Browsing by Author "Duarte, Yorley"
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- ItemA physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII(2021) Lopez, Ximena; Palacios-Prado, Nicolas; Guiza, Juan; Escamilla, Rosalba; Fernandez, Paola; Vega, Jose L.; Rojas, Maximiliano; Marquez-Miranda, Valeria; Chamorro, Eduardo; Cardenas, Ana M.; Maldifassi, Maria Constanza; Martinez, Agustin D.; Duarte, Yorley; Gonzalez-Nilo, Fernando D.; Saez, Juan C.Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation- measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1EGFP and rPanx1S394D-EGFP channels showed current at all voltages between +/- 100 mV, similar single channel currents with outward rectification, and unitary conductance (similar to 30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.
- ItemDifferent Classes of Antidepressants Inhibit the Rat α7 Nicotinic Acetylcholine Receptor by Interacting within the Ion Channel: A Functional and Structural Study(2021) Duarte, Yorley; Rojas, Maximiliano; Canan, Jonathan; Perez, Edwin G.; Gonzalez-Nilo, Fernando; Garcia-Colunga, JesusSeveral antidepressants inhibit nicotinic acetylcholine receptors (nAChRs) in a non-competitive and voltage-dependent fashion. Here, we asked whether antidepressants with a different structure and pharmacological profile modulate the rat alpha 7 nAChR through a similar mechanism by interacting within the ion-channel. We applied electrophysiological (recording of the ion current elicited by choline, I-Ch, which activates alpha 7 nAChRs from rat CA1 hippocampal interneurons) and in silico approaches (homology modeling of the rat alpha 7 nAChR, molecular docking, molecular dynamics simulations, and binding free energy calculations). The antidepressants inhibited I-Ch with the order: norfluoxetine similar to mirtazapine similar to imipramine < bupropion similar to fluoxetine similar to venlafaxine similar to escitalopram. The constructed homology model of the rat alpha 7 nAChR resulted in the extracellular vestibule and the channel pore is highly negatively charged, which facilitates the permeation of cations and the entrance of the protonated form of antidepressants. Molecular docking and molecular dynamics simulations were carried out within the ion-channel of the alpha 7 nAChR, revealing that the antidepressants adopt poses along the receptor channel, with slightly different binding-free energy values. Furthermore, the inhibition of I-Ch and free energy values for each antidepressant-receptor complex were highly correlated. Thus, the alpha 7 nAChR is negatively modulated by a variety of antidepressants interacting in the ion-channel.
- ItemDirect Oral FXa Inhibitors Binding to Human Serum Albumin: Spectroscopic, Calorimetric, and Computational Studies(2023) Mariño Ocampo, Nory Johana; Rodríguez Sánchez, Diego Fernando; Daniel Sebastian, Guerra Diaz; Zúñiga Núñez, Daniel; Duarte, Yorley; Fuentealba Patiño, Denis Alberto; Zacconi, Flavia C. M.Direct FXa inhibitors are an important class of bioactive molecules (rivaroxaban, apixaban,edoxaban, and betrixaban) applied for thromboprophylaxis in diverse cardiovascular pathologies.The interaction of active compounds with human serum albumin (HSA), the most abundant protein inblood plasma, is a key research area and provides crucial information about drugs’ pharmacokineticsand pharmacodynamic properties. This research focuses on the study of the interactions betweenHSA and four commercially available direct oral FXa inhibitors, applying methodologies includingsteady-state and time-resolved fluorescence, isothermal titration calorimetry (ITC), and moleculardynamics. The HSA complexation of FXa inhibitors was found to occur via static quenching, and thecomplex formation in the ground states affects the fluorescence of HSA, with a moderate bindingconstant of 104 M−1. However, the ITC studies reported significantly different binding constants (103 M−1) compared with the results obtained through spectrophotometric methods. The suspectedbinding mode is supported by molecular dynamics simulations, where the predominant interactionswere hydrogen bonds and hydrophobic interactions (mainly π–π stacking interactions between thephenyl ring of FXa inhibitors and the indole moiety of Trp214). Finally, the possible implications ofthe obtained results regarding pathologies such as hypoalbuminemia are briefly discussed.
- ItemEndogenous pannexin1 channels form functional intercellular cell-cell channels with characteristic voltage-dependent properties(2022) Palacios-Prado, Nicolas; Soto, Paola A.; Lopez, Ximena; Choi, Eun Ju; Marquez-Miranda, Valeria; Rojas, Maximiliano; Duarte, Yorley; Lee, Jinu; Gonzalez-Nilo, Fernando D.; Saez, Juan C.The occurrence of intercellular channels formed by pannexin1 has been challenged for more than a decade. Here, we provide an electrophysiological characterization of exoge-nous human pannexin1 (hPanx1) cell-cell channels expressed in HeLa cells knocked out for connexin45. The observed hPanx1 cell-cell channels show two phenotypes: O-state and S-state. The former displayed low transjunctional voltage (Vj) sensitivity and single -channel conductance of -175 pS, with a substate of -35 pS; the latter showed a pecu-liar dynamic asymmetry in Vj dependence and single-channel conductance identical to the substate conductance of the O-state. S-state hPanx1 cell-cell channels were also iden-tified between TC620 cells, a human oligodendroglioma cell line that endogenously expresses hPanx1. In these cells, dye and electrical coupling increased with temperature and were strongly reduced after hPanx1 expression was knocked down by small interfer-ing RNA or inhibited with Panx1 mimetic inhibitory peptide. Moreover, cell-cell cou-pling was augmented when hPanx1 levels were increased with a doxycycline-inducible expression system. Application of octanol, a connexin gap junction (GJ) channel inhibi-tor, was not sufficient to block electrical coupling between HeLa KO Cx45-hPanx1 or TC620 cell pairs. In silico studies suggest that several arginine residues inside the chan-nel pore may be neutralized by hydrophobic interactions, allowing the passage of DAPI, consistent with dye coupling observed between TC620 cells. These findings demonstrate that endogenously expressed hPanx1 forms intercellular cell-cell channels and their unique properties resemble those described in innexin-based GJ channels. Since Panx1 is ubiquitously expressed, finding conditions to recognize Panx1 cell-cell channels in different cell types might require special attention.
- ItemGreen by Design: Convergent Synthesis, Computational Analyses, and Activity Evaluation of New FXa Inhibitors Bearing Peptide Triazole Linking Units(2022) Rodriguez, Diego F.; Duran Osorio, Francisca; Duarte, Yorley; Olivares, Pedro; Moglie, Yanina; Dua, Kamal; Zacconi, Flavia C. M.Green chemistry implementation has led to promising results in waste reduction in the pharmaceutical industry. However, the early sustainable development of pharmaceutically active compounds and ingredients remains a considerable challenge. Herein, we wish to report a green synthesis of new pharmaceutically active peptide triazoles as potent factor Xa inhibitors, an important drug target associated with the treatment of diverse cardiovascular diseases. The new inhibitors were synthesized in three steps, featuring cycloaddition reactions (high atom economy), microwave-assisted organic synthesis (energy efficiency), and copper nanoparticle catalysis, thus featuring Earth-abundant metals. The molecules obtained showed FXa inhibition, with IC50-values as low as 17.2 mu M and no associated cytotoxicity in HEK293 and HeLa cells. These results showcase the environmental potential and chemical implications of the applied methodologies for the development of new molecules with pharmacological potential.
- ItemInnovative Three-Step Microwave-Promoted Synthesis of N-Propargyltetrahydroquinoline and 1,2,3-Triazole Derivatives as a Potential Factor Xa (FXa) Inhibitors: Drug Design, Synthesis, and Biological Evaluation(2020) Santana-Romo, Fabian; Lagos, Carlos F.; Duarte, Yorley; Castillo, Francisco; Moglie, Yanina; Maestro, Miguel A.; Charbe, Nitin; Zacconi, Flavia C.The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (mu), chemical hardness (eta), and electrophilicity (omega) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC50) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.
- ItemMicrowave-mediated synthesis of N-allyl/propargyl derivatives : enzymatic analysis as a potential factor Xa (FXa) inhibitor, theoretical and computational molecular docking(2020) Santana Romo, Fabián Mauricio; Duarte, Yorley; Castillo Suzarte, Francisco Javier; Maestro, Miguel A.; Zacconi, Flavia C. M.
- ItemNovel N-benzoylimidazolium ionic liquids derived from benzoic and hydroxybenzoic acids as therapeutic alternative against Biofilm-forming bacteria in skin and soft-tissue infections(2022) Forero-Doria, Oscar; Parra-Cid, Cristobal; Venturini, Whitney; Espinoza, Carolina; Araya-Maturana, Ramiro; Valenzuela-Riffo, Felipe; Saldias, Cesar; Leiva, Angel; Duarte, Yorley; Echeverria, Javier; Guzman, LuisThe skin and soft tissue infections (SSTIs) -producing pathogens have acquired resistance to a wide range of antimicrobials, thus it is highly relevant to have new treatment alternatives. In this study, we report the synthesis, characterization, and antibacterial activity of three novel series of ionic liquids (ILs) derived from benzoic and hydroxybenzoic acids, with different lengths of the alkyl chain. The minimum inhibitory concentration (MIC) were tested in Gram positive: Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes, and Gram negative: Acinetobacter baumannii and Escherichia coli, showing a MIC range of 0.01562 2.0 mM, with the activity varying according to the aromatic ring functionalization and the length of the alkyl chains. Regarding the antibiofilm activity, different efficacy was observed among the different ILs, some of them presenting antibiofilm activities close to 80% as in the case of those derived from syringic acid with an alkyl chain of six carbon atoms against Pseudomonas aeruginosa. Furthermore, the cell viability in HaCaT cells was determined, showing a half maximal effective concentration (EC50) values higher than the MIC values. The antimicrobial and antibiofilm results, along with not producing cellular toxicity at the MIC values shows that these ILs could be a promising alternative against SSTIs.
- ItemThe TGA Transcription Factors from Clade II Negatively Regulate the Salicylic Acid Accumulation in Arabidopsis(2022) Fonseca, Alejandro; Urzua, Tomas; Jelenska, Joanna; Sbarbaro, Christopher; Seguel, Aldo; Duarte, Yorley; Greenberg, Jean T.; Holuigue, Loreto; Blanco-Herrera, Francisca; Herrera-Vasquez, ArielSalicylic acid (SA) is a hormone that modulates plant defenses by inducing changes in gene expression. The mechanisms that control SA accumulation are essential for understanding the defensive process. TGA transcription factors from clade II in Arabidopsis, which include the proteins TGA2, TGA5, and TGA6, are known to be key positive mediators for the transcription of genes such as PR-1 that are induced by SA application. However, unexpectedly, stress conditions that induce SA accumulation, such as infection with the avirulent pathogen P. syringae DC3000/AvrRPM1 and UV-C irradiation, result in enhanced PR-1 induction in plants lacking the clade II TGAs (tga256 plants). Increased PR-1 induction was accompanied by enhanced isochorismate synthase-dependent SA production as well as the upregulation of several genes involved in the hormone's accumulation. In response to avirulent P. syringae, PR-1 was previously shown to be controlled by both SA-dependent and -independent pathways. Therefore, the enhanced induction of PR-1 (and other defense genes) and accumulation of SA in the tga256 mutant plants is consistent with the clade II TGA factors providing negative feedback regulation of the SA-dependent and/or -independent pathways. Together, our results indicate that the TGA transcription factors from clade II negatively control SA accumulation under stress conditions that induce the hormone production. Our study describes a mechanism involving old actors playing new roles in regulating SA homeostasis under stress.
- ItemToward the cholinesterase inhibition potential of TADDOL derivatives: Seminal biological and computational studies(2022) Constantino, Andrea R.; Charbe, Nitin Bharat; Duarte, Yorley; Gutierrez, Margarita; Giordano Villatoro, Ady; Prasher, Parteek; Dua, Kamal; Mandolesi, Sandra; Zacconi, Flavia C. M.Alzheimer's disease (AD) is a degenerative neurological disease characterized by gradual loss of cognitive skills and memory. The exact pathogenesis involved still remains unrevealed, but several studies indicate the involvement of an array of different enzymes, underlining the multifactorial character of the disease. Inhibition of these enzymes is therefore a powerful approach in the development of AD treatments, with promising candidates, including acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and monoamine oxidase. Interestingly, AChE is the target of a major pesticide family (organophosphates), with several reports indicating an intersection between the pesticide's activity and AD. In this study, various TADDOL derivatives were synthesized and their in vitro activities as AChE/BuChE inhibitors as well as their antioxidant activities were studied. Molecular modeling studies revealed the capability of TADDOL derivatives to bind to AChE and induce inhibition, especially compounds 2b and 3c furnishing IC50 values of 36.78 +/- 8.97 and 59.23 +/- 5.31 mu M, respectively. Experimental biological activities and molecular modeling studies clearly demonstrate that TADDOL derivatives with specific stereochemistry have an interesting potential for the design of potent AChE inhibitors. The encouraging results for compounds 2b and 3c indicate them as promising scaffolds for selective and potent AChE inhibitors.