Applications of NMR in drug design: Sructure-activity relationships in disulfide-rich peptides

NMR is a powerful technique for determining structures of biologically active molecules in solution. In recent years. our laboratory has focussed on the structure determination of small disulfide-rich proteins from both plants and animals which are valuable targets in drug design applications. This article will review these structural studies and their implications in drug design.

Structure activity relationship, acute toxicity and cytotoxicity of antimycobacterial neolignan analogues

Objectives The study`s aims were to evaluate the antimycobacterial activity of 13 synthetic neolignan analogues and to perform structure activity relationship analysis (SAR). The cytotoxicity of the compound 2-phenoxy-1-phenylethanone (LS-2, 1) in mammalian cells, such as the acute toxicity in mice, was also evaluated. Methods The extra and intracellular antimycobacterial activity was evaluated on Mycobacterium tuberculosis H37Rv. Cytotoxicity studies were performed using V79 cells, J774 macrophages and rat hepatocytes. Additionally, the in-vivo acute toxicity was tested in mice. The SAR analysis was performed by Principal Component Analysis (PCA). Key findings Among the 13 analogues tested, LS-2 (1) was the most effective, showing promising antimycobacterial activity and very low cytotoxicity in V79 cells and in J774 macrophages, while no toxicity was observed in rat hepatocytes. The selectivity index (SI) of LS-2 (1) was 91 and the calculated LD50 was 1870 mg/kg, highlighting the very low toxicity in mice. SAR analysis showed that the highest electrophilicity and the lowest molar volume are physical-chemical characteristics important for the antimycobacterial activity of the LS-2 (1). Conclusions LS-2 (1) showed promising antimycobacterial activity and very weak cytotoxicity in cell culture, as well as an absence of toxicity in primary culture of hepatocytes. In the acute toxicity study there was an indication of absence of toxicity on murine models, in vivo.

Diffusion and efficiency of ISO 9001 in Portugal: a qualitative and quantitative study from a holistic theoretical perspective

Purpose – the aim of this paper is to analyse the diffusion and efficiency of ISO 9001 on different sectors of activity Design/methodology/approach – for that purpose, a holistic an integrative theoretical approach was based on the scope of the Contingency theory, the Institutional theory and the Resources-Based View (RBV). This theorethical perspective was used in a broad empirical study, using a qualitative and quantitative methodology, concerning Portuguese companies from different sectors of activity. Findings – according to the findings from both perspectives, a ranked combination of the named theoretical frame was constructed. Research limitations/implications – as to the analysis of the efficiency of ISO 9000, one of the limitations of this study lays in the consideration of just two sectors of activity, and another relates to its domestic geographical placement. Practical implications – this study used the ISO 9001 structure for the interviews and this has revealed very useful for the organizations to grasp the matters inquired. Originality/value – a relevant contribution to the state of art is achieved through the considered theoretical scope of analysis

Structure-function relationships in a glycosyltransferase, a phosphatase and an oxidoreductase

Dissertation presented to obtain the Ph.D degree in Biochemistry

Structure-properties relationships in thermoplastic polyurethane elastomer nanocomposites: Interactions between polymer phases and nanofillers

Polyurethane thermoplastic elastomer (TPU) nanocomposites were prepared by the incorporation of 1 wt% of high-structured carbon black (HSCB), carbon nanofibers (CNF), nanosilica (NS) and nanoclays (NC), following a proper high-shear blending procedure. The TPU nanofilled mechanical properties and morphology was assessed. The nanofillers interact mainly with the TPU hard segments (HS) domains, determining their glass transition temperature, and increasing their melting temperature and enthalpy. A significant improvement upon the modulus, sustained stress levels and deformation capabilities is evidenced. The relationships between the morphology and the nanofilled TPU properties are established, evidencing the role of HS domains on the mechanical response, regardless the nanofiller type.

The contribution of phenolic acids to the anti-inflammatory activity of mushrooms: Screening in phenolic extracts, individual parent molecules and synthesized glucuronated and methylated derivatives

In the present study, the ethanolic extracts of fourteen edible mushrooms were investigated for their anti-inflammatory potential in LPS (lipopolysaccharide) activated RAW 264.7 macrophages. Furthermore the extracts were chemically characterized in terms of phenolic acids and related compounds. The identified molecules (p-hydroxybenzoic, p-coumaric and cinnamic acids) and their glucuronated and methylated derivatives obtained by chemical synthesis were also evaluated for the same bioactivity, in order to establish structure-activity relationships and to comprehend the effects of in vivo metabolism reactions in the activity of the compounds. The extracts of Pleurotus ostreatus, Macrolepiota procera, Boletus impolitus and Agaricus bisporus revealed the strongest anti-inflammatory potential (EC50 values 96 ± 1 to 190 ± 6 µg/mL, and also the highest concentration of cinnamic acid (656 to 156 µg/g), which was also the individual compound with the highest anti-inflammatory activity. The derivatives of p-coumaric acid revealed the strongest properties, specially the derivative methylated in the carboxylic group (CoA-M1) that exhibited similar activity to the one showed by dexamethaxone used as anti-inflammatory standard; by contrast, the derivatives of p-hydroxybenzoic revealed the lowest inhibition of NO production. All in all, whereas the conjugation reactions change the chemical structure of phenolic acids and may increase or decrease their activity, the glucuronated and methylated derivatives of the studied compounds are still displaying anti-inflammatory activity.

Evaluación del efecto del láser y magnetoterapia en miopatía experimental valorando biomarcadores de estrés oxidativo, histomorfometría y actividad enzimática mitocondrial. Evaluation of the effect of laser and magnetic therapy in experimental myopathy assessing oxidative stress biomarkers, histomorphometry and mitochondrial enzyme activity

El láser de baja y media energía y la magnetoterapia son utilizados en desórdenes osteomioarticulares por sus efectos analgésico, antiinflamatorio y trófico, entre los más destacados. Sin embargo, son insuficientes las investigaciones sobre su mecanismo de acción y antecedentes científicos que avalen sus efectos. Es por ello, que la determinación de acontecimientos celulares y moleculares que ocurren durante la interacción de estos tipos de energía con el sistema muscular, sería relevante para el conocimiento y optimización de tales terapias en las ciencias biomédicas. En las miopatías inflamatorias idiopáticas, se encuentra afectada la estructura, morfología y bioquímica del tejido muscular. La energía que éste requiere para el normal funcionamiento es generada en la mitocondria. Esta organela también es la responsable de la generación de especies oxidantes provocando estrés oxidativo y el inicio de los procesos de apoptosis. Por lo antes dicho, consideramos que la determinación de los biomarcadores inflamatorios asociados a estrés oxidativo, realizando el análisis histomorfométrico ultraestructural y valorando la actividad de los complejos enzimáticos mitocondriales, permitiría una evaluación de la acción terapéutica del láser y la magnetoterapia en un modelo experimental de miopatía. Para ello se propone evaluar el efecto de la magnetoterapia y del láser de baja energía (He-Ne y As.Ga) en miopatía experimental determinando indicadores inflamatorios asociados a estrés oxidativo, análisis histomorfométrico y valoración de la actividad enzimática mitocondrial. Específicamente: -Determinar indicadores inflamatorios y de estrés oxidativo: Oxido Nítrico, Grupos carbonilos, L-citrulina, Fibrinógeno, Superóxido dismutasa, Glutation peroxidasa y Catalasa por espectrofotometría. -Identificar los cambios anatomopatológicos del músculo esquelético por microscopía óptica (MO): cuantificación del infiltrado inflamatorio; MO de alta resolución (MOAR) y por microscopía electrónica: histomorfometría de la ultraestructura miofibrilar y mitocondrial. -Valorar las actividades enzimáticas de la citrato sintasa y de los complejos: I (NADH-ubiquinona reductasa), II (succinato-ubiquinona-reductasa) III (ubiquinona-citocromo c-reductasa) y IV (citocromo c-oxidasa); en mitocondrias de tejido muscular por espectrofotometría. -Evaluar la actividad apoptótica en las fibras musculares de los diferentes grupos por ténica de T.U.N.E.L. Las mediciones mitocondriales (por ME) y de infiltrado inflamatorio (por MO) se realizarán en un total de 5 fotos de aumentos similares en forma aleatoria por grupo estudiado (n=10). Los cambios estructurales observados se analizarán en el programa Axiovision 4.8, para cuantificar el área total ocupada, número total y grado de alteración de las mitocondrias y el porcentaje de infiltrado inflamatorio determinando el grado de inflamación. Los resultados de los datos cuantitativos se analizarán aplicando ANAVA (test de Fisher para comparaciones múltiples); y para los datos categóricos se utilizará Chi cuadrado (test de Pearson), estableciéndose un nivel de significación de p < 0.05 para todos los casos. Importancia del Proyecto: La salud y el bienestar del hombre son los logros perseguidos por las ciencias de la salud. La obtención de terapias curativas o paliativas con un mínimo de efectos colaterales para el enfermo se incluye en estos logros. Por esto y todo lo anteriormente expuesto es que consideramos de gran importancia poder esclarecer desde las ciencias básicas los efectos celulares y moleculares en modelos experimentales la acción de la terapia con láser y magnetoterapia para una aplicación clínica con base científica en todas las áreas de las Ciencias Médicas. In the idiopathic inflammatory myopathies, is affected the structure, morphology and biochemistry of muscle tissue. The mitochondria is responsible for the generation of oxidizing species leading to oxidative stress and the beginning of the process of apoptosis. As said before, we consider the determination of inflammatory biomarkers related to oxidative stress, by ultrastructural morphometric analysis and assessing the activity of mitochondrial enzyme complexes, permit an evaluation of the therapeutic action of laser and magnetic therapy in an experimental model myopathy. We propose to evaluate the effect of the treatment identifying indicators in experimental inflammatory myopathy associated with oxidative stress, histomorphometric analysis and assessment of mitochondrial enzyme activity. Specifically -determining: Nitric oxide, carbonyl groups, L-citrulline, fibrinogen, superoxide dismutase, glutathione peroxidase and catalase by spectrophotometry. -Identify the pathological changes in skeletal muscle by optical microscopy (OM): quantification of the inflammatory infiltrate, OM high resolution (MOAR) and electron microscopy, histomorphometry of myofibrillar and mitochondrial ultrastructure. -Evaluate the enzymatic activity of citrate synthase and complexes: I, II, III and IV in mitochondria muscle tissue by spectrophotometry. -Evaluate apoptotic activity in muscle fibers by TUNEL technique of Mitochondrial measurements and inflammatory infiltration (by OM) was performed in a total of 5 photos of similar increases in random by the study group (n = 10). The structural changes observed are discussed in the program Axiovision 4.8, to quantify number, degree of alteration of mitochondria and the percentage of inflammatory infiltrate determining the degree of inflammation. The results of the quantitative data were analyzed using ANOVA (Fisher test), and categorical data with Chi-square (Pearson test), establishing a significance level of p <0.05.

Structure-function relationships of the alpha1b-adrenergic receptor.

The alpha1b-adrenergic receptor (AR) is a member of the large superfamily of seven transmembrane domain (TMD) G protein-coupled receptors (GPCR). Combining site-directed mutagenesis of the alpha1b-AR with computational simulations of receptor dynamics, we have explored the conformational changes underlying the process of receptor activation, i.e. the transition between the inactive and active states. Our findings suggest that the structural constraint stabilizing the alpha1b-AR in the inactive form is a network of H-bonding interactions amongst conserved residues forming a polar pocket and R143 of the DRY sequence at the end of TMDIII. We have recently reported that point mutations of D142, of the DRY sequence and of A293 in the distal portion of the third intracellular loop resulted in ligand-independent (constitutive) activation of the alpha1b-AR. These constitutively activating mutations could induce perturbations resulting in the shift of R143 out of the polar pocket. The main role of R143 may be to mediate receptor activation by triggering the exposure of several basic amino acids of the intracellular loops towards the G protein. Our investigation has been extended also to the biochemical events involved in the desensitization process of alpha1b-AR. Our results indicate that immediately following agonist-induced activation, the alpha1b-AR can undergo rapid agonist-induced phosphorylation and desensitization. Different members of the G protein coupled receptor kinase family can play a role in agonist-induced regulation of the alpha1b-AR. In addition, constitutively active alpha1b-AR mutants display different phosphorylation and internalization features. The future goal is to further elucidate the molecular mechanism underlying the complex equilibrium between activation and inactivation of the alpha1b-AR and its regulation by pharmacological substances. These findings can help to elucidate the mechanism of action of various agents displaying properties of agonists or inverse agonists at the adrenergic system.

The activation process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.

In this study, a quantitative approach was used to investigate the role of D142, which belongs to the highly conserved E/DRY sequence, in the activation process of the alpha1B-adrenergic receptor (alpha1B-AR). Experimental and computer-simulated mutagenesis were performed by substituting all possible natural amino acids at the D142 site. The resulting congeneric set of proteins together with the finding that all the receptor mutants show various levels of constitutive (agonist-independent) activity enabled us to quantitatively analyze the relationships between structural/dynamic features and the extent of constitutive activity. Our results suggest that the hydrophobic/hydrophilic character of D142, which could be regulated by protonation/deprotonation of this residue, is an important modulator of the transition between the inactive (R) and active (R*) state of the alpha1B-AR. Our study represents an example of quantitative structure-activity relationship analysis of the activation process of a G protein-coupled receptor.

PPAR alpha structure-function relationships derived from species-specific differences in responsiveness to hypolipidemic agents.

The nuclear receptor PPAR alpha is a key regulatory transcription factor in lipid homeostasis, some liver detoxification processes and the control of inflammation. Recent findings suggest that many hypolipidemic drugs and anti-inflammatory agents can potentially act by binding to PPAR alpha and inducing its activity. Here, we identify some structure-function relationships in PPAR alpha, by using the species-specific responsiveness to the two hypolipidemic agents, Wy 14,643 and 5,8,11,14-eicosatetraynoic acid (ETYA). We first show that the species-specific differences are mediated primarily via the ligand binding domain of the receptor and that these two drugs are indeed ligands of PPAR alpha. By mutagenesis analyses we identify amino acid residues in the ligand binding domains of Xenopus, mouse and human PPAR alpha, that confer preferential responsiveness to ETYA and Wy 14,643. These findings will aid in the development of new synthetic PPAR alpha ligands as effective therapeutics for lipid-related diseases and inflammatory disorders.

Evolutionary aspects of population structure for molecular and quantitative traits in the freshwater snail Radix balthica.

Detecting the action of selection in natural populations can be achieved using the QST-FST comparison that relies on the estimation of FST with neutral markers, and QST using quantitative traits potentially under selection. QST higher than FST suggests the action of directional selection and thus potential local adaptation. In this article, we apply the QST-FST comparison to four populations of the hermaphroditic freshwater snail Radix balthica located in a floodplain habitat. In contrast to most studies published so far, we did not detect evidence of directional selection for local optima for any of the traits we measured: QST calculated using three different methods was never higher than FST. A strong inbreeding depression was also detected, indicating that outcrossing is probably predominant over selfing in the studied populations. Our results suggest that in this floodplain habitat, local adaptation of R. balthica populations may be hindered by genetic drift, and possibly altered by uneven gene flow linked to flood frequency.

Synthesis and structure - Activity relationship study of potent cytotoxic analogues of the marine alkaloid Lamellarin D

The marine alkaloid, Lamellarin D (Lam-D), has shown potent cytotoxicity in numerous cancer cell lines, and was recently identified as a potent topoisomerase I inhibitor. A library of open lactone analogs of Lam-D was prepared from a methyl 5,6-dihydropyrrolo[2,1-a]isoquinoline-3- carboxylate scaffold (1) by introducing various aryl groups through sequential and regioselective bromination, followed by Pd(0)-catalyzed Suzuki cross-coupling chemistry. The compounds were obtained in a 24-44% overall yield, and tested in a panel of three human tumor cell lines, MDA-MB- 231 (breast), A-549 (lung), and HT-29 (colon), to evaluate their cytotoxic potential. From these data the SAR study concluded that more than 75% of the open-chain Lam-D analogs tested showed cytotoxicity in a low micromolar GI50 range.

Total synthesis and antiproliferative activity screening of (±)-aplicyanins A, B and E and related analogs

The first total synthesis of the indole alkaloids ()-aplicyanins A, B and E, plus seventeen analogs, all in racemic form is reported. Modifications to the parent compound included changing the number of bromine substituents on the indole, the groups on the indole nitrogen (H, Me or OMe), and/or the oxidation level of the heterocyclic core tetrahydropyrimidine. Each compound was screened against three human tumor cell lines, and fourteen of the newly synthesized compounds showed considerable cytotoxicity. The assay results were used to establish structure-activity relationships. These results suggest that the acetyl group moiety on the imine nitrogen, and the bromine at position 5 of the indole, are both critical to activity.

Synthesis and structure - Activity relationship study of potent cytotoxic analogues of the marine alkaloid Lamellarin D

The marine alkaloid, Lamellarin D (Lam-D), has shown potent cytotoxicity in numerous cancer cell lines, and was recently identified as a potent topoisomerase I inhibitor. A library of open lactone analogs of Lam-D was prepared from a methyl 5,6-dihydropyrrolo[2,1-a]isoquinoline-3- carboxylate scaffold (1) by introducing various aryl groups through sequential and regioselective bromination, followed by Pd(0)-catalyzed Suzuki cross-coupling chemistry. The compounds were obtained in a 24-44% overall yield, and tested in a panel of three human tumor cell lines, MDA-MB- 231 (breast), A-549 (lung), and HT-29 (colon), to evaluate their cytotoxic potential. From these data the SAR study concluded that more than 75% of the open-chain Lam-D analogs tested showed cytotoxicity in a low micromolar GI50 range.