PRU.
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Antineoplásicos , Polímeros , Antineoplásicos/química , Antineoplásicos/farmacología , Humanos , Oxaliplatino/farmacología , Polímeros/química , Espectrometría de FluorescenciaRESUMEN
Microtubule stabilizers are a mainstay in the treatment of many solid cancers and continue to find utility in combination therapy with molecularly targeted anticancer agents and immunotherapeutics. However, innate and acquired resistance to microtubule stabilizers can limit their clinical efficacy. The taccalonolides are a unique class of microtubule stabilizers isolated from plants of Tacca that circumvent clinically relevant mechanisms of drug resistance. Although initial reports suggested that the microtubule-stabilizing activity of the taccalonolides was independent of direct tubulin binding, additional studies have identified that potent C-22, C-23 epoxidized taccalonolides covalently bind the Aspartate 226 residue of ß-tubulin and that this interaction is critical for their microtubule-stabilizing activity. The taccalonolides have distinct properties as compared to other microtubule stabilizers with regard to their biochemical effects on tubulin structure and dynamics that promote distinct cellular phenotypes. Some taccalonolides have demonstrated in vivo antitumor efficacy in drug-resistant tumor models with exquisite potency and long-lasting antitumor efficacy as a result of their irreversible target engagement. The recent identification of a site on the taccalonolide scaffold that is amenable to modification has provided evidence of the specificity of the taccalonolide-tubulin interaction. This also affords an opportunity to further optimize the targeted delivery of the taccalonolides to further improve their anticancer efficacy and potential for clinical development.
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Dioscoreaceae , Microtúbulos , Esteroides , Dioscoreaceae/química , Dioscoreaceae/metabolismo , Microtúbulos/efectos de los fármacos , Esteroides/química , Esteroides/farmacología , Relación Estructura-Actividad , Tubulina (Proteína)/metabolismoRESUMEN
The taccalonolide microtubule stabilizers covalently bind ß-tubulin and overcome clinically relevant taxane resistance mechanisms. Evaluations of the target specificity and detailed drug-target interactions of taccalonolides, however, have been limited in part by their irreversible target engagement. In this study, we report the synthesis of fluorogenic taccalonolide probes that maintain the native biological properties of the potent taccalonolide, AJ. These carefully optimized, cell-permeable probes outperform commercial taxane-based probes and enable direct visualization of taccalonolides in both live and fixed cells with dramatic microtubule colocalization. The specificity of taccalonolide binding to ß-tubulin is demonstrated by immunoblotting, which allows for determination of the relative contribution of key tubulin residues and taccalonolide moieties for drug-target interactions by activity-based protein profiling utilizing site-directed mutagenesis and computational modeling. This combinatorial approach provides a generally applicable strategy for investigating the binding specificity and molecular interactions of covalent binding drugs in a cellular environment.
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Microtúbulos/química , Esteroides/química , Línea Celular Tumoral , Humanos , Cinética , Microtúbulos/metabolismo , Modelos Moleculares , Esteroides/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Natural products have served as inspirational scaffolds for the design and synthesis of novel antineoplastic agents. Here we present our preliminary efforts on the synthesis and biological evaluation of a new class of electrophilic steroids inspired by the naturally occurring taccalonolides. We demonstrate that these simplified analogs exhibit highly persistent antiproliferative properties similar to the taccalonolides and retain activity against resistant cancer cell lines that warrants further preclinical development.
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RATIONALE: Androgen deprivation therapy (ADT) is an effective treatment for prostate cancer, but induces profound cognitive impairment. Little research has addressed mechanisms underlying these deficits or potential treatments. This is an unmet need to improve quality of life for prostate cancer survivors. OBJECTIVES: We investigated mechanisms of cognitive impairment after ADT in rats and potential utility of the multimodal serotonin-targeting drug, vortioxetine, to improve the impairment, as vortioxetine has specific efficacy against cognitive impairment in depression. METHODS: Male Sprague-Dawley rats were surgically castrated. Vortioxetine (28 mg/kg/day) was administered in the diet. The attentional set-shifting test was used to assess medial prefrontal cortex (mPFC) executive function. Afferent-evoked field potentials were recorded in the mPFC of anesthetized rats after stimulating the ventral hippocampus (vHipp) or medial dorsal thalamus (MDT). Gene expression changes were assessed by microarray. Effects of vortioxetine on growth of prostate cancer cells were assessed in vitro. RESULTS: ADT impaired cognitive set shifting and attenuated responses evoked in the mPFC by the vHipp afferent, but not the MDT. Both the cognitive impairment and attenuated vHipp-evoked responses were reversed by chronic vortioxetine treatment. Preliminary investigation of gene expression in the mPFC indicates that factors involved in neuronal plasticity and synaptic transmission were down-regulated by castration and up-regulated by vortioxetine in castrated animals. Vortioxetine neither altered the growth of prostate cancer cells in vitro nor interfered with the antiproliferative effects of the androgen antagonist, enzalutamide. CONCLUSIONS: These results suggest that vortioxetine may be useful in mitigating cognitive impairment associated with ADT for prostate cancer.