An Alternative Method for the Selective Synthesis of Ortho-nitro Anilines Using Bismuth Nitrate Pentahydrate.

BACKGROUND: Nitroaromatic compounds are important scaffolds used for the syn-thesis of a variety of compounds, such as explosives, herbicides, dyes, perfumes and phar-maceuticals. Bismuth nitrate pentahydrate is a widely used reagent in organic synthesis; how-ever, its utility as a nitrating agent for anilines is underexplored. OBJECTIVE: The aim of this work is to propose and find the proper reaction conditions of an alternative nitrating agent constituted by a mixture of bismuth nitrate / acetic anhydride in DCM with a series of substituted anilines under mild reflux. METHODS: Several anilines having both activating and deactivating substituents in the ortho, meta and para positions were the substrate for the nitration reaction. Experimental conditions were performed in "one-pot" conditions before product purification. RESULTS: Bi(NO3)3•5H2O demonstrated to be effective and somehow regioselective when it came to the nitration of anilines in the ortho position. Although other products were also identified under these conditions, in most cases, the ortho derivative was the major or even the only product obtained with moderate to high yields in the range of 50% - 96%. CONCLUSION: Bi(NO3)3•5H2O is an efficient and safe nitrating agent since the use of concen-trated and corrosive acids like sulfuric and nitric is avoided; furthermore, bismuth nitrate is low-priced and no special care nor equipment is required.

Cytotoxicity Activity of Some meso-Dihydroguaiaretic Acid Derivatives and Mode of Action of the Most Active Compound.

The aim of this study was to screen sixteen meso-1 semi-synthetic derivatives bearing ether, esther, carbamate, phosphate or aminoether functional groups against five cancer cell lines: MCF-7 (breast), A549 (lung), HepG2 (liver), HeLa (cervix), and DU145 (prostate) at 25 µM using the MTT assay. Results from the screening showed that two derivatives had the lowest percentage of cell viability at 25 µM, the aminoether derivative meso-11 and the esther derivative meso-20 against A549 (44.15±0.78 %) and MCF-7 (41.60±0.92 %), respectively. Then, it was determined the IC50 value of each compound against their most sensitive cancer cell line. Results showed that aminoether derivative meso-11 showed potent cytotoxicity against A549 (IC50 =17.11±2.11 µM), whereas it resulted more cytotoxic against the LL-47 lung normal cell line (IC50 =9.49±1.19 µM) having a Selective Index (SI) of 0.55. On the other hand, the esther derivative meso-20 exhibited potent activity against MCF-7 (IC50 =18.20±1.98 µM), whereas it displayed moderate cytotoxicity against the MCF-10 breast normal cell line (IC50 =41.22±2.17 µM) with a SI of 2.2. Finally, studies on the mechanism of action of meso-20 indicated disruption of MCF-7 plasma membrane in vitro and the AMPK activation in silico.

Evaluation of the Antioxidant Activity of Levetiracetam in a Temporal Lobe Epilepsy Model.

Epilepsy is a neurological disorder in which it has been shown that the presence of oxidative stress (OS) is implicated in epileptogenesis. The literature has shown that some antiseizure drugs (ASD) have neuroprotective properties. Levetiracetam (LEV) is a drug commonly used as an ASD, and in some studies, it has been found to possess antioxidant properties. Because the antioxidant effects of LEV have not been demonstrated in the chronic phase of epilepsy, the objective of this study was to evaluate, for the first time, the effects of LEV on the oxidant-antioxidant status in the hippocampus of rats with temporal lobe epilepsy (TLE). The in vitro scavenging capacity of LEV was evaluated. LEV administration in rats with TLE significantly increased superoxide dismutase (SOD) activity, increased catalase (CAT) activity, but did not change glutathione peroxidase (GPx) activity, and significantly decreased glutathione reductase (GR) activity in comparison with epileptic rats. LEV administration in rats with TLE significantly reduced hydrogen peroxide (H2O2) levels but did not change lipoperoxidation and carbonylated protein levels in comparison with epileptic rats. In addition, LEV showed in vitro scavenging activity against hydroxyl radical (HO•). LEV showed significant antioxidant effects in relation to restoring the redox balance in the hippocampus of rats with TLE. In vitro, LEV demonstrated direct antioxidant activity against HO•.

The Proliferating Cell Nuclear Antigen (PCNA) Transcript Variants as Potential Relapse Markers in B-Cell Acute Lymphoblastic Leukemia.

Leukemia is the most common childhood malignancy in Mexico, representing more than 50% of all childhood cancers. Although treatment leads to a survival of up to 90% in developing countries, in our country, it is less than 65%. Additionally, ~30% of patients relapse with poor prognosis. Alternative splicing plays an important role in transcriptome diversity and cellular biology. This mechanism promotes an increase in the assortment of proteins with potentially distinct functions from a single gene. The proliferating cell nuclear antigen (PCNA) gene encodes two transcripts for the same protein of 261 amino acids, which is associated with several important cellular processes and with several types of cancer. However, the diversity of the transcript variants expressed in this condition is not clear. Then, we used microarray gene expression to identify changes in the exon expression level of PCNA. The data were validated using RT-PCR and Sanger sequencing, and three additional transcripts (PCNA_V3, PCNA_V4, and PCNA_V5) were identified. Computational analyses were used to determine the potential proteins resulting, their structure, and interactions with PCNA native protein and themselves. Additionally, the PCNA transcript variants were inhibited using specific siRNA, determining that their inhibition contributes to the malignant characteristics in vitro. Finally, we quantified the PCNA transcript variants in acute lymphoblastic leukemia samples and identified their expression in this disease. Based on the clinical characteristics, we determined that PCNA_V2 and PCNA_V4 are expressed at significantly low levels in relapsed B-ALL patients. We conclude that the low expression of PCNA_V2 and PCNA_V4 could be a potential molecular marker of relapse in acute lymphoblastic leukemia patients.

Levetiracetam Mechanisms of Action: From Molecules to Systems.

Epilepsy is a chronic disease that affects millions of people worldwide. Antiepileptic drugs (AEDs) are used to control seizures. Even though parts of their mechanisms of action are known, there are still components that need to be studied. Therefore, the search for novel drugs, new molecular targets, and a better understanding of the mechanisms of action of existing drugs is still crucial. Levetiracetam (LEV) is an AED that has been shown to be effective in seizure control and is well-tolerable, with a novel mechanism of action through an interaction with the synaptic vesicle protein 2A (SV2A). Moreover, LEV has other molecular targets that involve calcium homeostasis, the GABAergic system, and AMPA receptors among others, that might be integrated into a single mechanism of action that could explain the antiepileptogenic, anti-inflammatory, neuroprotective, and antioxidant properties of LEV. This puts it as a possible multitarget drug with clinical applications other than for epilepsy. According to the above, the objective of this work was to carry out a comprehensive and integrative review of LEV in relation to its clinical uses, structural properties, therapeutical targets, and different molecular, genetic, and systemic action mechanisms in order to consider LEV as a candidate for drug repurposing.

Structure-Activity Relationship of N-Phenylthieno[2,3-b]pyridine-2-carboxamide Derivatives Designed as Forkhead Box M1 Inhibitors: The Effect of Electron-Withdrawing and Donating Substituents on the Phenyl Ring.

We report synthesis, characterization, biological evaluation, and molecular-docking studies of 18 thieno[2,3-b]pyridines with a phenylacetamide moiety at position 2, which is disubstituted with F, Cl, Br, or I at position 4, and with electron-withdrawing and electron-donating groups (-CN, -NO2, -CF3, and -CH3) at position 2, to study how the electronic properties of the substituents affected the FOXM1-inhibitory activity. Among compounds 1-18, only those bearing a -CN (regardless of the halogen) decreased FOXM1 expression in a triple-negative breast cancer cell line (MDA-MB-231), as shown by Western blotting. However, only compounds 6 and 16 decreased the relative expression of FOXM1 to a level lower than 50%, and hence, we determined their anti-proliferative activity (IC50) in MDA-MB-231 cells using the MTT assay, which was comparable to that observed with FDI-6, in contrast to compound 1, which was inactive according to both Western blot and MTT assays. We employed molecular docking to calculate the binding interactions of compounds 1-18 in the FOXM1 DNA-binding site. The results suggest a key role for residues Val296 and Leu289 in this binding. Furthermore, we used molecular electrostatic potential maps showing the effects of different substituents on the overall electron density.

Benzimidazole Derivatives as New and Selective Inhibitors of Arginase from Leishmania mexicana with Biological Activity against Promastigotes and Amastigotes.

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 µM and 82 µM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.

Endothelial TRP channels and cannabinoid receptors are involved in affinin-induced vasodilation.

Affinin is mainly recognized by its antinociceptive effect. Recently, our research group demonstrated that this compound produces vasodilation via activation of the gasotransmitters signaling pathways. However, the molecular targets of affinin were not identified. Considering the structural similarity of this alkamide with anandamide, we hypothesized that affinin-induced vasodilation could involve participation of TRP channels and cannabinoid receptors. In this work, by using the isolated rat aorta assay, we assessed involvement of TRP channels, the cannabinoid system, and the HNO-CGRP-TRPA1 pathway on the mechanism of action of affinin. Additionally, we measured NO and H2S levels elicited by affinin on rat aorta homogenates and carried out computer simulations of molecular interactions between affinin and the TRPA1 and TRPV1 channels and the CB1 receptor. Our results indicated that affinin induces an increase in aortic NO and H2S levels. We found evidence that the vasodilator effect induced by affinin involves activation of TRPA1 and TRPV1 channels and the CB1 and eCB receptors. In silico analyses showed that affinin is able to bind with high affinity to these molecular targets. Moreover, we also proved that affinin-induced vasodilation is partly mediated via activation of the HNO-TRPA1-CGRP pathway. Based on these results we propose a novel mechanism of action to explain the vasodilatory effect of affinin, which could be developed as an alternative drug to treat cardiovascular diseases.

Behavior of Chemokine Receptor 6 (CXCR6) in Complex with CXCL16 Soluble form Chemokine by Molecular Dynamic Simulations: General Protein‒Ligand Interaction Model and 3D-QSAR Studies of Synthetic Antagonists.

The CXCR6‒CXCL16 axis is involved in several pathological processes, and its overexpression has been detected in different types of cancer, such as prostate, breast, ovary, and lung cancer, along with schwannomas, in which it promotes invasion and metastasis. Moreover, this axis is involved in atherosclerosis, type 1 diabetes, primary immune thrombocytopenia, vitiligo, and other autoimmune diseases, in which it is responsible for the infiltration of different immune system cells. The 3D structure of CXCR6 and CXCL16 has not been experimentally resolved; therefore, homology modeling and molecular dynamics simulations could be useful for the study of this signaling axis. In this work, a homology model of CXCR6 and a soluble form of CXCL16 (CXCR6‒CXCL16s) are reported to study the interactions between CXCR6 and CXCL16s through coarse-grained molecular dynamics (CG-MD) simulations. CG-MD simulations showed the two activation steps of CXCR6 through a decrease in the distance between the chemokine and the transmembrane region (TM) of CXCR6 and transmembrane rotational changes and polar interactions between transmembrane segments. The polar interactions between TM3, TM5, and TM6 are fundamental to functional conformation and the meta-active state of CXCR6. The interactions between D77-R280 and T243-TM7 could be related to the functional conformation of CXCR6; alternatively, the interaction between Q195-Q244 and N248 could be related to an inactive state due to the loss of this interaction, and an arginine cage broken in the presence of CXCL16s allows the meta-active state of CXCR6. A general protein‒ligand interaction supports the relevance of TM3‒TM5‒TM6 interactions, presenting three relevant pharmacophoric features: HAc (H-bond acceptor), HDn (H-bond donator), and Hph (hydrophobic), distributed around the space between extracellular loops (ECLs) and TMs. The HDn feature is close to TM3 and TM6; likewise, the HAc and Hph features are close to ECL1 and ECL2 and could block the rotation and interactions between TM3‒TM6 and the interactions of CXCL16s with the ECLs. Tridimensional quantitative structure-activity relationships (3D-QSAR) models show that the positive steric (VdW) and electrostatic fields coincide with the steric and positive electrostatic region of the exo-azabicyclo[3.3.1]nonane scaffold in the best pIC50 ligands. This substructure is close to the E274 residue and therefore relevant to the activity of CXCR6. These data could help with the design of new molecules that inhibit chemokine binding or antagonize the receptor based on the activation mechanism of CXCR6 and provoke a decrease in chemotaxis caused by the CXCR6‒CXCL16 axis.

Cytotoxic Fractions from Hechtia glomerata Extracts and p-Coumaric Acid as MAPK Inhibitors.

Preliminary bioassay-guided fractionation was performed to identify cytotoxic compounds from Hechtia glomerata, a plant that is used in Mexican ethnomedicine. Organic and aqueous extracts were prepared from H. glomerata's leaves and evaluated against two cancer cell lines. The CHCl3/MeOH (1:1) active extract was fractionated, and the resulting fractions were assayed against prostate adenocarcinoma PC3 and breast adenocarcinoma MCF7 cell lines. Active fraction 4 was further analyzed by high-performance liquid chromatography-quadrupole time-of-flight-mass spectrometry analysis to identify its active constituents. Among the compounds that were responsible for the cytotoxic effects of this fraction were flavonoids, phenolic acids, and aromatic compounds, of which p-coumaric acid (p-CA) and its derivatives were abundant. To understand the mechanisms that underlie p-CA cytotoxicity, a microarray assay was performed on PC3 cells that were treated or not with this compound. The results showed that mitogen-activated protein kinases (MAPKs) that regulate many cancer-related pathways were targeted by p-CA, which could be related to the reported effects of reactive oxygen species (ROS). A molecular docking study of p-CA showed that this phenolic acid targeted these protein active sites (MAPK8 and Serine/Threonine protein kinase 3) at the same binding site as their inhibitors. Thus, we hypothesize that p-CA produces ROS, directly affects the MAPK signaling pathway, and consequently causes apoptosis, among other effects. Additionally, p-CA could be used as a platform for the design of new MAPK inhibitors and re-sensitizing agents for resistant cancers.

Study of Endogen Substrates, Drug Substrates and Inhibitors Binding Conformations on MRP4 and Its Variants by Molecular Docking and Molecular Dynamics.

Multidrug resistance protein-4 (MRP4) belongs to the ABC transporter superfamily and promotes the transport of xenobiotics including drugs. A non-synonymous single nucleotide polymorphisms (nsSNPs) in the ABCC4 gene can promote changes in the structure and function of MRP4. In this work, the interaction of certain endogen substrates, drug substrates, and inhibitors with wild type-MRP4 (WT-MRP4) and its variants G187W and Y556C were studied to determine differences in the intermolecular interactions and affinity related to SNPs using protein threading modeling, molecular docking, all-atom, coarse grained, and umbrella sampling molecular dynamics simulations (AA-MDS and CG-MDS, respectively). The results showed that the three MRP4 structures had significantly different conformations at given sites, leading to differences in the docking scores (DS) and binding sites of three different groups of molecules. Folic acid (FA) had the highest variation in DS on G187W concerning WT-MRP4. WT-MRP4, G187W, Y556C, and FA had different conformations through 25 ns AA-MD. Umbrella sampling simulations indicated that the Y556C-FA complex was the most stable one with or without ATP. In Y556C, the cyclic adenosine monophosphate (cAMP) and ceefourin-1 binding sites are located out of the entrance of the inner cavity, which suggests that both cAMP and ceefourin-1 may not be transported. The binding site for cAMP and ceefourin-1 is quite similar and the affinity (binding energy) of ceefourin-1 to WT-MRP4, G187W, and Y556C is greater than the affinity of cAMP, which may suggest that ceefourin-1 works as a competitive inhibitor. In conclusion, the nsSNPs G187W and Y556C lead to changes in protein conformation, which modifies the ligand binding site, DS, and binding energy.

Molecular docking, SAR analysis and biophysical approaches in the study of the antibacterial activity of ceramides isolated from Cissus incisa.

The developing of antibacterial resistance is becoming in crisis. In this sense, natural products play a fundamental role in the discovery of antibacterial agents with diverse mechanisms of action. Phytochemical investigation of Cissus incisa leaves led to isolation and characterization of the ceramides mixture (1): (8E)-2-(tritriacont-9-enoyl amino)-1,3,4-octadecanetriol-8-ene (1-I); (8E)-2-(2',3'-dihydroxyoctacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-II); (8E)-2-(2'-hydroxyheptacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-III); and (8E)-2-(-2'-hydroxynonacosanoyl amino)-1,3,4-octadecanetriol-8-ene (1-IV). Until now, this is the first report of the ceramides (1-I), (1-II), and (1-IV). The structures were elucidated using NMR and mass spectrometry analyses. Antibacterial activity of ceramides (1) and acetylated derivates (2) was evaluated against nine multidrug-resistant bacteria by Microdilution method. (1) showed the best results against Gram-negatives, mainly against carbapenems-resistant Acinetobacter baumannii with MIC = 50 µg/mL. Structure-activity analysis and molecular docking revealed interactions between plant ceramides with membrane proteins, and enzymes associated with biological membranes of Gram-negative bacteria, through hydrogen bonding of functional groups. Vesicular contents release assay showed the capacity of (1) to disturb membrane permeability detected by an increase of fluorescence probe over time. The membrane disruption is not caused for ceramides lytic action on cell membranes, according in vitro hemolyticactivity results. Combining SAR analysis, bioinformatics and biophysical techniques, and also experimental tests, it was possible to explain the antibacterial action of these natural ceramides.

In-silico drug repurposing study: Amprenavir, enalaprilat, and plerixafor, potential drugs for destabilizing the SARS-CoV-2 S-protein-angiotensin-converting enzyme 2 complex.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that leads to coronavirus disease (COVID-19) has put public health at risk in 2020. The spike protein (SP) in SARS-CoV-2 is primarily responsible for the attachment and entry of the virus into the cell, which binds to the angiotensin-converting enzyme 2 (ACE2). Owing to the lack of an effective therapy, drug repositioning is an opportunity to search for molecules with pharmacological potential for the treatment of COVID-19. In this study, three candidates with the potential to destabilize the SP-ACE2 complex are reported. Through molecular docking, 147 drugs were evaluated and their possible binding sites in the interface region of the SP-ACE2 complex and the SP of SARS-CoV-2 were identified. The five best candidate molecules were selected for molecular dynamics studies to observe changes in interactions between SP-ACE2 and ligands with the SP-ACE2 complex. Using umbrella sampling molecular dynamics simulations, the binding energy of SP with ACE2 (-29.58 kcal/mol) without ligands, and in complex with amprenavir (-20.13 kcal/mol), enalaprilat (-23.84 kcal/mol), and plerixafor (-19.72 kcal/mol) were calculated. These drugs are potential candidates for the treatment of COVID-19 as they destabilize the SP-ACE2 complex; the binding energy of SP is decreased in the presence of these drugs and may prevent the virus from entering the cell. Plerixafor is the drug with the greatest potential to destabilize the SP-ACE2 complex, followed by amprenavir and enalaprilat; thus, these three drugs are proposed for future in vitro and in vivo evaluations.

Computational Study of C-X-C Chemokine Receptor (CXCR)3 Binding with Its Natural Agonists Chemokine (C-X-C Motif) Ligand (CXCL)9, 10 and 11 and with Synthetic Antagonists: Insights of Receptor Activation towards Drug Design for Vitiligo.

Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8+ cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gαi/0ßγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the ßγ complex from the α subunit in the G-protein. Additionally, a general protein-ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.

"Effect of valerenic acid on neuroinflammation in a MPTP-induced mouse model of Parkinson's disease".

Parkinson´s disease is the most important neuromotor pathology due to the prominent loss of dopaminergic neurons in the substantia nigra pars compacta. There is an inherent deficiency of dopamine in Parkinson´s disease, which is aggravated when neuroinflammatory processes are present. Several biomolecules are interesting candidates for the regulation of inflammation and possible neuroprotection, such as valerenic acid, one of the main components of Valeriana officinalis. A 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP)-induced mouse model of Parkinson's disease was developed to evaluate the motor effects of valerenic acid. The evaluation was carried out with four tests (an invert screen test for muscle strength, cross beam test, open field mobility test and lifting on hind legs test). Subsequently, the neuroinflammatory process was evaluated through ELISA of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and IFN-γ). The decreases in the inflammatory and neurodegenerative processes were evaluated by Western blot and immunohistochemistry analyses of the tissues, which included an evaluation of the tyrosine hydroxylase and GFAP proteins. Finally, the predicted mechanism of action of valerenic acid was supported by molecular docking calculations with the 5-HT5A receptor. The results indicate that the use of valerenic acid as a co-treatment decreases the neuroinflammation in Parkinson's disease induced by MPTP and provides evidence of a decrease in the evaluated pro-inflammatory cytokines and in the amount of GFAP in the mesencephalic area. Valerenic acid prevents neuroinflammation in a Parkinson's disease mouse model, which might reflect the neuroprotection of dopaminergic neurons with the recovery of motor ability.

Identification and molecular characterization of the tubulin-podophyllotoxin-type lignans binding site on Giardia lamblia.

There are several drugs for the treatment of giardiasis; however, there is a tendency for patients to abandon treatment because of drug-related adverse effects, resulting in relapses, acquired resistance, and higher rates of treatment failure. Recently, we reported some podophyllotoxin-type lignans from Bursera fagaroides var. fagaroides showing antigiardial activity. In the present work, we demonstrated that 5'-desmethoxy-peltatin-A-methylether (5-DES), acetylpodophyllotoxin (APOD), and podophyllotoxin (POD) affect the distribution and staining pattern of microtubular structures on Giardia trophozoites. Virtual screening results revealed that the lignans act via binding in a hydrophobic pocket in the heterodimer interface of tubulin in Giardia. This study provides useful insight to understand the action mechanism of 5DES, APOD, and POD on Giardia lamblia. The optimization of these podophyllotoxin-type lignans will lead to promising candidates for antigiardial drugs.

Vasodilation Elicited by Isoxsuprine, Identified by High-Throughput Virtual Screening of Compound Libraries, Involves Activation of the NO/cGMP and H2S/KATP Pathways and Blockade of α1-Adrenoceptors and Calcium Channels.

Recently, our research group demonstrated that uvaol and ursolic acid increase NO and H2S production in aortic tissue. Molecular docking studies showed that both compounds bind with high affinity to endothelial NO synthase (eNOS) and cystathionine gamma-lyase (CSE). The aim of this study was to identify hits with high binding affinity for the triterpene binding-allosteric sites of eNOS and CSE and to evaluate their vasodilator effect. Additionally, the mechanism of action of the most potent compound was explored. A high-throughput virtual screening (HTVS) of 107,373 compounds, obtained from four ZINC database libraries, was performed employing the crystallographic structures of eNOS and CSE. Among the nine top-scoring ligands, isoxsuprine showed the most potent vasodilator effect. Pharmacological evaluation, employing the rat aorta model, indicated that the vasodilation produced by this compound involved activation of the NO/cGMP and H2S/KATP signaling pathways and blockade of α1-adrenoceptors and L-type voltage-dependent Ca2+ channels. Incubation of aorta homogenates in the presence of isoxsuprine caused 2-fold greater levels of H2S, which supported our preliminary in silico data. This study provides evidence to propose that the vasodilator effect of isoxsuprine involves various mechanisms, which highlights its potential to treat a wide variety of cardiovascular diseases.

Molecular dynamic simulations of the catalytic subunit of calpains 1, 2, 5, and 10: Structural analysis with an aim toward drug design.

Calpains are cysteine proteases involved in the development of several human chronic illnesses such as neurodegenerative diseases, cardiovascular ailments, diabetes, and obesity which constitutes them into possible therapeutic targets. Here, using molecular dynamic simulations and docking, we studied the binding of known inhibitors to representative members of classical and nonclassical calpains. Our aim is to gain better understanding on the inhibition mechanism of calpains and to develop better and more specific inhibitors. Our atomistic models confirmed the importance of calcium ions for the structure of calpains and, as a consequence, their functionality. With these models and their subsequent use in molecular docking, essential structural requirements were identified for the binding of ligands to the calpain catalytic site that provide useful information for the design of new selective calpain inhibitors.

In search of AKT kinase inhibitors as anticancer agents: structure-based design, docking, and molecular dynamics studies of 2,4,6-trisubstituted pyridines.

The AKT isoforms are a group of key kinases that play a critical role in tumorigenesis. These enzymes are overexpressed in different types of cancers, such as breast, colon, prostate, ovarian, and lung. Because of its relevance the AKT isoforms are attractive targets for the design of anticancer molecules. However, it has been found that AKT1 and AKT3 isoforms have a main role in tumor progression and metastasis; thus, the identification of AKT isoforms specific inhibitors seems to be a challenge. Previously, we identified an ATP binding pocket pan-AKT inhibitor, this compound is a 2,4,6-trisubstituted pyridine (compound 11), which represents a new interesting scaffold for the developing of AKT inhibitors. Starting from the 2,4,6-trisubstituted pyridine scaffold, and guided by structure-based design technique, 42 new inhibitors were designed and further evaluated in the three AKT isoforms by multiple docking approach and molecular dynamics. Results showed that seven compounds presented binding selectivity for AKT1 and AKT3, better than for AKT2. The binding affinities of these seven compounds on AKT1 and AKT3 isoforms were mainly determined by hydrophobic contributions between the aromatic portion at position 4 of the pyridine ring with residues Phe236/234, Phe237/235, Phe438/435, and Phe442/439 in the ATP binding pocket. Results presented in this work provide an addition knowledge leading to promising selective AKT inhibitors.

meso-Dihydroguaiaretic acid derivatives with antibacterial and antimycobacterial activity.

Thirty-three meso-dihydroguaiaretic acid (meso-DGA) derivatives bearing esters, ethers, and amino-ethers were synthesized. All derivatives were tested against twelve drug-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including sensitive (H37Rv) and multidrug-resistant Mycobacterium tuberculosis strains. Among the tested compounds, four esters (7, 11, 13, and 17), one ether (23), and three amino-ethers (30, 31, and 33) exhibited moderate activity against methicillin-resistant Staphylococcus aureus, whereas 30 and 31 showed better results than levofloxacin against vancomycin-resistant Enterococcus faecium. Additionally, nineteen meso-DGA derivatives displayed moderate to potent activity against M. tuberculosis H37Rv with minimum inhibitory concentration (MIC) values ranging from 3.125 to 50µg/mL. Seven meso-DGA derivatives bearing amino-ethers (26-31 and 33) exhibited the lowest MICs against M. tuberculosis H37Rv and G122 strains, with 31 being as potent as ethambutol (MICs of 3.125 and 6.25µg/mL). The presence of positively charged group precursors possessing steric and hydrophobic features (e.g. N-ethylpiperidine moieties in meso-31) resulted essential to significantly increase the antimycobacterial properties of parent meso-DGA as supported by the R-group pharmacophoric and field-based QSAR analyses. To investigate the safety profile of the antimycobacterial compounds, cytotoxicity on Vero cells was determined. The amino-ether 31 exhibited a selectivity index value of 23, which indicate it was more toxic to M. tuberculosis than to mammalian cells. Therefore, 31 can be considered as a promising antitubercular agent for further studies.