Angélica Fierro

Synthesis, Biological Evaluation, and Molecular Simulation of Chalcones and Aurones as Selective MAO-B Inhibitors

A series of chalcones and aurones were synthesized and evaluated in vitro as monoamine oxidase inhibitors (MAOi). Our results show that aurones, which had not been previously reported as MAOi, are MAO‐B inhibitors. Thus, both families inhibited selectively the B isoform of MAO in the micromolar range, offering novel scaffolds for the design of new and potent MAO inhibitors. The main structural requirements for their activity were characterized with the aid of 3D‐QSAR and docking studies.

Revealing Monoamine Oxidase B Catalytic Mechanisms by Means of the Quantum Chemical Cluster Approach.

Two of the possible catalytic mechanisms for neurotransmitter oxidative deamination by monoamine oxidase B (MAO B), namely, polar nucleophilic and hydride transfer, were addressed in order to comprehend the nature of their rate-determining step. The Quantum Chemical Cluster Approach was used to obtain transition states of MAO B complexed with phenylethylamine (PEA), benzylamine (BA), and p-nitrobenzylamine (NBA). The choice of these amines relies on their importance to address MAO B catalytic mechanisms so as to help us to answer questions such as why BA is a better substrate than NBA or how para-substitution affects substrates reactivity. Transition states were later validated by comparison with the experimental free energy barriers. From a theoretical point of view, and according to the our reported transition states, their calculated barriers and structural and orbital differences obtained by us among these compounds, we propose that good substrates such as BA and PEA might follow the hydride transfer pathway while poor substrates such as NBA prefer the polar nucleophilic mechanism, which might suggest that MAO B can act by both mechanisms. The low free energy barriers for BA and PEA reflect the preference that MAO B has for hydride transfer over the polar nucleophilic mechanism when catalyzing the oxidative deamination of neurotransmitters.

2-Phenylaminonaphthoquinones and related compounds: Synthesis, trypanocidal and cytotoxic activities

A series of new 2-aminonaphthoquinones and related compounds were synthesized and evaluated in vitro as trypanocidal and cytotoxic agents. Some tested compounds inhibited epimastigote growth and trypomastigote viability. Several compounds showed similar or higher activity and selectivity as compared with current trypanocidal drug, nifurtimox. Compound 4l exhibit higher selectivity than nifurtimox against Trypanosoma cruzi in comparison with Vero cells. Some of the synthesized quinones were tested against cancer cells and normal fibroblasts, showing that certain chemical modifications on the naphthoquinone moiety induce and excellent increase the selectivity index of the cytotoxicity (4g and 10). The results presented here show that the anti-T. cruzi activity of 2-aminonaphthoquinones derivatives can be improved by the replacement of the benzene ring by a pyridine moiety. Interestingly, the presence of a chlorine atom at C-3 and a highly lipophilic alkyl group or aromatic ring are newly observed elements that should lead to the discovery of more selective cytotoxic and trypanocidal compounds.

Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.

Molecular characterization of the chalcone isomerase gene family in Deschampsia antarctica

Deschampsia antarctica (Poaceae) is one of the two vascular plants known to have colonized the Antarctic region. Studies examining the biosynthesis of flavonoids, compounds which plants use, for example, for protection against overexposure to UV light or as antioxidants that scavenge free radicals and other oxidative species, in D. antarctica may provide clues to its success in that extreme environment. We characterized the family of genes encoding chalcone isomerase (CHI EC 5.5.1.6), an important enzyme involved in flavonoid biosynthesis, in D. antarctica. Sequence analysis of the three family members revealed differences in numbers of introns and lengths of coding regions among the three and suggest that DaCHI3 is likely a pseudogene (ψDaChi2). Salinity stress resulted in differential mRNA expression of the DaCHI genes with ψDaCHI2 exhibiting the earliest response (3-h post-treatment), induced by as much as sevenfold, while DaCHI1 and DaCHI2 mRNAs accumulated later (3d and 5d post-treatment, respectively) and, in the case of DaCHI2, with a response of nearly sixfold. We discuss how differences in the proposed gene structures, deduced protein characteristics, and mRNA expression patterns suggest that the members of this gene family may have unique functions in the phenylpropanoid pathway in D. antarctica.

Influence of protonation on substrate and inhibitor interactions at the active site of human monoamine oxidase-A

Although substrate conversion mediated by human monoaminooxidase (hMAO) has been associated with the deprotonated state of their amine moiety, data regarding the influence of protonation on substrate binding at the active site are scarce. Thus, in order to assess protonation influence, steered molecular dynamics (SMD) runs were carried out. These simulations revealed that the protonated form of the substrate serotonin (5-HT) exhibited stronger interactions at the protein surface compared to the neutral form. The latter displayed stronger interactions in the active site cavity. These observations support the possible role of the deprotonated form in substrate conversion. Multigrid docking studies carried out to rationalize the role of 5-HT protonation in other sites besides the active site indicated two energetically favored docking sites for the protonated form of 5-HT on the enzyme surface. These sites seem to be interconnected with the substrate/inhibitor cavity, as revealed by the tunnels observed by means of CAVER program. pK(a) calculations in the surface loci pointed to Glu³²⁷, Asp³²⁸, His⁴⁸⁸, and Asp¹³² as candidates for a possible in situ deprotonation step. Docking analysis of a group of inhibitors (structurally related to substrates) showed further interactions with the same two docking access sites. Interestingly, the protonated/deprotonated amine moiety of almost all compounds attained different docking poses in the active site, none of them oriented to the flavin moiety, thus producing a more variable and less productive orientations to act as substrates. Our results highlight the role of deprotonation in facilitating substrate conversion and also might reflect the necessity of inhibitor molecules to adopt specific orientations to achieve enzyme inhibition.

Why p-OMe- and p-Cl-β-Methylphenethylamines Display Distinct Activities upon MAO-B Binding

Despite their structural and chemical commonalities, p-chloro-β-methylphenethylamine and p-methoxy-β-methylphenethylamine display distinct inhibitory and substrate activities upon MAO-B binding. Density Functional Theory (DFT) quantum chemical calculations reveal that β-methylation and para-substitution underpin the observed activities sustained by calculated transition state energy barriers, attained conformations and key differences in their interactions in the enzyme’s substrate binding site. Although both compounds meet substrate requirements, it is clear that β-methylation along with the physicochemical features of the para-substituents on the aromatic ring determine the activity of these compounds upon binding to the MAO B-isoform. While data for a larger set of compounds might lend generality to our conclusions, our experimental and theoretical results strongly suggest that the contrasting activities displayed depend on the conformations adopted by these compounds when they bind to the enzyme.

The role of solvent exclusion in the interaction between D124 and the metal site in SOD1: implications for ALS

Structural changes in the metal site of the copper–zinc superoxide dismutase (SOD1) are involved in the various mechanisms proposed for the pathogenesis of the SOD1-linked familial form of amyotrophic lateral sclerosis (ALS). Elucidating how the metal site of SOD1 can be disrupted by ALS-linked mutations is important for a better understanding of the pathogenesis of the disease and for developing more efficient treatments. Residue D124, a second-sphere ligand of the copper and zinc ions, is known from experimental studies to be essential for the integrity of the metal-site structure. In this work, we used density functional theory calculations and molecular dynamics simulations to elucidate which factors keep D124 attached to the metal site and how structural changes may disrupt the binding between D124 and the metal first-sphere ligands. The calculations show that D124 is kept attached to the metal site in a kinetic trap. The exclusion of solvent molecules by the electrostatic loop of the protein is found to create the binding of D124 to the metal site. The calculations also indicate that changes in the structure of the electrostatic loop of the protein can weaken the D124–metal site interaction, lowering the affinity of the zinc site for the metal. Destabilization of the electrostatic loop of SOD1 has been previously shown to be a common property of ALS-linked variants of the protein, but its role in the pathogenesis of SOD1-linked ALS has not been elucidated.

Inclusion of Ethyl Acetoacetate Bearing 7‐Hydroxycoumarin Dye by β‐Cyclodextrin and its Cooperative Assembly with Mercury(II) Ions: Spectroscopic and Molecular Modeling Studies

The inclusion of the fluorescent organic dye, ethyl 3-(7-hydroxy-2-oxo-2H-chromen-3-yl)-3-oxopropanoate (1) by the host β-cyclodextrin (β-CD), and its response toward mercuric ions (Hg2+ ), was studied by UV/Vis, fluorescence, and 1 H NMR spectroscopic analyses, mass spectrometry and molecular modeling studies. 1 H NMR measurements together with molecular modeling studies for dye 1 demonstrate that it exhibits two tautomeric forms (keto and enol); however, when the dye is included into the β-CD cavity, the enol form predominates. Moreover, by using spectroscopic and spectrometry techniques, a 1:1 stoichiometry was determined for the complexes formed between dye 1 (enol form) and β-CD, with a binding constant (Kb1 =1.8×104  m-1 ) and for the dye 1 (keto form)-Hg2+ (Kb2 =2.3×103  m-1 ). Interestingly, in the presence of 1-β-CD complex and mercuric ions, a ternary supramolecular system (Hg-1-β-CD complex) was established, with a 1:1:1 stoichiometry and a Kb3 value of 4.3×103  m-1 , with the keto form of the dye being the only one present in this assembly. The three-component system provides a starting point for the development of novel and directed supramolecular assemblies.

Controlled keto–enol tautomerism of coumarin containing β-ketodithioester by its encapsulation in cucurbit[7]uril

Conventional spectroscopy techniques (UV-Vis, fluorescence and NMR), mass spectrometry and molecular modeling studies are used to assess the inclusion of cucurbit[7]uril (CB7) with coumarin 7-N,N-diethylamino-2H-chromen-2-one derivatives bearing ethyl acetoacetate (CAM1) and methyl β-ketodithioester (CAM2) moieties. For the first time, it has been demonstrated that the macrocycle CB7 is able to stabilize the keto tautomeric form of both coumarin derivatives. More interestingly, it was also seen that for CAM2, the macrocycle CB7 shifts the keto–enol equilibrium from its enol form to the keto tautomer after its inclusion, establishing important differences with inclusion in β-CD, while for CAM1, the macrocycle CB7 maintains the original keto form.