RESUMEN
Two new series of complexes with pyridine-containing Schiff bases, [VV O(SALIEP)L] and [VV O(Cl-SALIEP)L] (SALIEP=N-(salicylideneaminato)-2-(2-aminoethylpyridine; Cl-SALIEP=N-(5-chlorosalicylideneaminato)-2-(2-aminoethyl)pyridine, L=catecholato(2-) ligand) have been synthesized. Characterization by 1 H and 51 Vâ NMR and UV-Vis spectroscopies confirmed that: 1)â most complexes form two major geometric isomers in solution, and [VV O(SALIEP)(DTB)] (DTB=3,5-di-tert-butylcatecholato(2-)) forms two isomers that equilibrate in solution; and 2)â tert-butyl substituents were necessary to stabilize the reduced VIV species (EPR spectroscopy and cyclic voltammetry). The pyridine moiety within the Schiff base ligands significantly changed their chemical properties with unsubstituted catecholate ligands compared with the parent HSHED (N-(salicylideneaminato)-N'-(2-hydroxyethyl)-1,2-ethanediamine) Schiff base complexes. Immediate reduction to VIV occurred for the unsubstituted-catecholato VV complexes on dissolution in DMSO. By contrast, the pyridine moiety within the Schiff base significantly improved the hydrolytic stability of [VV O(SALIEP)(DTB)] compared with [VV O(HSHED)(DTB)]. [VV O(SALIEP)(DTB)] had moderate stability in cell culture media. There was significant cellular uptake of the intact complex by T98G (human glioblastoma) cells and very good anti-proliferative activity (IC50 6.7±0.9â µM, 72â h), which was approximately five times higher than for the non-cancerous human cell line, HFF-1 (IC50 34±10â µM). This made [VV O(SALIEP)(DTB)] a potential drug candidate for the treatment of advanced gliomas by intracranial injection.
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Antineoplásicos , Complejos de Coordinación , Glioblastoma , Compuestos Organometálicos , Humanos , Vanadio/química , Bases de Schiff/química , Compuestos Organometálicos/química , Glioblastoma/tratamiento farmacológico , Antineoplásicos/química , Piridinas/química , Espectroscopía de Resonancia por Spin del Electrón , Oxidación-Reducción , Ligandos , Complejos de Coordinación/farmacologíaRESUMEN
Confining water to nanosized spaces creates a unique environment that can change water's structural and dynamic properties. When ions are present in these nanoscopic spaces, the limited number of water molecules and short screening length can dramatically affect how ions are distributed compared to the homogeneous distribution assumed in bulk aqueous solution. Here, we demonstrate that the chemical shift observed in 19F NMR spectroscopy of fluoride anion, F-, probes the location of sodium ions, Na+, confined in reverse micelles prepared from AOT (sodium dioctyl sulfosuccinate) surfactants. Our measurements show that the nanoconfined environment of reverse micelles can lead to extremely high apparent ion concentrations and ionic strength, beyond the limit in bulk aqueous solutions. Most notably, the 19F NMR chemical shift trends we observe for F- in the reverse micelles indicate that the AOT sodium counterions remain at or near the interior interface between surfactant and water, thus providing the first experimental support for this hypothesis.
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Limited stability of most transition-metal complexes in biological media has hampered their medicinal applications but also created a potential for novel cancer treatments, such as intratumoral injections of cytotoxic but short-lived anticancer drugs. Two related V(V) complexes, [VO(Hshed)(dtb)] (1) and [VO(Hshed)(cat)] (2), where H2shed = N-(salicylideneaminato)-N'-(2-hydroxyethyl)-1,2-ethanediamine, H2dtb = 3,5-di-tert-butylcatechol, and H2cat = 1,2-catechol, decomposed within minutes in cell culture medium at 310 K (t1/2 = 43 and 9 s for 1 and 2, respectively). Despite this, both complexes showed high antiproliferative activities in triple-negative human breast cancer (MDA-MB-231) cells, but the mechanisms of their activities were radically different. Complex 1 formed noncovalent adducts with human serum albumin, rapidly entered cells via passive diffusion, and was nearly as active in a short-term treatment (IC50 = 1.9 ± 0.2 µM at 30 min) compared with a long-term treatment (IC50 = 1.3 ± 0.2 µM at 72 h). The activity of 1 decreased about 20-fold after its decomposition in cell culture medium for 30 min at 310 K. Complex 2 showed similar activities (IC50 ≈ 12 µM at 72 h) in both fresh and decomposed solutions and was inactive in a short-term treatment. The activity of 2 was mainly due to the reactions among V(V) decomposition products, free catechol, and O2 in cell culture medium. As a result, the activity of 1 was less sensitive than that of 2 to the effects of hypoxic conditions that are characteristic of solid tumors and to the presence of apo-transferrin that acts as a scavenger of V(V/IV) decomposition products in blood serum. In summary, complex 1, but not 2, is a suitable candidate for further development as an anticancer drug delivered via intratumoral injections. These results demonstrate the importance of fine-tuning the ligand properties for the optimization of biological activities of metal complexes.
Asunto(s)
Complejos de Coordinación , Compuestos Organometálicos , Humanos , Complejos de Coordinación/farmacología , Vanadio/farmacología , Compuestos Organometálicos/farmacología , Transferrina , Albúminas , Hipoxia , Catecoles/farmacologíaRESUMEN
Lipid peroxidation (LPO), a process that affects human health, can be induced by exposure to vanadium salts and compounds. LPO is often exacerbated by oxidation stress, with some forms of vanadium providing protective effects. The LPO reaction involves the oxidation of the alkene bonds, primarily in polyunsaturated fatty acids, in a chain reaction to form radical and reactive oxygen species (ROS). LPO reactions typically affect cellular membranes through direct effects on membrane structure and function as well as impacting other cellular functions due to increases in ROS. Although LPO effects on mitochondrial function have been studied in detail, other cellular components and organelles are affected. Because vanadium salts and complexes can induce ROS formation both directly and indirectly, the study of LPO arising from increased ROS should include investigations of both processes. This is made more challenging by the range of vanadium species that exist under physiological conditions and the diverse effects of these species. Thus, complex vanadium chemistry requires speciation studies of vanadium to evaluate the direct and indirect effects of the various species that are present during vanadium exposure. Undoubtedly, speciation is important in assessing how vanadium exerts effects in biological systems and is likely the underlying cause for some of the beneficial effects reported in cancerous, diabetic, neurodegenerative conditions and other diseased tissues impacted by LPO processes. Speciation of vanadium, together with investigations of ROS and LPO, should be considered in future biological studies evaluating vanadium effects on the formation of ROS and on LPO in cells, tissues, and organisms as discussed in this review.
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Sales (Química) , Vanadio , Humanos , Especies Reactivas de Oxígeno/farmacología , Peroxidación de Lípido , Vanadio/toxicidad , Sales (Química)/farmacología , Estrés OxidativoRESUMEN
A comprehensive understanding of the mechanisms involved in epigenetic changes in gene expression is essential to the clinical management of diseases linked to the SMYD family of lysine methyltransferases. The five known SMYD enzymes catalyze the transfer of donor methyl groups from S-adenosylmethionine (SAM) to specific lysines on histones and non-histone substrates. SMYDs family members have distinct tissue distributions and tissue-specific functions, including regulation of development, cell differentiation, and embryogenesis. Diseases associated with SMYDs include the repressed transcription of SMYD1 genes needed for the formation of ion channels in the heart leading to heart failure, SMYD2 overexpression in esophageal squamous cell carcinoma (ESCC) or p53-related cancers, and poor prognosis associated with SMYD3 overexpression in more than 14 types of cancer including breast cancer, colon cancer, prostate cancer, lung cancer, and pancreatic cancer. Given the importance of epigenetics in various pathologies, the development of epigenetic inhibitors has attracted considerable attention from the pharmaceutical industry. The pharmacologic development of the inhibitors involves the identification of molecules regulating both functional SMYD SET (Suppressor of variegation, Enhancer of Zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF1) domains, a process facilitated by available X-ray structures for SMYD1, SMYD2, and SMYD3. Important leads for potential pharmaceutical agents have been reported for SMYD2 and SMYD3 enzymes, and six epigenetic inhibitors have been developed for drugs used to treat myelodysplastic syndrome (Vidaza, Dacogen), cutaneous T-cell lymphoma (Zoinza, Isrodax), and peripheral T-cell lymphoma (Beleodag, Epidaza). The recently demonstrated reversal of SMYD histone methylation suggests that reversing the epigenetic effects of SMYDs in cancerous tissues may be a desirable target for pharmacological development.
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Epigénesis Genética , N-Metiltransferasa de Histona-Lisina , Humanos , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Factores de Transcripción/metabolismo , Neoplasias/genéticaRESUMEN
A hydrophobic Schiff base catecholate vanadium complex was recently discovered to have anticancer properties superior to cisplatin and suited for intratumoral administration. This [VO(HSHED)(DTB)] complex, where HSHED is N-(salicylideneaminato)-N'-(2-hydroxyethyl)-1,2-ethanediamine and the non-innocent catecholato ligand is di-t-butylcatecholato (DTB), has higher stability compared to simpler catecholato complexes. Three new chloro-substituted Schiff base complexes of vanadium(V) with substituted catecholates as co-ligands were synthesized for comparison with their non-chlorinated Schiff base vanadium complexes, and their properties were characterized. Up to four geometric isomers for each complex were identified in organic solvents using 51V and 1H NMR spectroscopies. Spectroscopy was used to characterize the structure of the major isomer in solution and to demonstrate that the observed isomers are exchanged in solution. All three chloro-substituted Schiff base vanadium(V) complexes with substituted catecholates were also characterized by UV-vis spectroscopy, mass spectrometry, and electrochemistry. Upon testing in human glioblastoma multiforme (T98g) cells as an in vitro model of brain gliomas, the most sterically hindered, hydrophobic, and stable compound [t1/2 (298 K) = 15 min in cell medium] was better than the two other complexes (IC50 = 4.1 ± 0.5 µM DTB, 34 ± 7 µM 3-MeCat, and 19 ± 2 µM Cat). Furthermore, upon aging, the complexes formed less toxic decomposition products (IC50 = 9 ± 1 µM DTB, 18 ± 3 µM 3-MeCat, and 8.1 ± 0.6 µM Cat). The vanadium complexes with the chloro-substituted Schiff base were more hydrophobic, more hydrolytically stable, more easily reduced compared to their corresponding parent counterparts, and the most sterically hindered complex of this series is only the second non-innocent vanadium Schiff base complex with a potent in vitro anticancer activity that is an order of magnitude more potent than cisplatin under the same conditions.
Asunto(s)
Complejos de Coordinación , Vanadio , Humanos , Vanadio/farmacología , Vanadio/química , Cisplatino , Bases de Schiff/farmacología , Bases de Schiff/química , Agua , Espectroscopía de Resonancia Magnética , Complejos de Coordinación/farmacología , LigandosRESUMEN
Lipoquinones are the topic of this review and are a class of hydrophobic lipid molecules with key biological functions that are linked to their structure, properties, and location within a biological membrane. Ubiquinones, plastoquinones, and menaquinones vary regarding their quinone headgroup, isoprenoid sidechain, properties, and biological functions, including the shuttling of electrons between membrane-bound protein complexes within the electron transport chain. Lipoquinones are highly hydrophobic molecules that are soluble in organic solvents and insoluble in aqueous solution, causing obstacles in water-based assays that measure their chemical properties, enzyme activities and effects on cell growth. Little is known about the location and ultimately movement of lipoquinones in the membrane, and these properties are topics described in this review. Computational studies are particularly abundant in the recent years in this area, and there is far less experimental evidence to verify the often conflicting interpretations and conclusions that result from computational studies of very different membrane model systems. Some recent experimental studies have described using truncated lipoquinone derivatives, such as ubiquinone-2 (UQ-2) and menaquinone-2 (MK-2), to investigate their conformation, their location in the membrane, and their biological function. Truncated lipoquinone derivatives are soluble in water-based assays, and hence can serve as excellent analogs for study even though they are more mobile in the membrane than the longer chain counterparts. In this review, we will discuss the properties, location in the membrane, and syntheses of three main classes of lipoquinones including truncated derivatives. Our goal is to highlight the importance of bridging the gap between experimental and computational methods and to incorporate properties-focused considerations when proposing future studies relating to the function of lipoquinones in membranes.
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Plastoquinona , Ubiquinona , Vitamina K 2 , Ubiquinona/metabolismo , Quinonas/metabolismo , Conformación Molecular , AguaRESUMEN
The interpeptidic CuII exchange rate constants were measured for two Cu amyloid-ß complexes, Cu(Aß1-16) and Cu(Aß1-28), to fluorescent peptides GHW and DAHW using a quantitative tryptophan fluorescence quenching methodology. The second-order rate constants were determined at three pH values (6.8, 7.4, and 8.7) important to the two Cu(Aß) coordination complexes, components Cu(Aß)I and Cu(Aß)II. The interpeptidic CuII exchange rate constant is approximately 104 M-1 s-1 but varies in magnitude depending on many variables. These include pH, length of the Aß peptide, location of the anchoring histidine ligand in the fluorescent peptide, number of amide deprotonations required in the tryptophan peptide to coordinate CuII, and interconversion between Cu(Aß)I and Cu(Aß)II. We also present EPR data probing the CuII exchange between peptides and the formation of ternary species between Cu(Aß) and GHW. As the nonfluorescent GHK and DAHK peptides are important motifs found in the blood and serum, their ability to sequester CuII ions from Cu(Aß) complexes may be relevant for the metal homeostasis and its implication in Alzheimer's disease. Thus, their kinetic CuII interpeptidic exchange rate constants are important chemical rate constants that can help elucidate the complex CuII trafficking puzzle in the synaptic cleft.
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Péptidos beta-Amiloides/metabolismo , Cobre/metabolismo , Fluorescencia , Péptidos/metabolismo , Triptófano/metabolismo , Péptidos beta-Amiloides/química , Cobre/química , Espectroscopía de Resonancia por Spin del Electrón , Fluorometría , Conformación Molecular , Péptidos/química , Espectrofotometría Ultravioleta , Triptófano/químicaRESUMEN
Menaquinones (MK) are hydrophobic molecules that consist of a naphthoquinone headgroup and a repeating isoprenyl side chain and are cofactors used in bacterial electron transport systems to generate cellular energy. We have previously demonstrated that the folded conformation of truncated MK homologues, MK-1 and MK-2, in both solution and reverse micelle microemulsions depended on environment. There is little information on how MKs associate with phospholipids in a model membrane system and how MKs affect phospholipid organization. In this manuscript, we used a combination of Langmuir monolayer studies and molecular dynamics (MD) simulations to probe these questions on truncated MK homologues, MK-1 through MK-4 within a model membrane. We observed that truncated MKs reside farther away from the interfacial water than ubiquinones are are located closer to the phospholipid tails. We also observed that phospholipid packing does not change at physiological pressure in the presence of truncated MKs, though a difference in phospholipid packing has been observed in the presence of ubiquinones. We found through MD simulations that for truncated MKs, the folded conformation varied, but MKs location and association with the bilayer remained unchanged at physiological conditions regardless of side chain length. Combined, this manuscript provides fundamental information, both experimental and computational, on the location, association, and conformation of truncated MK homologues in model membrane environments relevant to bacterial energy production.
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Membrana Dobles de Lípidos/química , Lípidos/química , Vitamina K 2/química , Membrana Dobles de Lípidos/metabolismo , Conformación Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Fosfolípidos/química , Temperatura , Termodinámica , Vitamina K 2/metabolismoRESUMEN
In this study, the binding to lysozyme (Lyz) of four important VIV compounds with antidiabetic and/or anticancer activity, [VIVO(pic)2(H2O)], [VIVO(ma)2], [VIVO(dhp)2], and [VIVO(acac)2], where pic-, ma-, dhp-, and acac- are picolinate, maltolate, 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate, and acetylacetonate anions, and of the vanadium-containing natural product amavadin ([VIV(hidpa)2]2-, with hidpa3- N-hydroxyimino-2,2'-diisopropionate) was investigated by ElectroSpray Ionization-Mass Spectrometry (ESI-MS). Moreover, the interaction of [VIVO(pic)2(H2O)], chosen as a representative VIVO2+ complex, was examined with two additional proteins, myoglobin (Mb) and ubiquitin (Ub), to compare the data. The examined vanadium concentration was in the range 15-150 µM, i.e., very close to that found under physiological conditions. With pic-, dhp-, and hidpa3-, the formation of adducts n[VIVOL2]-Lyz or n[VIVL2]-Lyz is favored, while with ma- and acac- the species n[VIVOL]-Lyz are detected, with n dependent on the experimental VIV/protein ratio. The behavior of the systems with [VIVO(pic)2(H2O)] and Mb or Ub is very similar to that of Lyz. The results suggested that under physiological conditions, the moiety cis-VIVOL2 (L = pic-, dhp-) is bound by only one accessible side-chain protein residue that can be Asp, Glu, or His, while VIVOL+ (L = ma-, acac-) can interact with the two equatorial and axial sites. If the VIV complex is thermodynamically stable and does not have available coordination positions, such as amavadin, the protein cannot interact with it through the formation of coordination bonds and, in such cases, noncovalent interactions are predicted. The formation of the adducts is dependent on the thermodynamic stability and geometry in aqueous solution of the VIVO2+ complex and affects the transport, uptake, and mechanism of action of potential V drugs.
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Alanina/análogos & derivados , Antineoplásicos/química , Complejos de Coordinación/química , Ácidos Hidroxámicos/química , Hipoglucemiantes/química , Proteínas/química , Alanina/química , Animales , Bovinos , Pollos , Caballos , Muramidasa/química , Mioglobina/química , Espectrometría de Masa por Ionización de Electrospray , Ubiquitina/química , Vanadio/químicaRESUMEN
The light-induced photolysis of [Zn(NTAdeCage)]- generates a temporally controlled burst of Zn2+, which is rapidly chelated in situ by the free ligand Zincon2-. The [Zn(Zincon)]2- coordination progress is monitored using absorption spectroscopy in bulk aqueous buffer and reverse micelle environments. The [Zn(NTAdeCage)]- photocage and free ligand Zincon2- have different reverse micelle locations that affect the [Zn(Zincon)]2- formation at the nanoscale compared to the bulk aqueous buffer. The formation of [Zn(Zincon)]2- in a bulk aqueous buffer is more efficient despite the released Zn2+ and Zincon2- being physically closer within reverse micelles. The observed reduction of complex formation is attributed to the interfacial partitioning of Zincon2-, distinct from the Zn2+ photocage in the water pool, requiring diffusion for the species to meet to form [Zn(Zincon)]2-. This work introduces a proof-of-concept methodology to experimentally measure fast chelation reactions in confined spaces and thus provides an approach to exploring cellular responses.
RESUMEN
SMYD3 is a lysine methyltransferase that regulates the expression of over 80 genes and is required for the uncontrolled proliferation of most breast, colorectal, and hepatocellular carcinomas. The elimination of SMYD3 restores normal expression patterns of these genes and halts aberrant cell proliferation, making it a promising target for small molecule inhibition. In this study, we sought to establish a proof of concept for our in silico/in vitro hit-to-lead enzyme inhibitor development platform and to identify a lead small molecule candidate for SMYD3 inhibition. We used Schrodinger® software to screen libraries of small molecules in silico and the five compounds with the greatest predicted binding affinity within the SMYD3 binding pocket were purchased and assessed in vitro in direct binding assays and in breast cancer cell lines. We have confirmed the ability of one of these inhibitors, Inhibitor-4, to restore normal rates of cell proliferation, arrest the cell cycle, and induce apoptosis in breast cancer cells without affecting wildtype cell behavior. Our results provide a proof of concept for this fast and affordable small molecule hit-to-lead methodology as well as a promising candidate small molecule SMYD3 inhibitor for the treatment of human cancer.
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Descubrimiento de Drogas , Inhibidores de Histona Desacetilasas/química , N-Metiltransferasa de Histona-Lisina/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Descubrimiento de Drogas/métodos , Femenino , Inhibidores de Histona Desacetilasas/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via 1H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.
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Glicina/metabolismo , Glicilglicina/metabolismo , Membranas/metabolismo , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Agua/metabolismo , Glicina/química , Glicilglicina/química , Membranas/química , Micelas , Modelos Teóricos , Oligopéptidos/química , Fragmentos de Péptidos/química , Agua/químicaRESUMEN
Menaquinones are a class of isoprenoid molecules that have important roles in human biology and bacterial electron transport, and multiple methods have been developed for their synthesis. These compounds consist of a methylnaphthoquinone (MK) unit and an isoprene side chain, such as found in vitamin K1 (phylloquinone), K2, and other lipoquinones. The most common naturally occurring menaquinones contain multiple isoprene units and are very hydrophobic, rendering it difficult to evaluate the biological activity of these compounds in aqueous assays. One way to overcome this challenge has been the application of truncated MK-derivatives for their moderate solubility in water. The synthesis of such derivatives has been dominated by Friedel-Crafts alkylation with BF3âOEt2. This attractive method occurs over two steps from commercially available starting materials, but it generally produces low yields and a mixture of isomers. In this review, we summarize reported syntheses of both truncated and naturally occurring MK-derivatives that encompass five different synthetic strategies: Nucleophilic ring methods, metal-mediated reactions, electrophilic ring methods, pericyclic reactions, and homologation and side chain extensions. The advantages and disadvantages of each method are discussed, identifying methods with a focus on high yields, regioselectivity, and stereochemistry leading to a detailed overview of the reported chemistry available for preparation of these compounds.
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Naftoquinonas/síntesis química , Vitamina K 2/síntesis química , Vitamina K/análogos & derivados , Alquilación , Catálisis , Naftoquinonas/química , Oxidación-Reducción , Vitamina K/química , Vitamina K 2/químicaRESUMEN
The chemistry and short lifetimes of metal-based anti-cancer drugs can be turned into an advantage for direct injections into tumors, which then allow the use of highly cytotoxic drugs. The release of their less toxic decomposition products into the blood will lead to decreased toxicity and can even have beneficial effects. We present a ternary VV complex, 1 ([VOL1 L2 ], where L1 is N-(salicylideneaminato)-N'-(2-hydroxyethyl)ethane-1,2-diamine and L2 is 3,5-di-tert-butylcatechol), which enters cells intact to induce high cytotoxicity in a range of human cancer cells, including T98g (glioma multiforme), while its decomposition products in cell culture medium were ≈8-fold less toxic. 1 was 12-fold more toxic than cisplatin in T98g cells and 6-fold more toxic in T98g cells than in a non-cancer human cell line, HFF-1. Its high toxicity in T98g cells was retained in the presence of physiological concentrations of the two main metal-binding serum proteins, albumin and transferrin. These properties favor further development of 1 for brain cancer treatment by intratumoral injections.
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Antineoplásicos/química , Neoplasias Encefálicas/tratamiento farmacológico , Complejos de Coordinación/química , Compuestos de Vanadio/química , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Complejos de Coordinación/uso terapéutico , Medios de Cultivo , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Espectroscopía de Protones por Resonancia Magnética , Compuestos de Vanadio/uso terapéuticoRESUMEN
Deoxynucleotide misincorporation efficiencies can span a wide 104-fold range, from â¼10-2 to â¼10-6, depending principally on polymerase (pol) identity and DNA sequence context. We have addressed DNA pol fidelity mechanisms from a transition-state (TS) perspective using our "tool-kit" of dATP- and dGTP-ß,γ substrate analogues in which the pyrophosphate leaving group (p Ka4 = 8.9) has been replaced by a series of bisphosphonates covering a broad acidity range spanning p Ka4 values from 7.8 (CF2) to 12.3 [C(CH3)2]. Here, we have used a linear free energy relationship (LFER) analysis, in the form of a Brønsted plot of log( kpol) versus p Ka4, for Y-family error-prone pol η and X-family pols λ and ß to determine the extent to which different electrostatic active site environments alter kpol values. The apparent chemical rate constant ( kpol) is the rate-determining step for the three pols. The pols each exhibit a distinct catalytic signature that differs for formation of right (A·T) and wrong (G·T) incorporations observed as changes in slopes and displacements of the Brønsted lines, in relation to a reference LFER. Common to this signature among all three pols is a split linear pattern in which the analogues containing two halogens show kpol values that are systematically lower than would be predicted from their p Ka4 values measured in aqueous solution. We discuss how metal ions and active site amino acids are responsible for causing "effective" p Ka4 values that differ for dihalo and non-dihalo substrates as well as for individual R and S stereoisomers for CHF and CHCl.
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ADN Polimerasa beta/metabolismo , ADN Polimerasa gamma/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Emparejamiento Base , Dominio Catalítico , ADN Polimerasa beta/química , ADN Polimerasa gamma/química , ADN Polimerasa Dirigida por ADN/química , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Humanos , Cinética , Especificidad por Sustrato , TermodinámicaRESUMEN
Menaquinones (MKs) are essential for electron transport in prokaryotes, and importantly, partially saturated MKs represent a novel virulence factor. However, little is known regarding how the degree of saturation in the isoprenyl side chain influences conformation or quinone redox potential. MenJ is an enzyme that selectively reduces the second isoprene unit on MK-9 and is contextually essential for the survival of Mycobacterium tuberculosis in J774A.1 macrophage-like cells, suggesting that MenJ may be a conditional drug target for pathogenic mycobacteria. Therefore, fundamental information about the properties of this system is important, and we synthesized the simplest MKs, unsaturated MK-1 and the saturated analogue, MK-1(H2). Using two-dimensional nuclear magnetic resonance spectroscopy, we established that MK-1 and MK-1(H2) adopted similar folded-extended conformations (i.e., the isoprenyl side chain folds upward) in each solvent examined but the folded-extended conformations differed slightly between organic solvents. Saturation of the isoprenyl side chain slightly altered the MK-1 analogue conformation in each solvent. We used molecular mechanics to illustrate the MK-1 analogue conformations. The measured quinone redox potentials of MK-1 and MK-1(H2) differed between organic solvents (presumably due to differences in dielectric constants), and remarkably, an â¼20 mV semiquinone redox potential difference was observed between MK-1 and MK-1(H2) in pyridine, acetonitrile, and dimethyl sulfoxide, demonstrating that the degree of saturation in the isoprenyl side chain of MK-1 influences the quinone redox potential. Finally, MK-1 and MK-1(H2) interacted with Langmuir phospholipid monolayers and Aerosol-OT reverse micelle (RM) model membrane interfaces, where MK-1 adopted a slightly different folded conformation within the RM model membrane interface.
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Vitamina K 2/química , 1,2-Dipalmitoilfosfatidilcolina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/química , Proteínas Bacterianas/química , Membranas Artificiales , Micelas , Modelos Moleculares , Conformación Molecular , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Fosfatidiletanolaminas/química , Espectroscopía de Protones por Resonancia Magnética , Vitamina K 2/síntesis químicaRESUMEN
Oncolytic viruses rewire the immune system and can lead to long-lasting antitumor defenses against primary and metastatic tumors. However, results from clinical studies have shown heterogeneity in responses suggesting that multiplexed approaches may be necessary to consistently generate positive outcomes in patients. To this end, we explored the combination of oncolytic rhabdovirus VSV∆51 with vanadium(V) dipicolinate derivatives, which have already been explored for their antidiabetic properties in animal models. The combination of vanadium-based dipicolinate compounds with VSV∆51 significantly increased viral replication and cytotoxicity in the human renal cell carcinoma cell line 786-0. The effects of three vanadium(V)-dipicolinate coordination complexes ([VO2dipic]-, [VO2dipic-OH]- and [VO2dipic-Cl]- with -OH or -Cl in the para position) were compared to that of the simple salts using spectroscopy and speciation profiles. Like the vanadate salts and the vanadyl cation, all dioxovanadium(V) dipicolinate complexes tested were found to increase viral infection and cytotoxicity when used in combination with VSV∆51. Viral sensitization is dependent on the vanadium since free dipicolinate ligands exerted no effect on viral infection and viability. The ability of these complexes to interact with interfaces and the stability of the complexes were evaluated under physiological conditions. Results indicate that these complexes undergo hydrolysis in cell culture media thereby generating vanadate. The vanadium dipicolinate derivatives in the context of immunovirotherapy shares similarities with previous studies exploring the antidiabetic properties of the compounds. The synergy between vanadium compounds and the oncolytic virus suggests that these compounds may be valuable in the development of novel and effective pharmaco-viral therapies.
Asunto(s)
Antivirales/farmacología , Complejos de Coordinación/farmacología , Viroterapia Oncolítica , Virus Oncolíticos/efectos de los fármacos , Ácidos Picolínicos/farmacología , Compuestos de Vanadio/farmacología , Virosis/terapia , Antivirales/síntesis química , Antivirales/química , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Ácidos Picolínicos/química , Relación Estructura-Actividad , Células Tumorales Cultivadas , Compuestos de Vanadio/química , Virosis/tratamiento farmacológicoRESUMEN
Oncolytic viruses (OV) are an emerging class of anticancer bio-therapeutics that induce antitumor immunity through selective replication in tumor cells. However, the efficacy of OVs as single agents remains limited. We introduce a strategy that boosts the therapeutic efficacy of OVs by combining their activity with immuno-modulating, small molecule protein tyrosine phosphatase inhibitors. We report that vanadium-based phosphatase inhibitors enhance OV infection in vitro and ex vivo, in resistant tumor cell lines. Furthermore, vanadium compounds increase antitumor efficacy in combination with OV in several syngeneic tumor models, leading to systemic and durable responses, even in models otherwise refractory to OV and drug alone. Mechanistically, this involves subverting the antiviral type I IFN response toward a death-inducing and pro-inflammatory type II IFN response, leading to improved OV spread, increased bystander killing of cancer cells, and enhanced antitumor immune stimulation. Overall, we showcase a new ability of vanadium compounds to simultaneously maximize viral oncolysis and systemic anticancer immunity, offering new avenues for the development of improved immunotherapy strategies.
Asunto(s)
Vectores Genéticos/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Compuestos de Vanadio/farmacología , Animales , Biomarcadores , Quimiocina CXCL9/metabolismo , Terapia Combinada , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Terapia Genética/métodos , Humanos , Inmunoterapia , Mediadores de Inflamación/metabolismo , Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Activación de Linfocitos/inmunología , Ratones , Mortalidad , Especies Reactivas de Oxígeno/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pyridine-based small-molecule drugs, vitamins, and cofactors are vital for many cellular processes, but little is known about their interactions with membrane interfaces. These specific membrane interactions of these small molecules or ions can assist in diffusion across membranes or reach a membrane-bound target. This study explores how minor differences in small molecules (isoniazid, benzhydrazide, isonicotinamide, nicotinamide, picolinamide, and benzamide) can affect their interactions with model membranes. Langmuir monolayer studies of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylethanolamine (DPPE), in the presence of the molecules listed, show that isoniazid and isonicotinamide affect the DPPE monolayer at lower concentrations than the DPPC monolayer, demonstrating a preference for one phospholipid over the other. The Langmuir monolayer studies also suggest that nitrogen content and stereochemistry of the small molecule can affect the phospholipid monolayers differently. To determine the molecular interactions of the simple N-containing aromatic pyridines with a membrane-like interface, 1H one-dimensional NMR and 1H-1H two-dimensional NMR techniques were utilized to obtain information about the position and orientation of the molecules of interest within aerosol-OT (AOT) reverse micelles. These studies show that all six of the molecules reside near the AOT sulfonate headgroups and ester linkages in similar positions, but nicotinamide and picolinamide tilt at the water-AOT interface to varying degrees. Combined, these studies demonstrate that small structural changes of small N-containing molecules can affect their specific interactions with membrane-like interfaces and specificity toward different membrane components.