RESUMEN
A genetic selection system for activity of HIV protease is described that is based on a synthetic substrate constructed as a modified AraC regulatory protein that when cleaved stimulate l-arabinose metabolism in an Escherichia coli araC strain. Growth stimulation on selective plates was shown to depend on active HIV protease and the scissile bond in the substrate. In addition, the growth of cells correlated well with the established cleavage efficiency of the sites in the viral polyprotein, Gag, when these sites were individually introduced into the synthetic substrate of the selection system. Plasmids encoding protease variants selected based on stimulation of cell growth in the presence of saquinavir or cleavage of a site not cleaved by wild-type protease, were indistinguishable with respect to both phenotypes. Also, both groups of selected plasmids encoded side chain substitutions known from clinical isolates or displayed different side chain substitutions but at identical positions. One highly frequent side chain substitution, E34V, not regarded as a major drug resistance substitution was found in variants obtained under both selective conditions and is suggested to improve protease processing of the synthetic substrate. This substitution is away from the substrate-binding cavity and together with other substitutions in the selected reading frames supports the previous suggestion of a substrate-binding site extended from the active site binding pocket itself.
Asunto(s)
Fármacos Anti-VIH/farmacocinética , Farmacorresistencia Viral/genética , Proteasa del VIH/genética , Sustitución de Aminoácidos , Factor de Transcripción de AraC/genética , Arabinosa/metabolismo , Quimosina/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Fusión gag-pol/metabolismo , Productos del Gen gag/metabolismo , Genes araC , Proteasa del VIH/química , Proteasa del VIH/aislamiento & purificación , Proteasa del VIH/metabolismo , Modelos Moleculares , Mutación Missense , Mutación Puntual , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saquinavir/antagonistas & inhibidores , Saquinavir/farmacología , Selección Genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
We report a first set of peptidomimetic ligands mimicking the adhesive interface identified by recent crystallographic structures of E- and N-cadherin. Compounds 2 and 3 inhibit adhesion of epithelial ovarian cancer (EOC) cells with improved efficacy compared to the ADH-1 peptide, a N-cadherin antagonist that is in early clinical trials in EOC patients.
Asunto(s)
Cadherinas/antagonistas & inhibidores , Diseño de Fármacos , Neoplasias Glandulares y Epiteliales/tratamiento farmacológico , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Peptidomiméticos , Bibliotecas de Moléculas Pequeñas/farmacología , Carcinoma Epitelial de Ovario , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Neoplasias Glandulares y Epiteliales/patología , Oligopéptidos/síntesis química , Oligopéptidos/química , Neoplasias Ováricas/patología , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Natural N-glycosylation involves a ß-anomeric linkage connecting the sugar to one asparagine residue of the protein. We herein report NMR- and modelling-based data on glycomimetics containing α-glycosidic linkages. The bioactivity of α-Gal-containing glycopeptides has been documented by revealing binding to two plant lectins, i.e. a potent ß-trefoil toxin (Viscum album agglutinin) and ß-sandwich lectin (Erythrina corallodendron agglutinin), by NMR protocols. Docking provided insights into the 3D structures of the resulting complexes. These results provide the basis to introduce α-substituted neoglycopeptides to the toolbox of scaffold for the design of potent lectin inhibitors.
Asunto(s)
Glicopéptidos/química , Glicopéptidos/metabolismo , Lectinas de Plantas/metabolismo , Erythrina/química , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Lectinas de Plantas/química , Conformación Proteica , Viscum album/químicaRESUMEN
In this work, we have studied in detail the binding of two α-fucosylamide-based mimics of Lewis(X) to DC-SIGN ECD (ECD = extracellular domain) using STD NMR and docking. We have concluded that the binding mode occurs mainly through the fucose moiety, in the same way as Lewis(X). Similarly to other mimics containing mannose or fucose previously studied, we have shown that both compounds bind to DC-SIGN ECD in a multimodal fashion. In this case, the main contact is the interaction of two hydroxyl groups one equatorial and the other one axial (O3 and O4) of the fucose with the Ca(2+) as Lewis(X) and similarly to mannose-containing mimics (in this case the interacting groups are both in the equatorial position). Finally, we have measured the K(D) of one mimic that was 0.4 mM. Competitive STD NMR experiments indicate that the aromatic moiety provides additional binding contacts that increase the affinity.
Asunto(s)
Biomimética/métodos , Moléculas de Adhesión Celular/química , Células Dendríticas/química , Inmunidad Innata , Lectinas Tipo C/química , Antígeno Lewis X/química , Receptores de Superficie Celular/química , Bibliotecas de Moléculas Pequeñas/química , Sitios de Unión , Conformación de Carbohidratos , Moléculas de Adhesión Celular/metabolismo , Células Dendríticas/inmunología , Fucosa/química , Fucosa/metabolismo , Humanos , Cinética , Lectinas Tipo C/metabolismo , Antígeno Lewis X/metabolismo , Espectroscopía de Resonancia Magnética , Manosa/química , Manosa/metabolismo , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores de Superficie Celular/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
DC-SIGN and Langerin are two C-type lectins involved in the initial steps of HIV infections: the former acts as a viral attachment factor and facilitates viral invasion of the immune system, the latter has a protective effect. Potential antiviral compounds targeted against DC-SIGN were synthesized using a common fucosylamide anchor. Their DC-SIGN affinity was tested by SPR and found to be similar to that of the natural ligand Lewis-X (Le(X)). The compounds were also found to be selective for DC-SIGN and to interact only weakly with Langerin. These molecules are potentially useful therapeutic tools against sexually transmitted HIV infection.
Asunto(s)
Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/metabolismo , Fucosa/química , Fucosa/farmacología , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/metabolismo , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Antígenos CD/metabolismo , Infecciones por VIH/tratamiento farmacológico , Humanos , Lectinas de Unión a Manosa/metabolismo , Modelos Moleculares , Unión ProteicaRESUMEN
The ruthenium nitrosyl complex trans-[Ru(NO)(NH(3))(4)(py)](PF(6))(3) (pyNO), a nitric oxide (NO) donor, was studied in regard to the release of NO and its impact both on isolated mitochondria and HepG2 cells. In isolated mitochondria, NO release from pyNO was concomitant with NAD(P)H oxidation and, in the 25-100 microM range, it resulted in dissipation of mitochondrial membrane potential, inhibition of state 3 respiration, ATP depletion and reactive oxygen species (ROS) generation. In the presence of Ca(2+), mitochondrial permeability transition (MPT), an unspecific membrane permeabilization involved in cell necrosis and some types of apoptosis, was elicited. As demonstrated by externalization of phosphatidylserine and activation of caspase-9 and caspase-3, pyNO (50-100 microM) induced HepG2 cell death, mainly by apoptosis. The combined action of the NO itself, the peroxynitrite yielded by NO in the presence of reactive oxygen species (ROS) and the oxidative stress generated by the NAD(P)H oxidation is proposed to be involved in cell death by pyNO, both via respiratory chain inhibition and ROS levels increase, or even via MPT, if Ca(2+) is present.
Asunto(s)
Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , Compuestos Organometálicos/farmacología , Rutenio/farmacología , Adenosina Trifosfato/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Tumoral , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Dilatación Mitocondrial/efectos de los fármacos , NADPH Oxidasas/metabolismo , Oxidación-Reducción , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Metallo-enzymes are a large class of biomolecules promoting specialized chemical reactions. Quantum-classical quantum mechanics/molecular mechanics molecular dynamics, describing the metal site at quantum mechanics level, while accounting for the rest of system at molecular mechanics level, has an accessible time-scale limited by its computational cost. Hence, it must be integrated with classical molecular dynamics and enhanced sampling simulations to disentangle the functions of metallo-enzymes. In this review, we provide an overview of these computational methods and their capabilities. In particular, we will focus on some systems such as CYP19A1 a Fe-dependent enzyme involved in estrogen biosynthesis, and on Mg2+-dependent DNA/RNA processing enzymes/ribozymes and the spliceosome, a protein-directed ribozyme. This information may guide the discovery of drug-like molecules and genetic manipulation tools.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Metales/química , Metales/metabolismo , Modelos Moleculares , Aromatasa/química , Aromatasa/metabolismo , Vías Biosintéticas , Dominio Catalítico , ADN/metabolismo , Teoría Funcional de la Densidad , Descubrimiento de Drogas , Unión Proteica , Conformación Proteica , ARN/metabolismo , ARN Catalítico/química , ARN Catalítico/metabolismo , Relación Estructura-Actividad , TermodinámicaRESUMEN
Silica gel bearing isonicotinamide groups was prepared by further modification of 3-aminopropyl-functionalized silica by a reaction with isonicotinic acid and 1,3-dicyclohexylcarbodiimide to yield 3-isonicotinamidepropyl-functionalized silica gel (ISNPS). This support was characterized by means of infrared spectroscopy, elemental analysis, and specific surface area. The ISNPS was used to immobilize the [Ru(NH(3))(4)SO(3)] moiety by reaction with trans-[Ru(NH(3))(4)(SO(2))Cl]Cl, yielding [Si(CH(2))(3)(isn)Ru(NH(3))(4)(SO(3))]. The related immobilized [Si(CH(2))(3)(isn)Ru(NH(3))(4)(L)](3+/2+) (L=SO(2), SO(2-)(4), OH(2), and NO) complexes were prepared and characterized by means of UV-vis and IR spectroscopy, as well as by cyclic voltammetry. Syntheses of the nitrosyl complex were performed by reaction of the immobilized ruthenium ammine [Si(CH(2))(3)(isn)Ru(NH(3))(4)(OH(2))](2+) with nitrite in acid or neutral (pH 7.4) solution. The similar results obtained in both ways indicate that the aqua complex was able to convert nitrite into coordinated nitrosyl. The reactivity of [Si(CH(2))(3)(isn)Ru(NH(3))(4)(NO)](3+) was investigated in order to evaluate the nitric oxide (NO) release. It was found that, upon light irradiation or chemical reduction, the immobilized nitrosyl complex was able to release NO, generating the corresponding Ru(III) or Ru(II) aqua complexes, respectively. The NO material could be regenerated from these NO-depleted materials obtained photochemically or by reduction. Regeneration was done by reaction with nitrite in aqueous solution (pH 7.4). Reduction-regeneration cycles were performed up to three times with no significant leaching of the ruthenium complex.
Asunto(s)
Aminas/síntesis química , Óxido Nítrico/química , Nitritos/química , Compuestos Organometálicos/síntesis química , Rutenio/química , Dióxido de Silicio , Aminas/química , Niacinamida , Compuestos Organometálicos/química , Gel de SíliceRESUMEN
OBJECTIVES: Vascular smooth muscle cell (VSMC) migration and proliferation at sites of vascular injury are both critical steps in the development of intimal hyperplasia (IH). Local delivery of nitric oxide (NO) largely prevents these events. Among the NO donors, tetraazamacrocyclic nitrosyl complexes, such as trans-[Ru(NO)Cl(cyclam)](PF6 )2 (cyclamNO), gained attention for their features, which include the possibility of being embedded in solid matrices, and ability to participate in a nitrite/NO catalytic conversion cycle. METHODS: Methods used to evaluate cyclamNO activity: safety margin by NR and MTT; cell proliferation by 3H-thymidine incorporation and proliferating cell nuclear antigen (PCNA) expression; antimigratory properties by transwell and wound healing; prevention of cell phenotypic switching under platelet-derived growth factor type BB (PDGF-BB) stimuli by analysis of alpha smooth muscle actin (α-SMA) expression. KEY FINDINGS: Cell proliferation and migration induced by PDGF-BB were significantly inhibited by cyclamNO. The ~60% reduction on expression of contractile protein α-SMA induced by PDGF-BB revealed VSMC phenotypic switching which is significantly prevented by cyclamNO. Compared to the NO donor sodium nitroprusside, cyclamNO showed to be significantly less cytotoxic. CONCLUSIONS: With great potential to maintain VSMC functionality and prevent IH-associated events, cyclamNO might be a promissory drug for several applications in cardiovascular medicine, as in stents.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/metabolismo , Compuestos de Rutenio/farmacología , Actinas/metabolismo , Animales , Becaplermina , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Nitroprusiato , Fenotipo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Proto-Oncogénicas c-sis/administración & dosificación , ConejosRESUMEN
Three-dimensional domain swapping is an important mode of protein association leading to the formation of stable dimers. Monomers associating via this mechanism mutually exchange a domain to form a homodimer. Classical cadherins, an increasingly important target for anticancer therapy, use domain swapping to mediate cell adhesion. However, despite its importance, the molecular mechanism of domain swapping is still debated. Here, we study the conformational changes that lead to activation and dimerization via domain swapping of E-cadherin. Using state-of-the-art enhanced sampling atomistic simulations, we reconstruct its conformational free energy landscape, obtaining the free energy profile connecting the inactive and active form. Our simulations predict that the E-cadherin monomer populates the open and closed forms almost equally, which is in agreement with the proposed "selected fit" mechanism in which monomers in an active conformational state bind to form a homodimer, analogous to the conformational selection mechanism often observed in ligand-target binding. Moreover, we find that the open state population is increased in the presence of calcium ions at the extracellular boundary, suggesting their possible role as allosteric activators of the conformational change.
Asunto(s)
Cadherinas/química , Adhesión Celular/fisiología , Simulación de Dinámica Molecular , Cadherinas/metabolismo , Calcio/química , Dimerización , Iones/química , Estructura Terciaria de Proteína , Propiedades de Superficie , TermodinámicaRESUMEN
Ruthenium nitrosyl complexes have received considerable attention due to the fact that they are able to store, transfer and release NO in a controlled manner. It is well-known that the NO reactivity of ruthenium nitrosyl complexes can be modulated with the judicious choice of equatorial and axial ligands. In this piece of research we elucidate the nature of the Ru-NO and Ru-NO2 bonding in a cis-[Ru(NO)(NO2)(bpy)2](2+) complex energy decomposition (Su-Li EDA) and topological (e.g., QTAIM) and natural bond orbital analysis. It was observed that the strength of these bonds is directly correlated with the relative stability of isomers involved in nitro-nitrito and nitrosyl-isonitrosyl isomerism, as described previously by Coppens and Ooyama.
Asunto(s)
2,2'-Dipiridil/química , Óxido Nítrico/química , Dióxido de Nitrógeno/química , Compuestos Organometálicos/química , Teoría Cuántica , Rutenio/química , EstereoisomerismoRESUMEN
Ruthenium complexes including nitrosyl or nitrite complexes are particularly interesting because they can not only scavenge but also release nitric oxide in a controlled manner, regulating the NO-level in vivo. The judicious choice of ligands attached to the [RuNO] core has been shown to be a suitable strategy to modulate NO reactivity in these complexes. In order to understand the influence of different equatorial ligands on the electronic structure of the Ru-NO chemical bonding, and thus on the reactivity of the coordinated NO, we propose an investigation of the nature of the Ru-NO chemical bond by means of energy decomposition analysis (EDA), considering tetraamine and tetraazamacrocycles as equatorial ligands, prior to and after the reduction of the {RuNO}(6) moiety by one electron. This investigation provides a deep insight into the Ru-NO bonding situation, which is fundamental in designing new ruthenium nitrosyl complexes with potential biological applications.
RESUMEN
Chemical reactivity, photolability, and computational studies of the ruthenium nitrosyl complex with a substituted cyclam, fac-[Ru(NO)Cl(2)(κ(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl·H(2)O ((1-carboxypropyl)cyclam = 3-(1,4,8,11-tetraazacyclotetradecan-1-yl)propionic acid)), (I) are described. Chloride ligands do not undergo aquation reactions (at 25 °C, pH 3). The rate of nitric oxide (NO) dissociation (k(obs-NO)) upon reduction of I is 2.8 s(-1) at 25 ± 1 °C (in 0.5 mol L(-1) HCl), which is close to the highest value found for related complexes. The uncoordinated carboxyl of I has a pK(a) of â¼3.3, which is close to that of the carboxyl of the non coordinated (1-carboxypropyl)cyclam (pK(a) = 3.4). Two additional pK(a) values were found for I at â¼8.0 and â¼11.5. Upon electrochemical reduction or under irradiation with light (λ(irr) = 350 or 520 nm; pH 7.4), I releases NO in aqueous solution. The cyclam ring N bound to the carboxypropyl group is not coordinated, resulting in a fac configuration that affects the properties and chemical reactivities of I, especially as NO donor, compared with analogous trans complexes. Among the computational models tested, the B3LYP/ECP28MDF, cc-pVDZ resulted in smaller errors for the geometry of I. The computational data helped clarify the experimental acid-base equilibria and indicated the most favourable site for the second deprotonation, which follows that of the carboxyl group. Furthermore, it showed that by changing the pH it is possible to modulate the electron density of I with deprotonation. The calculated NO bond length and the Ru/NO charge ratio indicated that the predominant canonical structure is [Ru(III)NO], but the Ru-NO bond angles and bond index (b.i.) values were less clear; the angles suggested that [Ru(II)NO(+)] could contribute to the electronic structure of I and b.i. values indicated a contribution from [Ru(IV)NO(-)]. Considering that some experimental data are consistent with a [Ru(II)NO(+)] description, while others are in agreement with [Ru(III)NO], the best description for I would be a linear combination of the three canonical forms, with a higher weight for [Ru(II)NO(+)] and [Ru(III)NO].
Asunto(s)
Complejos de Coordinación/química , Compuestos Heterocíclicos/química , Óxido Nítrico/química , Rutenio/química , Técnicas Electroquímicas , Concentración de Iones de Hidrógeno , Conformación Molecular , Oxidación-Reducción , Fotólisis , TermodinámicaRESUMEN
The complex fac-[Ru(NO)Cl2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O (1-carboxypropyl)cyclam=3-(1,4,8,11-tetraazacyclotetradecan-1-yl)propionic acid) was prepared in a one pot reaction by mixing equimolar amounts of RuNOCl 3 and (1-carboxypropyl)cyclam and was characterized by X-ray crystallography, electrospray ionization tandem mass spectrometry (ESI-MS/MS), elemental analysis, NMR, and electronic and vibrational (IR) spectroscopies. fac-[Ru(NO)Cl 2(kappa(3)N(4),N(8),N(11)(1-carboxypropyl)cyclam)]Cl.H2O crystallizes in the triclinic, space group P1, No. 2, with unit cell parameters of a=8.501(1) A, b=9.157(1) A, c=14.200(1) A, alpha=72.564(5) degrees , beta=82.512(5) degrees , gamma=80.308(5) degrees , and Z=2. The Ru-N interatomic distance and bond angle in the [Ru-NO] unit are 1.739(2) A and 167.7(2) degrees , respectively. ESI-MS/MS shows characteristic dissociation chemistry that initiates by HCl or NO loss. The IR spectrum displays a nu(NO) at 1881 cm(-1) indicating a nitrosonium character. The electronic spectrum shows absorptions bands at 264 nm (log epsilon=3.27), 404 nm (log epsilon=2.53), and 532 nm (log epsilon=1.88). (1)H and (13)C NMR are in agreement with the proposed molecular structure, which shows a very singular architecture where the cyclam ring N (with the carboxypropyl pendant arm) is not coordinated to the ruthenium resulting in a kappa(3) instead of the expected kappa(4) denticity.