Patricio Iturriaga-Vásquez

Aminochrome induces disruption of actin, alpha-, and beta-tubulin cytoskeleton networks in substantia-nigra-derived cell line.

In previous studies, we observed that cells treated with aminochrome obtained by oxidizing dopamine with oxidizing agents dramatically changed cell morphology, thus posing the question if such morphological changes were dependent on aminochrome or the oxidizing agents used to produce aminochrome. Therefore, to answer this question, we have now purified aminochrome on a CM-Sepharose 50–100 column and, using NMR studies, we have confirmed that the resulting aminochrome was pure and that it retained its structure. Fluorescence microscopy with calcein-AM and transmission electron microscopy showed that RCSN-3 cells presented an elongated shape that did not change when the cells were incubated with 50 μM aminochrome or 100 μM dicoumarol, an inhibitor of DT-diaphorase. However, the cell were reduced in size and the elongated shape become spherical when the cells where incubated with 50 μM aminochrome in the presence of 100 μM dicoumarol. Under these conditions, actin, alpha-, and beta-tubulin cytoskeleton filament networks became condensed around the cell membrane. Actin aggregates were also observed in cells processes that connected the cells in culture. These results suggest that aminochrome one-electron metabolism induces the disruption of the normal morphology of actin, alpha-, and beta-tubulin in the cytoskeleton, and that DT-diaphorase prevents these effects.

Molecular Determinants for Competitive Inhibition of α4β2 Nicotinic Acetylcholine Receptors

The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [3H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHβE or erysodine, which highlights β2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids.

2-Arylthiomorpholine derivatives as potent and selective monoamine oxidase B inhibitors

2-Arylthiomorpholine and 2-arylthiomorpholin-5-one derivatives, designed as rigid and/or non-basic phenylethylamine analogues, were evaluated as rat and human monoamine oxidase inhibitors. Molecular docking provided insight into the binding mode of these inhibitors and rationalized their different potencies. Making the phenylethylamine scaffold rigid by fixing the amine chain in an extended six-membered ring conformation increased MAO-B (but not MAO-A) inhibitory activity relative to the more flexible alpha-methylated derivative. The presence of a basic nitrogen atom is not a prerequisite in either MAO-A or MAO-B. The best K(i) values were in the 10(-8)M range, with selectivities towards human MAO-B exceeding 2000-fold.

Monoamine Oxidase Inhibitory Properties of Optical Isomers and N-substituted Derivatives of 4-methylthioamphetamine

(+/-)-4-Methylthioamphetamine (MTA) was resolved into its enantiomers, and a series of N-alkyl derivatives of the parent compound, as well as its alpha-ethyl analogue, were prepared. The monoamine oxidase (MAO) inhibitory properties of these substances were evaluated in vitro, using a crude rat brain mitochondrial suspension as the source of enzyme. All compounds produced a selective, reversible and concentration-related inhibition of MAO-A. (+)-MTA proved to be the most potent inhibitor studied, while all the other derivatives were less active than the parent compound, with (-)-MTA being about 18 times less potent than the (+) isomer. The analysis of structure-activity relationships indicates that the introduction of alkyl substituents on the amino group of MTA leads to a reduction in the potency of the derivatives as MAO-A inhibitors, an effect which increases with the size of the substituent.

Naphthylisopropylamine and N-benzylamphetamine derivatives as monoamine oxidase inhibitors.

A series of naphthylisopropylamine and N-benzyl-4-methylthioamphetamine derivatives were evaluated as monoamine oxidase inhibitors. Their potencies were compared with those of a series of amphetamine derivatives, to test if the increase of electron richness of the aromatic ring and overall size of the molecule might improve their potency as enzyme inhibitors. Molecular dockings were performed to gain insight regarding the binding mode of these inhibitors and rationalize their different potencies. In the case of naphthylisopropylamine derivatives, the increased electron-donating capacity and size of the aromatic moiety resulting from replacement of the phenyl ring of amphetamine derivatives by a naphthalene system resulted in more potent compounds. In the other case, extension of the arylisopropylamine molecule by N-benzylation of the amino group led to a decrease in potency as monoamine oxidase inhibitors.

Electrophysiological characterization of nicotinic acetylcholine receptors in cat petrosal ganglion neurons in culture: effects of cytisine and its bromo derivatives.

Petrosal ganglion neurons are depolarized and fire action potentials in response to acetylcholine and nicotine. However, little is known about the subtype(s) of nicotinic acetylcholine receptors involved, although alpha4 and alpha7 subunits have been identified in petrosal ganglion neurons. Cytisine, an alkaloid unrelated to nicotine, and its bromo derivatives are agonists exhibiting different affinities, potencies and efficacies at nicotinic acetylcholine receptors containing alpha4 or alpha7 subunits. To characterize the receptors involved, we studied the effects of these agonists and the nicotinic acetylcholine receptor antagonists hexamethonium and alpha-bungarotoxin in isolated petrosal ganglion neurons. Petrosal ganglia were excised from anesthetized cats and cultured for up to 16 days. Using patch-clamp technique, we recorded whole-cell currents evoked by 5-10 s applications of acetylcholine, cytisine or its bromo derivatives. Agonists and antagonists were applied by gravity from a pipette near the neuron surface. Neurons responded to acetylcholine, cytisine, 3-bromocytisine and 5-bromocytisine with fast inward currents that desensitized during application of the stimuli and were reversibly blocked by 1 microM hexamethonium or 10 nM alpha-bungarotoxin. The order of potency of the agonists was 3-bromocytisine >> acetylcholine approximately = cytisine >> 5-bromocytisine, suggesting that homomeric alpha7 neuronal nicotinic receptors predominate in cat petrosal ganglion neurons in culture.

Simplified Tetrandrine Congeners as Possible Antihypertensive Agents with a Dual Mechanism of Action

A series of O- and/or N-substituted derivatives of (+/-)-coclaurine (1a) were synthesized as simplified structural mimics of the antihypertensive alkaloid tetrandrine (2) and assayed for binding to brain cortical sites labeled with the alpha(1)-adrenergic radioligand [(3)H]prazosin or the calcium channel radioligand [(3)H]diltiazem. The introduction of O-benzyl groups on the coclaurine molecule, which exhibits only adrenergic antagonist activity, led to the appearance of calcium channel blocking activity comparable to that of tetrandrine while retaining adrenolytic activity in the same concentration range. Contraction of aortal rings with noradrenaline or KCl was relaxed more potently by some of these coclaurine derivatives than by tetrandrine, suggesting leads for the development of novel antihypertensive drugs with a dual mechanism of action.

Design, Synthesis, Binding and Docking-Based 3D-QSAR Studies of 2-Pyridylbenzimidazoles—A New Family of High Affinity CB1 Cannabinoid Ligands

A series of novel 2-pyridylbenzimidazole derivatives was rationally designed and synthesized based on our previous studies on benzimidazole 14, a CB1 agonist used as a template for optimization. In the present series, 21 compounds displayed high affinities with Ki values in the nanomolar range. JM-39 (compound 39) was the most active of the series (KiCB1 = 0.53 nM), while compounds 31 and 44 exhibited similar affinities to WIN 55212-2. CoMFA analysis was performed based on the biological data obtained and resulted in a statistically significant CoMFA model with high predictive value (q2 = 0.710, r2 = 0.998, r2pred = 0.823).

Similarities between the Binding Sites of SB-206553 at Serotonin Type 2 and Alpha7 Acetylcholine Nicotinic Receptors: Rationale for Its Polypharmacological Profile

Evidence from systems biology indicates that promiscuous drugs, i.e. those that act simultaneously at various protein targets, are clinically better in terms of efficacy, than those that act in a more selective fashion. This has generated a new trend in drug development called polypharmacology. However, the rational design of promiscuous compounds is a difficult task, particularly when the drugs are aimed to act at receptors with diverse structure, function and endogenous ligand. In the present work, using docking and molecular dynamics methodologies, we established the most probable binding sites of SB-206553, a drug originally described as a competitive antagonist of serotonin type 2B/2C metabotropic receptors (5-HT2B/2CRs) and more recently as a positive allosteric modulator of the ionotropic α7 nicotinic acetylcholine receptor (nAChR). To this end, we employed the crystal structures of the 5-HT2BR and acetylcholine binding protein as templates to build homology models of the 5-HT2CR and α7 nAChR, respectively. Then, using a statistical algorithm, the similarity between these binding sites was determined. Our analysis showed that the most plausible binding sites for SB-206553 at 5-HT2Rs and α7 nAChR are remarkably similar, both in size and chemical nature of the amino acid residues lining these pockets, thus providing a rationale to explain its affinity towards both receptor types. Finally, using a computational tool for multiple binding site alignment, we determined a consensus binding site, which should be useful for the rational design of novel compounds acting simultaneously at these two types of highly different protein targets.

Evaluation of benzyltetrahydroisoquinolines as ligands for neuronal nicotinic acetylcholine receptors

Effects of derivatives of coclaurine (C), which mimic the ‘eastern’ or the nonquaternary halves of the alkaloids tetrandrine or d‐tubocurarine, respectively, both of which are inhibitors of nicotinic acetylcholine receptors (nACh), were examined on recombinant, human α7, α4β2 and α4β4 nACh receptors expressed in Xenopus oocytes and clonal cell lines using two‐electrode voltage clamping and radioligand binding techniques. In this limited series, Cs have higher affinity and are most potent at α4 subunit‐containing‐nACh receptors and least potent at homomeric α7 receptors, and this trend is very marked for the N‐unsubstituted C and its O,O′‐bisbenzyl derivative. 7‐O‐Benzyl‐N‐methylcoclaurine (BBCM) and its 12‐O‐methyl derivative showed the highest affinities and potencies at all three receptor subtypes, and this suggests that lipophilicity at C7 and/or C12 increases potency. Laudanosine and armepavine (A) were noncompetitive and voltage‐dependent inhibitors of α7, α4β2 or α4β4 receptors, but the bulkier C7‐benzylated 7BNMC (7‐O‐benzyl‐N‐methylcoclaurine) and 7B12MNMC (7‐O‐benzyl‐N,12‐O‐dimethyl coclaurine) were voltage‐independent, noncompetitive inhibitors of nACh receptors. Voltage‐dependence was also lost on going from A to its N‐ethyl analogue. These studies suggest that C derivatives may be useful tools for studies characterising the antagonist and ion channel sites on human α7, α4β2 or α4β4 nACh receptors and for revealing structure–function relationships for nACh receptor antagonists.