Benzimidazole derivatives as new and selective inhibitors of arginase from leishmania mexicana with biological activity against promastigotes and amastigotes

17 pags, 6 figs, 3 tabs, 2 schs. -- Supplementary materials are available online at https://www.mdpi.com/article/10 .3390/ijms222413613/s1.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.This research was funded by the Consejo Nacional de Ciencia y Tecnología (CONACyT), grants 257848 (A.T.-V.) and 258694 (C.A.-D.). The work in Spain was funded by a grant from the Spanish Ministry of Science, Innovation and Competitiveness PID2020-115331GB-100 to J.A.-H.Peer reviewe

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/0βγ 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 βγ 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

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

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

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

<p>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 <b>11</b>), 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.</p

Structure&ndash;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&ndash;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&ndash;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

Role of Nitric Oxide and Hydrogen Sulfide in the Vasodilator Effect of Ursolic Acid and Uvaol from Black Cherry Prunus serotina Fruits

The present research aimed to isolate the non-polar secondary metabolites that produce the vasodilator effects induced by the dichloromethane extract of Prunus serotina (P. serotina) fruits and to determine whether the NO/cGMP and the H2S/KATP channel pathways are involved in their mechanism of action. A bioactivity-directed fractionation of the dichloromethane extract of P. serotina fruits led to the isolation of ursolic acid and uvaol as the main non-polar vasodilator compounds. These compounds showed significant relaxant effect on rat aortic rings in an endothelium- and concentration-dependent manner, which was inhibited by NG-nitro-l-arginine methyl ester (l-NAME), dl-propargylglycine (PAG) and glibenclamide (Gli). Additionally, both triterpenes increased NO and H2S production in aortic tissue. Molecular docking studies showed that ursolic acid and uvaol are able to bind to endothelial NOS and CSE with high affinity for residues that form the oligomeric interface of both enzymes. These results suggest that the vasodilator effect produced by ursolic acid and uvaol contained in P. serotina fruits, involves activation of the NO/cGMP and H2S/KATP channel pathways, possibly through direct activation of NOS and CSE

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

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.D.N.M, thanks financial support from CONACYT grant (No. 605522) and for Mobility Scholarships Abroad 2018 (291250).E.L. acknowledge research grants from the Ministry of Economy and Competitiveness, MINECO, Spain (No. FEDER CTQ2016-74881-P), and the Basque government (No. IT1045-16). C.R.M. thanks to a) the Collaborative Project in Genomic Data Integration (CICLOGEN) PI17/01826 funded by the Carlos III Health Institute from the Spanish National plan for Scientific and Technical Research and Innovation 2013-2016 and the European Regional Development Funds (FEDER)-“A way to build Europe”; b) the Spanish Ministry of Economy and Competitiveness for its support through the funding of the unique installation BIOCAI (UNLC08-1E-002, UNLC13-13-3503) and the European Regional Development Funds (FEDER) by the European Union; c) the Consolidation and Structuring of Competitive Research Units - Competitive Reference Groups (ED431C 2018/49), funded by the Ministry of Education, University and Vocational Training of the Xunta de Galicia endowed with EU FEDER funds.Peer reviewe