RESUMEN
Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar-simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization, and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1, and MMP-9. This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.
Asunto(s)
Carcinogénesis/metabolismo , Carcinoma de Células Escamosas/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Neoplasias Cutáneas/enzimología , Animales , Valerato de Betametasona/farmacología , Carcinogénesis/efectos de los fármacos , Carcinoma de Células Escamosas/patología , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones Pelados , Rocuronio/farmacología , Neoplasias Cutáneas/patología , Rayos Ultravioleta/efectos adversosRESUMEN
The biological functions of the p53-related protein kinase (PRPK) remain unclear. We have previously demonstrated that PRPK is phosphorylated by the T-LAK cell-originated protein kinase (TOPK) and that phosphorylated PRPK (p-PRPK) promotes colon cancer metastasis. Here, we analyzed colon adenocarcinomas from 87 patients and found that higher expression levels of p-PRPK were associated with later stages of metastatic dissemination (stage III and IV) as compared with earlier stages (stages I and II). Indeed, levels of p-PRPK were higher in metastatic versus malignant human colon adenocarcinomas. Knocking down PRPK expression attenuated colorectal liver and lung metastasis of colon cancer cells in vivo An in vitro kinase assay indicated that active PRPK does not phosphorylate p53 directly. We found that PRPK phosphorylates survivin, a regulator of colon cancer metastasis. PRPK phosphorylates survivin at Thr34, which is important for survivin stability. Taken together, our data strongly suggest that the PRPK signaling pathway promotes colon cancer metastasis by modulating survivin stability, and that PRPK could be a new prognostic marker for the survival of colon cancer patients. In addition, we identified an FDA-approved bacteriostatic antibiotic, fusidic acid sodium salt (fusidic acid or FA) as an inhibitor of PRPK, and show that FA combined with 5-fluorouracil (5-FU) inhibited PRPK activity and colon cancer metastasis to the lung in mice. We contend that the combination of FA with 5-FU could be an alternative therapeutic strategy to traditional chemotherapy for colon cancer patients with poor prognosis. Mol Cancer Ther; 17(5); 1101-13. ©2018 AACR.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Fluorouracilo/administración & dosificación , Ácido Fusídico/administración & dosificación , Células HCT116 , Células HT29 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Mutación , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/genética , Neoplasias Experimentales/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Survivin/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a representative J-series cyclopentenone prostaglandin, has biphasic roles in cell proliferation and apoptosis. Hypoxia inducible factor-1 (HIF-1) regulates expression of various genes involved in tumor growth and angiogenesis. In the present study, treatment of human breast cancer (MCF-7) cells with 15d-PGJ2 resulted in the accumulation of the α-subunit of HIF-1. Pretreatment with zinc protoporphyrin IX, a pharmacological inhibitor of heme oxygenase-1 (HO-1), as well as siRNA knockdown of HO-1 gene in MCF-7 cells attenuated 15d-PGJ2-mediated HIF-1α accumulation. 15d-PGJ2 treatment increased intracellular production of reactive oxygen species (ROS), which was mediated by HO-1 induction. Preincubation of MCF-7 cells with trolox, a water-soluble form of vitamin E, attenuated 15d-PGJ2-induced HIF-1α expression although HO-1 expression was unchanged. This finding suggests that ROS accumulated as a consequence of HO-1 up-regulation can enhance HIF-1α expression in MCF-7 cells treated with 15d-PGJ2. Alternatively, 15d-PGJ2 was found to covalently bind to HIF-1α prolyl-4-hydroxylase 2 (PHD2) in MCF-7 cells, which hampers the proline hydroxylation of HIF-1α, thereby disrupting ubiquitin-dependent proteasomal degradation of this transcription factor. Pretreatment with thiol reducing agents blunted 15d-PGJ2-induced HIF-1α stabilization, indicative of a cysteine residue as a direct target of 15d-PGJ2. Molecular docking analysis suggests that 15d-PGJ2 preferentially binds to PHD2 in the vicinity of the Cys201 residue based on binding energies and carbon-sulfur distances. In summary, 15d-PGJ2 stabilizes HIF-1α in MCF-7 cells through HO-1 induction with subsequent ROS generation and also through direct modification of PHD2.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolil Hidroxilasas/metabolismo , Hemo-Oxigenasa 1/metabolismo , HumanosRESUMEN
Although polyazine extractants have been extensively studied as agents for partitioning trivalent actinides from lanthanides, an explanation for why certain azine compositions succeed and others fail is lacking. To address this issue, density functional theory calculations were used to evaluate fundamental properties (intrinsic binding affinity for a representative trivalent f-block metal, basicity, and hardness) for prototype azine donors pyridine, pyridazine, pyrimidine, pyrazine, 1,2,3-triazine, 1,2,4-triazine, and 1,3,5-triazine, as well as perform conformational analyses of bisazine chelates formed by directly connecting two donors together. The results provide criteria that both rationalize the behavior of known extractants, TERPY, TPTZ, hemi-BTP, BTP, BTBP, and BTPhen, and predict a new class of extractants based on pyridazine donor groups.
RESUMEN
Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.
Asunto(s)
Antibacterianos/farmacología , Ceftriaxona/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Aurora Quinasa B , Aurora Quinasas , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , RatonesRESUMEN
Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived compounds are now a major area of investigation. Thousands of phytochemicals have been isolated, and a large number of them have shown protective activities or effects in different disease models. Using conventional approaches to select the best single or group of best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by phytochemicals. Computational methods involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel phytochemicals.
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Anticarcinógenos/metabolismo , Anticarcinógenos/farmacología , Biología Computacional , Flavonoides/metabolismo , Flavonoides/farmacología , Terapia Molecular Dirigida , Anticarcinógenos/química , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Flavonoides/química , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Conformación Proteica , Interfaz Usuario-ComputadorRESUMEN
Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins ßTrCP1 or ßTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Animales , Línea Celular , Células Madre Embrionarias/citología , Humanos , Factor 4 Similar a Kruppel , Sistema de Señalización de MAP Quinasas , Ratones , Modelos Moleculares , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Especificidad por SustratoRESUMEN
Indole-3-carbinol (I3C) is produced in Brassica vegetables such as broccoli and cabbage and has been shown to inhibit proliferation and induce apoptosis in various cancer cells, including breast, prostate, colon, and leukemia. However, only high doses of I3C were shown to inhibit cell proliferation (IC(50) = 200-300 µmol/L). Our goal here was to develop a more potent antitumor agent by modifying the structure of I3C. We created I3C derivatives and found that (3-chloroacetyl)-indole (3CAI) more strongly inhibited colon cancer cell growth than I3C. In addition, by screening 85 kinases in a competitive kinase assay, we found that 3CAI was a specific AKT inhibitor. AKT is a serine/threonine kinase that plays a pivotal role in promoting transformation and chemoresistance by inducing proliferation and inhibiting apoptosis. Therefore, AKT is regarded as a critical target for cancer therapy. 3ICA, a derivative of I3C, is a potent and specific AKT inhibitor. This compound showed significant inhibition of AKT in an in vitro kinase assay and suppressed expression of AKT direct downstream targets such as mTOR and GSK3ß as well as induced growth inhibition and apoptosis in colon cancer cells. In addition, oral administration of this potent AKT inhibitor suppressed cancer cell growth in an in vivo xenograft mouse model.
Asunto(s)
Regulación Alostérica , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Indoles/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Adhesión Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Humanos , Técnicas In Vitro , Ratones , Ratones Desnudos , Modelos Moleculares , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Thorough conformational analyses have been performed on representative sulfur, oxygen, and nitrogen mustards. A total of 23, 18, and 38 unique conformers have been located for SM, OM, and NM, respectively, at the MP2/aug-cc-pVDZ level of theory. Despite the fact that these molecules differ only in the identity of the central heteroatom, comparison of their low energy conformations reveals that the shapes they adopt are distinctive to each molecule. Potential energy surfaces for CH(2)-X (X = S, O, and N-CH(3)) and CH(2)-CH(2) bond rotations are presented and, where possible, compared with dihedral angle distributions observed in crystal structure data. These results were used to benchmark and improve the performance of the MM3 and MMFF94 force fields.
RESUMEN
The glycosphingolipid α-GalCer has been found to influence mammalian immune system significantly through the natural killer T cells. Unfortunately, the pre-clinical and clinical studies revealed several critical disadvantages that prevented the therapeutic application of α-GalCer in treating cancer and other diseases. Recently, the detailed illustration of the CD1d/α-GalCer/NKT TCR complex crystal structural, together with other latest structural and biological understanding on glycolipid ligands and NKT cells, provided a new platform for developing novel glycolipid ligands with optimized therapeutic effects. Here, we designed a series of novel aromatic group substituted α-GalCer analogues. The biological activity of these analogues was characterized and the results showed the unique substitution group manipulated the immune responses of NKT cells. Computer modeling and simulation study indicated the analogues had unique binding mode when forming CD1d/glycolipid/NKT TCR complex, comparing to original α-GalCer.
Asunto(s)
Citocinas/efectos de los fármacos , Galactosilceramidas/química , Células T Asesinas Naturales/inmunología , Antígenos CD1d/metabolismo , Citocinas/biosíntesis , Galactosilceramidas/farmacología , Glucolípidos , Humanos , Sistema Inmunológico/efectos de los fármacos , Ligandos , Células T Asesinas Naturales/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
RSK2 is a widely expressed serine/threonine kinase, and its activation enhances cell proliferation. Here, we report that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation. RSK2 phosphorylated ATF1 at Ser-63 and enhanced ATF1 transcriptional activity. Docking experiments using the crystal structure of the RSK2 N-terminal kinase domain combined with in vitro pulldown assays demonstrated that eriodictyol, a flavanone found in fruits, bound with the N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal kinase activity. In cells, eriodictyol inhibited phosphorylation of ATF1 but had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically suppresses RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation and tumor promoter-induced transformation of JB6 Cl41 cells. Eriodictyol or knockdown of RSK2 or ATF1 also suppressed Ras-mediated focus formation. Overall, these results indicate that RSK2-ATF1 signaling plays an important role in neoplastic cell transformation and that eriodictyol is a novel natural compound for suppressing RSK2 kinase activity.
Asunto(s)
Factor de Transcripción Activador 1/metabolismo , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Flavanonas/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Activador 1/genética , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Factor de Crecimiento Epidérmico/metabolismo , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Estructura Terciaria de Proteína , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Transducción de Señal/genética , Activación Transcripcional/efectos de los fármacos , Proteínas rasRESUMEN
The anticancer effects of red wine have attracted considerable attention. Resveratrol (3,5,4'-trihydroxy-trans -stilbene) is a well-known polyphenolic compound of red wine with cancer chemopreventive activity. However, the basis for this activity is unclear. We studied leukotriene A(4) hydrolase (LTA(4)H) as a relevant target in pancreatic cancer. LTA(4)H knockdown limited the formation of leukotriene B(4) (LTB(4)), the enzymatic product of LTA(4)H, and suppressed anchorage-independent growth of pancreatic cancer cells. An in silico shape similarity algorithm predicted that LTA(4)H might be a potential target of resveratrol. In support of this idea, we found that resveratrol directly bound to LTA(4)H in vitro and in cells and suppressed proliferation and anchorage-independent growth of pancreatic cancer by inhibiting LTB(4) production and expression of the LTB(4) receptor 1 (BLT(1)). Notably, resveratrol exerted relatively stronger inhibitory effects than bestatin, an established inhibitor of LTA(4)H activity, and the inhibitory effects of resveratrol were reduced in cells where LTA(4)H was suppressed by shRNA-mediated knockdown. Importantly, resveratrol inhibited tumor formation in a xenograft mouse model of human pancreatic cancer by inhibiting LTA(4)H activity. Our findings identify LTA(4)H as a functionally important target for mediating the anticancer properties of resveratrol.
Asunto(s)
Epóxido Hidrolasas/antagonistas & inhibidores , Neoplasias Pancreáticas/prevención & control , Estilbenos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/química , Epóxido Hidrolasas/genética , Flavonoides/metabolismo , Flavonoides/farmacología , Células Hep G2 , Humanos , Leucotrieno B4/metabolismo , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fenoles/metabolismo , Fenoles/farmacología , Polifenoles , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Interferencia de ARN , Resveratrol , Estilbenos/metabolismo , Factores de Tiempo , VinoRESUMEN
The human CD1d protein presents a wide range of lipids to the TCR of invariant natural killer T cells (iNKT). Alpha-GalCer is one of the most potent iNKT stimulatory ligands presented by CD1d. The lipid portion of this ligand has been extensively investigated over the course of the past few years; however, the sugar portion of the ligand has received minimal attention. The following research focuses on computationally analyzing the recently crystallized CD1d/alpha-GalCer/TCR tertiary complex by molecular dynamics simulations using AMBER along with studying the structure activity relationship of the sugar headgroup also by simulation and docking using Autodock for a variety of alpha-GalCer analogs. The results show that the crystal structure is stable under simulation making it an accurate representation of the CD1d/alpha-GalCer/TCR complex and that modifications to the C2' and C3' positions of the sugar are not tolerated by the tertiary complex, whereas modifications to the C4' position are tolerated.
Asunto(s)
Antígenos CD1d/química , Glucolípidos/química , Receptores de Antígenos de Linfocitos T/química , Enlace de Hidrógeno , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Invariant natural killer T (iNKT) cells are innate T lymphocytes that express T cell receptors binding to exogenous and endogenous glycosphingolpid antigens presented by a nonpolymorphic, non-MHC antigen presenting molecule, CD1d. The endogenous glycosphingolipid metabolite, isoglobotrihexosylceramide (iGb3), is the first known natural ligand for both human and mouse iNKT cells, whose activity has been confirmed in a variety of iNKT cell clones generated by different investigators, representing the majority of the iNKT cell population. The signaling pathway mediated by T cell receptor is largely influenced by the structural variation of glycosphingolpid antigens, leading to multiple and varied biological functions of iNKT cells. In order to investigate the structural requirements behind iGb3 triggered iNKT cell activation, the structure-activity relationship (SAR) of iGb3 needs to be characterized. In this study, iGb3 analogues containing 2' '', 3' '', 4' '' and 6' '' deoxy terminal galactose were synthesized for probing the SAR between iGb3 and TCR. The biological assays on the synthetic iGb3 analogues were performed with use of the murine iNKT cell hybridoma DN32.D3. The results showed that the 2' '' and 3' '' hydroxyl groups of terminal galactose play more important roles for the recognition of iGb3 by TCR; while 4' '' and 6' '' hydroxyl groups were not as crucial for this recognition. These studies might help to understand the general structural requirements for natural endogenous ligands recognized by iNKT cells.
Asunto(s)
Globósidos/síntesis química , Globósidos/farmacología , Hibridomas/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/efectos de los fármacos , Trihexosilceramidas/síntesis química , Trihexosilceramidas/farmacología , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Globósidos/química , Hibridomas/inmunología , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Relación Estructura-Actividad , Trihexosilceramidas/químicaRESUMEN
S-Nitrosothiols (RSNOs) are important exogenous and endogenous sources of nitric oxide (NO) in biological systems. A series of 4-aryl-1,3,2-oxathiazolylium-5-olates derivatives with varying aryl para-substituents (-CF3, -H, -Cl, and -OCH3) were synthesized. These compounds were found to release NO under acidic condition (pH = 5). The decomposition pathway of the aryloxathiazolyliumolates proceeded via an acid-catalyzed ring-opening mechanism after which NO was released and an S-centered radical was generated. Electron paramagnetic resonance (EPR) spin trapping studies were performed to detect NO and the S-centered radical using the spin traps of iron(II) N-methyl-D-glucamine dithiocarbamate [(MGD)2-FeII] and 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Also, EPR spin trapping and UV-vis spectrophotometry were used to analyze the effect of aryl para substitution on the NO-releasing property of aryloxathiazolyliumolates. The results showed that the presence of an electron-withdrawing substituent such as -CF3 enhanced the NO-releasing capability of the aryloxathiazolyliumolates, whereas an electron-donating substituent like methoxy (-OCH3) diminished it. Computational studies using density functional theory (DFT) at the PCM/B3LYP/6-31+G**//B3LYP/6-31G* level were used to rationalize the experimental observations. The aryloxathiazolyliumolates diminished susceptibility to reduction by ascorbate or gluthathione, and their capacity to cause vasodilation as compared to other S-nitrosothiols suggests potential application in biological systems.
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Donantes de Óxido Nítrico/química , Oxazoles/química , S-Nitrosotioles/química , Tiazoles/química , Vasodilatadores/química , Animales , Fenómenos Químicos , Química Física , Biología Computacional , Electroquímica , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Indicadores y Reactivos , Modelos Moleculares , Óxido Nítrico/química , Donantes de Óxido Nítrico/síntesis química , Donantes de Óxido Nítrico/farmacología , Oxazoles/síntesis química , Oxazoles/farmacología , Ratas , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría Ultravioleta , Termodinámica , Tiazoles/síntesis química , Tiazoles/farmacología , Vasodilatadores/síntesis química , Vasodilatadores/farmacologíaRESUMEN
Anthracyclines are widely used in patients for anticancer activity. However, one of the limitations for their clinical use is P-gp-mediated drug resistance in cancer therapy. We hypothesize that modified anthracyclines will retain their anticancer activity, avert P-gp binding, and thus overcome P-gp-mediated drug resistance. Twenty-five daunorubicin analogues were synthesized with slight structure modifications in sugar moieties. Molecular docking, cytotoxicity, and P-gp inhibition assays in drug-resistant leukemia cells (K562/Dox) were used to identify several candidates that avert binding to multidrug-resistant protein (MsbA) and overcome drug resistance. Molecular docking showed that daunorubicin bound to the cavity between the intracellular domain (ICD) and nucleoside binding domain (NBD) of MsbA, which might be the "entry site" for the transport of its substrate. The molecular docking accurately predicted the substrates of multidrug-resistant protein. Several aspects are important for daunorubicin analogue binding to MsbA: (1) the substitution pattern and stereochemistry of the tetracyclic ring and sugar moiety; (2) the hydrogen bond donor or acceptor capability of the substituent at C'-3 and C'-4. Molecular docking, cytotoxicity, and P-gp inhibition assays identified ADNR, ADNR-1, and ADNR-3 for averting P-gp binding and overcoming drug resistance. The replacement of C'-3-NH2 with azido group in daunorubicin not only abolishes the hydrogen bond between the sugar moiety and MsbA but also completely changes the overall binding conformation, and thus averts the binding to MsbA. Cytotoxicity assays confirmed that these compounds showed high sensitivity against drug-resistant cancer cells (K562/Dox) with P-gp overexpression. P-gp inhibition assay indeed confirms that these appropriately modified compounds avert P-gp binding and thus overcome P-gp-mediated drug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Metabolismo de los Hidratos de Carbono , Daunorrubicina/metabolismo , Resistencia a Múltiples Medicamentos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Ciclosporina/farmacología , Citotoxicidad Inmunológica , Daunorrubicina/análogos & derivados , Daunorrubicina/química , Disacáridos/metabolismo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Concentración 50 Inhibidora , Células K562 , Monosacáridos/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Anthracyclines are considered to be some of the most effective anticancer drugs for cancer therapy. However, drug resistance and cardiotoxicity of anthracyclines limit their clinical application. We hypothesize that direct modifications of the sugar moiety of anthracyclines avert P-glycoprotein (P-gp) recognition and efflux, increase drug intracellular concentration in cancer cells, and thus overcome P-gp-mediated drug resistance. Daunorubicin (DNR) analogues with sugar modifications were synthesized by directly transforming the amino group of DNR to an azido group or triazole group. Molecular docking showed that the lead compound (3'-azidodaunorubicin, ADNR) averts P-gp binding, while daunorubicin (DNR) extensively interacts with multidrug-resistance (MDR) protein through H-bonds and electrostatic interactions. FACS assay demonstrated that these new compounds abolished P-gp drug efflux and accumulated high intracellular concentration in the drug-resistant leukemia K562/Dox. P-gp inhibition by CsA confirmed that these new analogues are no longer P-gp substrates. ADNR exhibited potent anticancer activity in both drug-sensitive (K562) and drug-resistant leukemia cells (K562/Dox), with a 25-fold lower drug resistance index than DNR. An in vivo xenograft model demonstrated that ADNR showed more than 2.5-fold higher maximum growth inhibition rate against drug-resistant cancers and significant improvement for animal survival rate versus DNR. No significant body weight reduction in mice was observed for ADNR at the maximum tolerable dose, as compared to more than 70% body weight reduction for DNR. These data suggest that sugar modifications of anthracyclines avert P-gp binding, abolish P-gp-mediated drug efflux, increase intracellular drug concentration, and thus overcome P-gp-mediated drug resistance in cancer therapy.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Antineoplásicos/síntesis química , Azidas/síntesis química , Daunorrubicina/análogos & derivados , Daunorrubicina/síntesis química , Resistencia a Antineoplásicos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Animales , Antineoplásicos/farmacología , Azidas/química , Azidas/farmacología , Línea Celular Tumoral , Daunorrubicina/química , Daunorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Conformación Molecular , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
Anthracyclines have received significant attention due to their effectiveness and extensive use as anticancer agents. At present, the clinical use of these drugs is offset by drug resistance in tumours and cardiotoxicity. Therefore, a relentless search for the 'better anthracycline' has been ongoing since the inception of these drugs > 30 years ago. This review focuses on the most recent pharmacology and medicinal chemistry developments on the design and use of anthracyclines. Based on their crystal structures as well as molecular modelling, a more detailed mechanism of topoisomerase poisoning by these new anthracyclines has emerged. Chemical modifications of anthracyclines have been found to possibly change the target selectivity among various topoisomerases and, thus, vary their anticancer activity. Additionally, recent sugar modifications of anthracyclines have also been found to overcome P-glycoprotein-mediated drug resistance in cancer therapy. The continued improvement of anthracycline clinical applications so far and the clinical trials of the 'third generation' of anthracyclines (such as sabarubicin) are also discussed. To finally find the 'better' anthracycline, further areas of research still need to be explored such as: the elucidation of the topoisomerase and P-glycoprotein crystal structures, molecular modelling based on crystal structure in order to design the next generation of better anthracycline drugs, the continued modifications of the anthracycline sugar moieties, and the further improvement of anthracycline drug delivery methods.
RESUMEN
N-Acetyl-D-neuraminic acid (NeuNAc) aldolase is an important enzyme for the metabolic engineering of cell-surface NeuNAc using chemically modified D-mannosamines. To explore the optimal substrates for this application, eight N-acyl derivatives of D-mannosamine were prepared, and their accessibility to NeuNAc aldolase was quantitatively investigated. The N-propionyl-, N-butanoyl-, N-iso-butanoyl-, N-pivaloyl-, and N-phenylacetyl-D-mannosamines proved to be as good substrates as, or even better than, the natural N-acetyl-D-mannosamine, while the N-trifluoropropionyl and benzoyl derivatives were poor. It was proposed that the electronic effects might have a significant influence on the enzymatic aldol condensation reaction of D-mannosamine derivatives, with electron-deficient acyl groups having a negative impact. The results suggest that N-propionyl-, N-butanoyl-, N-iso-butanoyl-, and N-phenylacetyl-D-mannosamines may be employed to bioengineer NeuNAc on cells.