Molecular docking study of naturallyoccurring compounds as inhibitors of N-myristoyl transferase towards antifungal agents discovery / Acoplamiento molecular de compuestos de origen natural como inhibidores de la N-miristoil transferasa en el descubrimiento de nuevos agentes antifúngicos

Fungal infections currently remain as a common problem in public health. Actually, drug discovery programs are oriented to the searching for lead structures. Virtual screening and molecular docking constitute great alternatives in order to find hit compounds. Novel infection targets can also be defined and employed together with molecular docking tools in drug discovery programs. Thus, thirty-two natural compounds were docked within the active site of N-myristoyl transferase (NMT) as antifungal enzyme target. From tested compounds, alkaloids, flavonoids, xanthones, and quinones exhibited strongest mean interaction with NMT than terpenoids, coumarins and phenolics. Particularly, affinities for one aporphine alkaloid, a prenylated flavonoid and two xanthones resulted to be comparable with that of previously reported synthetic inhibitor. Several hydrophobic and polar contacts were demonstrated by comparing different computational tools. The present results let to establish three possible lead structures to develop antifungal drugs although subsequent SAR analyses are still required.

Las infecciones causadas por hongos continúan siendo un problema de salud pública en la actualidad. De hecho, existen diversos programas para el descubrimiento de fármacos enfocados en la búsqueda de estructuras plantilla. El mapeo virtual junto con docking molecular constituye una alternativa importante para encontrar potenciales estructuras promisorias. Mediante herramientas de docking molecular se pueden definir nuevos blancos terapéuticos para combatir diversas infecciones. Por tanto, se llevó a cabo el estudio del acoplamiento molecular a treinta y dos compuestos de origen natural, empleando la N-miristoil transferasa (NMT) como blanco enzimático antifúngico. De los compuestos ensayados, alcaloides, flavonoides, xantonas y quinonas mostraron interacción media más fuerte con la NMT que los terpenos, cumarinas y fenólicos. Particularmente, la afinidad encontrada para un alcaloide aporfínico, un flavonoide prenilado y dos xantonas resultó comparable con la encontrada para un inhibidor sintético reportado. En el presente trabajo se demostraron varias interacciones tanto hidrofóbicas como hidrofílicas mediante diversas herramientas computacionales. Los resultados encontrados permiten establecer tres posibles estructuras promisorias para el desarrollo de fármacos antifúngicos, aunque se requiere aún de estudios de relación estructura-actividad.

Cinerin C: a macrophyllin-type bicyclo[3.2.1]octane neolignan from Pleurothyrium cinereum (Lauraceae).

The structure of naturally-occurring cinerin C [systematic name: (7S,8R,3'R,4'S,5'R)-Δ(8')-4'-hydroxy-5,5',3'-trimethoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan], isolated from the ethanol extract of leaves of Pleurothyrium cinereum (Lauraceae), has previously been established by NMR and HRMS spectroscopy, and its absolute configuration established by circular dichroism measurements. For the first time, its crystal structure has now been established by single-crystal X-ray analysis, as the monohydrate, C(22)H(26)O(7)·H(2)O. The bicyclooctane moiety comprises fused cyclopentane and cyclohexenone rings which are almost coplanar. An intermolecular O-H···O hydrogen bond links the 4'-OH and 5'-OCH(3) groups along the c axis.

In vitro anti-inflammatory effects of naturally-occurring compounds from two Lauraceae plants

The in vitro anti-inflammatory effects of seven known lignans and one dihydrochalcone isolated from the leaves of two Lauraceae species (Pleurothyrium cinereum and Ocotea macrophylla), were evaluated through the inhibition of COX-1, COX-2, 5-LOX and the aggregation of rabbit platelets induced by PAF, AA and ADP. (+)-de-4"-O--methylmagnolin 4 was found to be a potent COX-2/5-LOX dual inhibitor and PAF-antagonist (COX-2 IC50 2.27 µM; 5-LOX IC50 5.05 µM; PAF IC50 2.51 µM). However, all compounds exhibited an activity at different levels, indicating good anti-inflammatory properties to be considered in further structural optimization studies.

Os efeitos anti-inflamatórios in vitro de sete conhecidos lignanos e uma dihidrocalcona isolados das folhas de duas espécies da família Lauraceae (Pleurothyrium cinereum e Ocotea macrophylla) foram avaliados por meio da inibição da COX1, COX-2, 5-LOX e agregação de plaquetas de coelhos induzida por PAF, AA e ADP. A (+)-4"-O-metilmagnolina-4 foi encontrada como mais potente inibidora tanto da COX-2 quanto de 5-LOX e antagonista de PAF (COX-2 IC50 2,27 µM; 5- LOX IC50 5,05 µM; PAF IC50 2,51 µM). Entretanto, todos compostos mostram uma atividade em intensidades diferentes, indicando boas propriedades anti-inflamátorias a serem consideradas para futuros estudos de modificações e otimização estruturais.

In vitro anti-inflammatory effects of naturally-occurring compounds from two Lauraceae plants.

The in vitro anti-inflammatory effects of seven known lignans and one dihydrochalcone isolated from the leaves of two Lauraceae species (Pleurothyrium cinereum and Ocotea macrophylla), were evaluated through the inhibition of COX-1, COX-2, 5-LOX and the aggregation of rabbit platelets induced by PAF, AA and ADP. (+)-de-4"-O-methylmagnolin 4 was found to be a potent COX-2/5-LOX dual inhibitor and PAF-antagonist (COX-2 IC(50) 2.27 µM; 5-LOX IC(50) 5.05 µM; PAF IC(50) 2.51 µM). However, all compounds exhibited an activity at different levels, indicating good anti-inflammatory properties to be considered in further structural optimization studies.

NOVEL FUROCARBAZOLE A LKALOIDS AND ANTIBACTERIAL ACTIVITY OF ETHANOL EXTRACT FROM Zanthoxylum fagara (L.) Sargent / NUEVOS ALCALOIDES FUROCARBAZÓLICOS Y ACTIVIDAD ANTIBACTERIANA DEL EXTRACTO ETANÓLICO DE Zanthoxylum fagara (L.) Sargent / NOVOS ALCALÓIDES FUROCARBAZÓLICOS E ATIVIDADE ANTIBACTERIANA DO EXTRATO ETANÓLICO DE Zanthoxylum fagara (L.) Sargent

From ethanol-soluble extract of the bark from Zanthoxylum fagara (L.) Sargent. were isolated two novel furocarbazole alkaloids, 4-methoxy-10ff-furo[3,2-a]car-bazole (1) and 10ff-furo[3,2-a]carbazole (2), whose structures were elucidated on the basis of IR, MS and NMR (including 1D and 2D) techniques. In addition, the antibacterial effect of the ethanol extract of bark was evaluated against Gram-negative bacteria Escherichia coli, Salmonella typhi, Shigella boydii, Vibrio chole-rae El Tor, and Vibrio cholerae clinical lysate; and Gram-positive bacteria Bacillus subtilis and Staphylococcus epidermidis, using the Agar-well diffusion method. In above-mentioned assay was found that the ethanol extract of bark exhibited inhibition against strains B. subti-lis (17mm), V. cholerae El Tor (11mm), V. cholerae clinical lysate (10mm), and S. epidermidis (9mm).

Del extracto etanólico de corteza de Zanthoxylum fagara (L.) Sargent se aislaron dos nuevos alcaloides de núcleo furocarbazólicos, 4-metoxi-10H-furo[3,2-a]carbazol (l)y10ff-furo[3,2-a]carbazol (2), cuyas estructuras fueron elucidadas utilizando técnicas de IR, EM y RMN (en una y dos dimensiones). Con el extracto etanólico de corteza se realizaron ensayos de actividad biológica antibacteriana con las cepas gram-negativas Escherichia coli, Salmonella typhi, Shigella boydii, Vibrio cholerae El Tor, Vibrio cholerae caso clínico; y gram-positivas Bacillus subtilis,y Staphylococcus epidermidis, utilizando el método de difusión en agar. En este ensayo se encontró que el extracto etanólico de corteza presentó inhibición para las cepas de B. subtilis (17 mm), V. cholerae El Tor (11 mm), V. cholerae caso clínico (10 mm) y S. epidermidis (9 mm).

O extrato etanólico da casca de Zanthoxylum fagara (L.) Sargent. foram isolados dois novos alcalóides furocarbazolicos, 4-metoxi-10H-furo[3,2-a]carbazol (l)e10ff-furo [3,2-a]carbazol (2), cujas estruturas foram elucidados usando técnicas de IR, EM e RMN (em uma e duas dimensões). Com o extrato etanólico da casca foi testada atividade biológica antibacteriana com Gram-negativas estirpes Escherichia coli, Salmonella typhi, Shigella boydii, Vibrio cholerae El Tor, Vibrio cholerae caso clínico; e Gram-positivas Bacillus subtilis e Staphylococcus epidermidis, utilizando o método de difusão em ágar. Neste ensaio foi encontrado que o extrato etanólico da casca apresentou inibição para as estirpes de B. subtilis (17mm), V. cholerae El Tor (11mm), V. cholerae caso clínico (10mm) e S. epidermidis (9mm).

The first diastereoselective synthesis of cinerins A-C, PAF-antagonistic macrophyllin-type bicyclo[3.2.1]octane neolignans, using a novel Pd-catalysed oxyarylation.

The first diastereoselective synthesis of PAF-antagonistic cinerins A-C, macrophyllin-type bicyclo[3.2.1]octane neolignans from Pleurothyrium cinereum, has been accomplished using a novel Pd-catalysed oxyarylation to afford a 2,3-dihydrobenzofuran as the key intermediate.

One-pot, Pd-catalyzed synthesis of trans-dihydrobenzofurans from o-aminophenols.

An efficient and facile synthesis of trans-dihydrobenzofurans has been accomplished from o-aminophenols and phenylpropenes via a novel (one-pot) diastereoselective Pd-catalyzed oxyarylation reaction. The development and optimization of this method is described.

PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae).

Di-nor-benzofuran neolignan aldehydes, Delta(7)-3,4-methylenedioxy-3'-methoxy-8',9'-dinor-4',7-epoxy-8,3'-neolignan-7'-aldehyde (ocophyllal A) 1, Delta(7)-3,4,5,3'-tetramethoxy-8',9'-dinor-4',7-epoxy-8,3'-neolignan-7'-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 3'S, 4'S, 5'R)-Delta(8)'-4'-hydroxy-5'-methoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan (ocophyllol A) 3, (7R, 8R, 3'S, 4'S, 5'R)-Delta(8)'-4'-hydroxy-3,4,5'-trimethoxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan (ocophyllol B) 4, (7R, 8R, 3'S, 4'S, 5'R)-Delta(8)'-4'-hydroxy-3,4,5,5'-tetramethoxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan (ocophyllol C) 5, as well as 2'-epi-guianin 6 and (+)-licarin B 7, were isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1-7. Although compound 6 was the most potent PAF-antagonist, compounds 3-5 showed some activity.

Macrophyllin-type bicyclo[3.2.1]octanoid neolignans from the leaves of Pleurothyrium cinereum.

Four new macrophyllin-type bicyclo[3.2.1]octanoid neolignans, (7S,8R,3'S,5'R)-Delta(8')-5,5',3'-trimethoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-2',4'-dioxo-7.3',8.5'-neolignan (cinerin A), 1, (7R,8R,3'S,4'R,5'R)-Delta(8')-4'-hydroxy-5,5'-dimethoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan (cinerin B), 2, (7S,8R,3'R,4'S,5'R)-Delta(8')-4'-hydroxy-5,5',3'-trimethoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-2'-oxo-7.3',8.5'-neolignan (cinerin C), 3, and (7S,8R,2'R,3'S,5'R)-Delta(8')-2'-hydroxy-5,5'-dimethoxy-3,4-methylenedioxy-2',3',4',5'-tetrahydro-4'-oxo-7.3',8.5'-neolignan (cinerin D), 4, along with the known diterpene kaurenoic acid 5, were isolated from the leaves of Pleurothyrium cinereum. The structures and configuration of these compounds were determined by extensive spectroscopic analysis. Cinerins A-D (1-4) were tested for their inhibition efficacy of platelet activating factor (PAF)-induced aggregation of rabbit platelets. Compound 3 was the most potent PAF antagonist. Compounds 1-5 were tested against Mycobacterium tuberculosis (H(37)Rv strain) using the MABA method. Compound 5 induced 91.3% growth inhibition at 50 microg mL(-1). Compounds 1-5 showed no significant inhibitory activity against some Gram-positive and Gram-negative bacteria by the agar-well diffusion method.

NUEVO ALCALOIDE OXOAPORFíNICO Y OTROS CONSTITUYENTES QUÍMICOS AISLADOS DE Pleurothyrium cinereum (LAURACEAE) / NOVEL OXOAPORPHINE ALKALOID AND OTHER CHEMICAL CONSTITUENTS ISOLATED FROM Pleurothyrium cinereum (LAURACEAE) / NOVO ALCALÓIDE OXOAPORFINÓIDE E OUTROS CONSTITUINTES QUÍMICOS ISOLADOS DE Pleurothyrium cinereum (LAURACEAE)

Del extracto etanílico de las hojas de Pleurothyrium cinereum (Lauraceae) fue aislado el nuevo alcaloide oxoaporfínico oxiprenilado 1,2-metilendioxi-9,10-dimetoxi- 3-isopreniloxi-7H-dibenzo&[de, g]quinolin-7-ona (Pleurotirina) , el cual fue purificado por métodos cromatográficos y cuya elucidación estructural se realizó mediante técnicas espectroscípicas (RMN-1H, RMN-13C, RMN-2D y EM). Junto al alcaloide fueron aislados los compuestos thalicminina , ácido (-)-ent-16-kauren-19-oico , alloxantoxiletina , xantiletina , dihydroflavokawina B , 3-metoxi-3,4-metilendioxi- 4,7-epoxi-nor-8.5-neolignan-7.8-dieno y friedelina , los cuales se reportan por primera vez para la especie y para el género.

A new oxyprenylated oxoaporphine alkaloid 1,2-methylendioxy-9,10-dimethoxy- 3-isopentenyloxy-7H-dibenzo[de,g]quinolin- 7-one (Pleurotirine) was isolated from the ethanolic extract of leaves of Pleurothyrium cinereum (Lauraceae), which was purified using chromatographic methods. Its structure was elucidated by means of spectroscopic techniques (1H-NMR, 13C-NMR, 2D-NMR and MS). Together with this alkaloid were isolated thalicminine , ent-kaurenoic acid , xanthyletin , alloxanthoxyletin , dihidroflavokawin B , 3-methoxy-3, 4-methylendioxy-4, 7-epoxynor- 8.5- neolignan-7.8-diene and friedelin 8 They are reported for the first time for this species and genus.

Do extrato etanólico das folhas de Pleurothyrium cinereum (Lauraceae) foi isolado o novo alcalóide oxoaporfinóide oxiprenilado 1,2-metilendioxi- 9,10- dimetoxi- 3-isopreniloxi-7H-dibenzo[de,g]quinolin- 7-ona (Pleurotirina) , quais foram isolados por métodos cromatograficos e o elucidación estrutural foi feito por meio das técnicas espectroscopicas (RMN-1H, RMN-13C, RMN-2D, EM). Ao lado do alcalóide foram isolados os compostos thalicminina , ácido (-)-ent-16-kauren- 19-óico , alloxantoxiletina , xantiletina , dihidroflavokawin B , 3’-metoxi- 3,4-metilendioxi-4’,7-epoxi-nor-8.5’- neolignan-7.8’-dieno e friedelina , quais são relatados para a primeira vez para a espécie e a sorte.

Signal amplification using "spot-on-a-chip" technology for the identification of proteins via MALDI-TOF MS.

The presented "spot-on-a-chip" technology enables easy enrichment of samples in the low nanomolar (1-5 nM) range and provides a fast and reliable automated sample preparation method for performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis with high sensitivity and throughput. Through microdispensing, which allows accurate deposition of 60-pL droplets, dilute samples were enriched by making multiple droplet depositions in nanovials. The sample was confined to a defined spot area (300 x 300 microm), and multiple depositions increase the surface density of analyte in the nanovial, thereby providing detection of low attomole levels. The impact of the nanovial geometry with respect to the MALDI-TOF MS resolution for peptides deposited in the microfabricated silicon vials was investigated and the optimal geometry and size were determined. The spot-on-a-chip technology, that is, the combination of microdispensing, micromachined silicon nanovials and on-spot enrichment provides a signal amplification of at least 10-50 times as compared to an ordinary sample preparation. The linearity of the enrichment effect is shown by the analysis of a peptide mixture at the 5 nM level. The signal amplification provided by the spot-on-a-chip enrichment is demonstrated by the analysis of relevant biological samples, interleukin-8 from a spiked cell supernatant, and by successful protein identification of an excised spot from a high-sensitivity silver-stained two-dimensional electrophoresis gel separation.

Downsizing proteolytic digestion and analysis using dispenser-aided sample handling and nanovial matrix-assisted laser/desorption ionization-target arrays.

An efficient technique for enzymatic digestion of proteins in nanovial arrays and identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization (MALDI-MS) is presented in this work. Through dispensing of a protein solution with simultaneous evaporation the protein (substrate) is concentrated up to 300 times in-vial. At higher substrate concentrations the catalytic turnover numbers increase according to the Michaelis-Menten kinetics. Therefore, the dispenser-aided nanodigestion is valuable for identification of low-level proteins (10 nM-500 nM) as well as for automatic high efficiency digestions performed in 0.2-10 min. As an example of low-level protein identification, a 10 nM solution of lysozyme C was unambiguously identified after 5 min of nanodigestion. Moreover, only 30 s nanodigestion was sufficient to identify hemoglobin (10 microM), exemplifying the fast catalysis of the nanodigestion technique. The developed silicon flow-through piezoelectric dispenser is adapted for low-volume and preconcentrated samples in the nL-microL range and provides fast, accurate and contact-free sample positioning into the nanovials. In this work, the properties of the nanodigestion concept regarding proteins of different characteristics are explored. Furthermore, the potential of automated protein identification using precoated proteolytic nanovial-arrays is demonstrated.