Antonio Entrena

Inhibition of neuronal nitric oxide synthase activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin.

We assessed the effects of melatonin, N1‐acetyl‐N 2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10−11−10−3 m), but not AFMK, inhibited nNOS activity in vitro in a dose–response manner. The IC50 value for AMK (70 µm) was significantly lower than for melatonin (>1 mm). A 20% nNOS inhibition was reached with either 10−9 m melatonin or 10−11 m AMK. AMK inhibits nNOS by a non‐competitive mechanism through its binding to Ca2+‐calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mm norharmane, an indoleamine‐2,3‐dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.

Melatonin and its brain metabolite N1‐acetyl‐5‐methoxykynuramine prevent mitochondrial nitric oxide synthase induction in parkinsonian mice

Melatonin prevents mitochondrial failure in models of sepsis through its ability to inhibit the expression and activity of both cytosolic (iNOS) and mitochondrial (i‐mtNOS) inducible nitric oxide synthases. Because Parkinsons disease (PD), like sepsis, is associated with iNOS induction, we assessed the existence of changes in iNOS/i‐mtNOS and their relation with mitochondrial dysfunction in the MPTP model of PD, which also displays increased iNOS expression. We also evaluated the role of melatonin (aMT) and its brain metabolite, N1‐acetyl‐5‐methoxykynuramine (AMK), in preventing i‐mtNOS induction and mitochondrial failure in this model of PD. Mitochondria from substantia nigra (SN) and, to a lesser extent, from striatum (ST) showed a significant increase in i‐mtNOS activity, nitrite levels, oxidative stress, and complex I inhibition after MPTP treatment. MPTP‐induced i‐mtNOS was probably related to mitochondrial failure, because its prevention by aMT and AMK reduced oxidative/nitrosative stress and restored complex I activity. These findings represent the first experimental evidence of a potential role for i‐mtNOS in the mitochondrial failure of PD and support a novel mechanism in the neuroprotective effects of aMT and AMK.

Synthesis, biological evaluation, and docking studies of novel heterocyclic diaryl compounds as selective COX-2 inhibitors.

Three novel series of diaryl heterocyclic derivatives bearing the 2-oxo-5H-furan, 2-oxo-3H-1,3-oxazole, and 1H-pyrazole moieties as the central heterocyclic ring were synthesized and their in vitro inhibitory activities on COX-1 and COX-2 isoforms were evaluated using a purified enzyme assay. The 2-oxo-5H-furan derivative 6b was identified as potent COX inhibitor with selectivity toward COX-1 (COX-1 IC(50)=0.061 microM and COX-2 IC(50)=0.325 microM; selectivity index (SI)=0.19). Among the 1H-pyrazole derivatives, 11b was found to be a potent COX-2 inhibitor, about 38 times more potent than Rofecoxib (COX-2 IC(50)=0.011 microM and 0.398 microM, respectively), but showed no selectivity for COX-2 isoform. Compound 11c demonstrated strong and selective COX-2 inhibitory activity (COX-1 IC(50)=1 microM, COX-2 IC(50)=0.011 microM; SI= approximately 92). Molecular docking studies of compounds 6b and 11b-d into the binding sites of COX-1 and COX-2 allowed to shed light on the binding mode of these novel COX inhibitors.

Medium benzene-fused oxacycles with the 5-fluorouracil moiety: synthesis, antiproliferative activities and apoptosis induction in breast cancer cells

Abstract On the basis of the increase in lipophilicity, novel benzannelated six- and seven-membered derivatives have been synthesized, starting from 2-hydroxybenzyl alcohols, 2-hydroxybenzaldehydes, and catechol. The X-ray structure of (RS)-1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-5-fluorouracil ( 5 ) is presented and compared with its conformational analysis at the semiempirical (AM1) and ab initio (6-31G∗) levels and NOE effects. A good agreement between both experimental and theoretical data was found showing a chair conformation for the 2,3-dihydro-5H-1,4-dioxepin ring and an axial orientation of the 5-FU moiety on the C3 position. Compounds 5 and (RS)-1-(7-methoxy-2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-5-fluorouracil ( 6 ) were found to be the most potent inhibitor of MCF-7 cells growth. (RS)-1-(2,3-Dihydrobenzoxepin-2-yl)-5-fluorouracil ( 8 ) induced apoptosis up to 57.33% of cell population after 24 h.

The Mechanism of Allosteric Coupling in Choline Kinase α1 Revealed by the Action of a Rationally Designed Inhibitor

Applying a CHOK hold: Combined experimental and computational studies of the binding mode of a rationally designed inhibitor of the dimeric choline kinase α1 (CHOKα1) explain the molecular mechanism of negative cooperativity (see scheme) and how the monomers are connected. The results give insight into how the symmetry of the dimer can be partially conserved despite a lack of conservation in the static crystal structures.

Phenylpyrrole derivatives as neural and inducible nitric oxide synthase (nNOS and iNOS) inhibitors.

We have previously described a series of 3-phenyl-4,5-dihydro-1H-pyrazole derivatives as moderately potent nNOS inhibitors. As a follow up of these studies, several new 5-phenyl-1H-pyrrole-2-carboxamide derivatives have been synthesized, and their biological evaluation as in vitro inhibitors of both neural and inducible Nitric Oxide Synthase (nNOS and iNOS) is described. Some of these compounds show good iNOS/nNOS selectivity and the more potent compounds 5-(2-aminophenyl)-1H-pyrrole-2-carboxilic acid methylamide (QFF205) and cyclopentylamide (QFF212) have been tested as regulators of the in vivo nNOS and iNOS activity. Both compounds prevented the increment of the inducible NOS activity in both cytosol (iNOS) and mitochondria (i-mtNOS) observed in the MPTP model of Parkinsons disease.

Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors (III).

We have previously described a series of 4,5-dihydro-1H-pyrazole as moderately potent nNOS inhibitors. As a follow up of these studies, we report here the preparation and the preliminary evaluation of a series of 1-alkyl-3-benzoyl-4,5-dihydro-1H-pyrazole and 1-alkyl-3-benzoyl-1H-pyrazole as potential inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS). None of the reported compounds exhibited significant iNOS or nNOS inhibition, although the 1-benzyl-3-(2-amino-5-chlorobenzoyl)-1H-pyrazole-5-carboxylic acid ethyl ester derivative (10l), which shows an inhibition of 50% versus iNOS at a 1mM final concentration and no activity against nNOS, is potentially amenable of further optimization. The reasons for the inactivity of the reported series are discussed on the basis of docking studies.

5-fluorouracil derivatives. 1. Acyclonucleosides through a tin (IV) chloride-mediated regiospecific ring opening of alkoxy-1,4-diheteroepanes

Abstract The reaction of 5-fluorouracil with the seven-membered acetals 1a-g in the presence of tin (IV) chloride, trimethylchlorosilane and hexamethyldisilazane at room temperature gives 1-{[3-(2-hydroxyethylhetero)-1-alkoxy]alkyl}-5-fluorouracils 2a-f and 1-{[2-(3-hydroxypropoxy)-1-isopropoxy]ethyl}-5-fluorouracil 2g in 31–86% yields. The presence of an heteroatom on the 1-position of the cycloacetal and the use of tin (IV) chloride, capable of a 1,4-chelation, seem to impose their influence in the regiospecific ring opening of 1a-g. The conformational analyses carried out on 2b and (1R,3R)-2e and (1R,3S)-2e clearly indicate that the N1(sp2)-C1-C2-C3 moiety tends to fold in a gauche conformation. The antitumour activities of compounds 2b-g were assessed against HEp human cells showing that 2c is 4-fold more active than 5-FU. The drugs studied do not show any clear toxicity in comparison with the toxic effect of 5-FU.

Discovery of a new binding site on human choline kinase α1: design, synthesis, crystallographic studies, and biological evaluation of asymmetrical bispyridinium derivatives.

Human choline kinase α (CKα) is a validated drug target for the treatment of cancer. In recent years, a large number of CK inhibitors have been synthesized, and one of them is currently being evaluated in Phase I clinical trials as a treatment for solid tumors. Here we have evaluated a new series of asymmetrical biscationic CK inhibitors by means of enzymatic, crystallographic, and antitumor studies. We demonstrate that one of these structures adopts a completely new binding mode not observed before inducing the aperture of an adjacent binding site. This compound shows antiproliferative and apoptotic effects on cancer cells through activation of caspase-3. Therefore, this study not only provides fruitful insights into the design of more efficient compounds that may target different regions in CKα1 but also explains how these compounds induce apoptosis in cancer cells.

Insight into the Inhibition of Human Choline Kinase: Homology Modeling and Molecular Dynamics Simulations

A homology model of human choline kinase (CK‐α) based on the X‐ray crystallographic structure of C. elegans choline kinase (CKA‐2) is presented. Molecular dynamics simulations performed on CK‐α confirm the quality of the model, and also support the putative ATP and choline binding sites. A good correlation between the MD results and reported CKA‐2 mutagenesis assays has been found for the main residues involved in catalytic activity. Preliminary docking studies performed on the CK‐α model indicate that inhibitors can bind to the binding sites of both substrates (ATP and choline). A possible reason for inhibition of choline kinase by Ca2+ ion is also proposed.