Browsing by Author "Lagos, Carlos F."

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    11β-hydroxysteroid dehydrogenase type 2 polymorphisms and activity in a Chilean essential hypertensive and normotensive cohort.
    (2012) Campino, Carmen; Quinteros, Hector; Owen, Gareth I.; Carvajal, Cristian A.; Morales, Mauricio; Olivieri, Oliviero; Guidi, Giancesare; Faccini, Giovanni; Pasini, Francesco; Baudrand, Rene; Padilla, Oslando; Valdivia, Carolina; Thichauer, Juan; Lagos, Carlos F.; Kalergis, Alexis M.; Fardella, Carlos E.
    BACKGROUND: 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) inactivates cortisol (F) to cortisone (E); its impairment is associated with hypertension. We reported that 15.7% of the Chilean essential hypertensives possessed a high F/E ratio suggesting a partial deficit in 11β-HSD2 activity. It has been reported that the G534A(Glu178/Glu) polymorphism in the HSD11B2 gene is associated with hypertension. Investigate the frequency of the G534A polymorphism and its correlation with the glucocorticoid profile in Chilean essential hypertensive and normotensive subjects. METHODS: Essential hypertensive outpatients (n = 232) and normotensive subjects (n = 74) were recruited. A change in the AluI restriction enzyme digest pattern, caused by the presence of the G534A polymorphism, was utilized to screen DNA isolated from leukocytes within the cohort before confirmation by sequencing. Plasma renin activity (PRA), serum aldosterone, F, and E were measured by radioimmunoassay. Urinary tetrahydrocortisol (THF), 5α-tetrahydrocortisol (5α-THF), and tetrahydrocortisone (THE) were measured by gas chromatography-mass spectrometry. RESULTS: G534A polymorphism frequency was similar between hypertensive patients (19 of 232; 8.2%) and normotensive subjects (7 of 74; 9.5%). When categorized by presence or absence of the G534A polymorphism, no significant differences in the serum F/E ratio or other measured biochemical variables were detected. Despite a previous report that the G534A polymorphism is associated with a neighboring C468A (Thr156/Thr) polymorphism, analysis within our cohort showed that only one patient in each group presented with this double polymorphism. CONCLUSIONS: We report the frequency of the G534A polymorphism in the Spanish-Amerindian population. No correlation was detected between this polymorphism and the presence of hypertension and biochemical parameters in this Chilean cohort.
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    2D-QSAR and 3D-QSAR/CoMSIA Studies on a Series of (R)-2-((2-(1H-Indol-2-yl)ethyl)amino)-1-Phenylethan-1-ol with Human β₃-Adrenergic Activity
    (2017) Apablaza H., Gastón E.; Montoya, Luisa; Morales Verdejo, César Aarón; Mellado, Marco; Cuellar Fritis, Mauricio Alcides; Lagos, Carlos F.; Soto Delgado, Jorge; Chung, Hery; Pessoa Mahana, Carlos David; Jaime Mella
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    Active acetylcholine receptors prevent the atrophy of skeletal muscles and favor reinnervation
    (2020) Cisterna, Bruno A.; Vargas, Anibal A.; Puebla, Carlos; Fernandez, Paola; Escamilla, Rosalba; Lagos, Carlos F.; Matus, Maria F.; Vilos, Cristian; Cea, Luis A.; Barnafi, Esteban; Gaete, Hugo; Escobar, Daniel F.; Cardozo, Christopher P.; Saez, Juan C.
    Denervation of skeletal muscles induces severe muscle atrophy, which is preceded by cellular alterations such as increased plasma membrane permeability, reduced resting membrane potential and accelerated protein catabolism. The factors that induce these changes remain unknown. Conversely, functional recovery following denervation depends on successful reinnervation. Here, we show that activation of nicotinic acetylcholine receptors (nAChRs) by quantal release of acetylcholine (ACh) from motoneurons is sufficient to prevent changes induced by denervation. Using in vitro assays, ACh and non-hydrolysable ACh analogs repressed the expression of connexin43 and connexin45 hemichannels, which promote muscle atrophy. In co-culture studies, connexin43/45 hemichannel knockout or knockdown increased innervation of muscle fibers by dorsal root ganglion neurons. Our results show that ACh released by motoneurons exerts a hitherto unknown function independent of myofiber contraction. nAChRs and connexin hemichannels are potential molecular targets for therapeutic intervention in a variety of pathological conditions with reduced synaptic neuromuscular transmission.
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    An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum
    (2008) Colombres, Marcela; Garate, Jose A.; Lagos, Carlos F.; Araya-Secchi, Raul; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Perez-Acle, Tomas; Eyzaguirre, Jaime
    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 angstrom resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.
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    Citosine-Adenine-Repeat Microsatellite of 11 beta-hydroxysteroid dehydrogenase 2 Gene in Hypertensive Children
    (OXFORD UNIV PRESS, 2016) Valdivia, Carolina; Carvajal, Cristian A.; Campino, Carmen; Allende, Fidel; Martinez Aguayo, Alejandro; Baudrand, Rene; Vecchiola, Andrea; Lagos, Carlos F.; Tapia Castillo, Alejandra; Fuentes, Cristobal A.; Aglony, Marlene; Solari, Sandra; Kalergis, Alexis M.; Garcia, Hernan; Owen, Gareth I.; Fardella, Carlos E.
    BACKGROUND
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    Design, synthesis, biological evaluation and binding mode modeling of benzimidazole derivatives targeting the cannabinoid receptor type 1
    (2015) Espinosa Bustos, Christian Marcelo; Lagos, Carlos F.; Romero Parra, Javier Hernán; Zárate Méndez, Ana María; Mella Raipán, Jaime Alberto; Pessoa Mahana, Hernán; Recabarren Gajardo, Gonzalo; Iturriaga Vásquez, Patricio; Tapia Apati, Ricardo; Pessoa Mahana, Carlos David
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    Innovative 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.
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    Novel N-Arylsulfonylindoles Targeted as Ligands of the 5-HT6 Receptor. Insights on the Influence of C-5 Substitution on Ligand Affinity
    (2021) Arrieta-Rodriguez, Loreto; Espinoza-Rosales, Daniela; Vera, Gonzalo; Cho, Young Hwa; Cabezas, David; Vasquez-Velasquez, David; Mella-Raipan, Jaime; Lagos, Carlos F.; Recabarren-Gajardo, Gonzalo
    A new series of twenty-two C-5 substituted N-arylsulfonylindoles was prepared with the aim of exploring the influence of C-5 substitution on 5-HT6 receptor affinity. Eleven compounds showed moderate to high affinity at the receptor (K-i = 58-403 nM), with compound 4d being identified as the most potent ligand. However, regarding C-5 substitution, both methoxy and fluorine were detrimental for receptor affinity compared to our previously published unsubstituted compounds. In order to shed light on these observations, we performed docking and molecular dynamics simulations with the most potent compounds of each series (4d and 4l) and PUC-10, a highly active ligand previously reported by our group. The comparison brings about deeper insight about the influence of the C-5 substitution on the binding mode of the ligands, suggesting that these replacements are detrimental to the affinity due to precluding a ligand from reaching deeper inside the binding site. Additionally, CoMFA/CoMSIA studies were performed to systematize the information of the main structural and physicochemical characteristics of the ligands, which are responsible for their biological activity. The CoMFA and CoMSIA models presented high values of q(2) (0.653; 0.692) and r(2) (0.879; 0.970), respectively. Although the biological activity of the ligands can be explained in terms of the steric and electronic properties, it depends mainly on the electronic nature.
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    PCSK9 conjugated liposomes for targeted delivery of paclitaxel to the cancer cell: a proof-of-concept study
    (ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER, 2022) Charbe, Nitin Bharat; Lagos, Carlos F.; Ortiz, Cristian Andres Vilos; Tambuwala, Murtaza; Palakurthi, Sushesh Srivatsa; Zacconi, Flavia C. M.
    Ligand-based targeting of the receptors that are overexpressed explicitly on cancer cells represents an effective drug delivery approach to enhance the chemotherapeutic efficacy. Proprotein convertase subtilisin/kexin type 9 (PCSK9) which is a serine protease enzyme primarily produced by the liver cells, can potentially be used as a targeting ligand. PCSK9 binds to the LDL-r on hepatocytes' surface, leading to endocytosis and endosomal degradation. High LDL-r expression, which is believed to meet the higher demand of the cholesterol and phospholipids to build proliferating cancer cell membrane, ensures selective uptake of the PCSK9 conjugated liposomes. In the present work, the PCSK9 conjugated liposomal system was developed to deliver paclitaxel (PTX) to cancer cells. The protein was conjugated by EDC and NHS in a two-step coupling reaction to the li-posomes containing COOH-PEG2000-COOH lipid. Conjugation was confirmed by NMR, and liposomes were further characterized by SEM and zeta sizer. PCSK9-conjugated liposomes showed high encapsulation efficiency of 69.1% with a diameter of 90.0 & PLUSMN; 4.9 nm. Long-term stability (30 days) study (Zeta potential:-9.88) confirmed excellent constancy and significant drug retention (58.2%). Invitro cytotoxicity and targeting effi-ciency was explored using MTS assay in human embryonic kidney cells (HEK293), liver hepatocellular cells (HEPG2), and a human colon cancer cell line (HCT116) for 24 h. PCSK9 conjugated liposomes exhibited significantly higher growth inhibition than the unconjugated (control) liposomes in HCT116 cell line (p < 0.001). The novel PCSK9 conjugated liposomes presented potent and precise in vitro anticancer activity and, therefore, are suggested for the first time as a promising targeted delivery system for cancer treatment.
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    Polymorphisms in the RAC1 Gene Are Associated With Hypertension Risk Factors in a Chilean Pediatric Population
    (OXFORD UNIV PRESS, 2014) Tapia Castillo, Alejandra; Carvajal, Cristian A.; Campino, Carmen; Vecchiola, Andrea; Allende, Fidel; Solari, Sandra; Garcia, Lorena; Lavanderos, Sergio; Valdivia, Carolina; Fuentes, Cristobal; Lagos, Carlos F.; Martinez Aguayo, Alejandro; Baudrand, Rene; Aglony, Marlene; Garcia, Hernan; Fardella, Carlos E.
    The GTPase Rac1 has been implicated in hypertension as a modulator of mineralocorticoid receptor activity. Our aim is to investigate the frequency of polymorphisms rs10951982 (intron 1, G > A) and rs836478 (intron 3, T > C) in the RAC1 gene and perform association studies with clinical and biochemical parameters in a Chilean pediatric cohort.
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    Structural Requirements of the Human Sodium-Dependent Bile Acid Transporter (hASBT): Role of 3-and 7-OH Moieties on Binding and Translocation of Bile Acids
    (2014) Gonzalez, Pablo M.; Lagos, Carlos F.; Ward, Weslyn C.; Polli, James E.
    Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3 beta-hydroxyl (-OH) group on the cholestane ring to the common 3 alpha-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3 beta-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3 alpha-OH group on BAs binding and translocation by hASBT. Ten 3 beta-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3 alpha-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT: Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3 alpha-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH "on-" or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3 alpha-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3 alpha-OH group. Results have implications for BA prodrug design.
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    Structure-activity relationships studies on weakly basic N-arylsulfonylindoles with an antagonistic profile in the 5-HT6 receptor
    (2017) Mella, Jaime; Villegas, Francisco; Morales Verdejo, César Aarón; Lagos, Carlos F.; Recabarren Gajardo, Gonzalo
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    Testosterona inhibe la actividad de la aldosterona sintasa silvestre y quimérica in vitro
    (Sociedad Medica de Santiago, 2021) Vecchiola Cardenas, Andrea Paola; Saldias Fuentes, Cristóbal Abraham; Carvajal Maldonado, Cristian Andrés; Campino Johnson, María Del Carmen; Allende Sanzana, Fidel Alejandro; Tapia-Castillo, Alejandra; Lagos, Carlos F.; Fardella Bello, Carlos Enrique
    © 2021 Sociedad Medica de Santiago. All rights reserved.Background: Familial hyperaldosteronism type I is caused by the generation of a chimeric aldosterone synthase enzyme (ASCE) which is regulated by ACTH instead of angiotensin II. We have reported that in vitro, the wild-type (ASWT) and chimeric aldosterone synthase (ASCE) enzymes are inhibited by progesterone and estradiol does not affect their activity. Aim: To explore the direct action of testosterone on ASWT and ASCE enzymes. Material and Methods: HEK-293 cells were transiently transfected with vectors containing the full ASWT or ASCE cDNAs. The effect of testosterone on AS enzyme activities was evaluated incubating HEK-cells transfected with enzyme vectors and adding deoxycorticosterone (DOC) alone or DOC plus increasing doses of testosterone. Aldosterone production was measured by HPLC-MS/MS. Docking of testosterone within the active sites of both enzymes was performed by modelling in silico. Results: In this system, testosterone inhibited ASWT (90% inhibition at five µM, 50% inhibitory concentration (IC50) =1.690 µM) with higher efficacy and potency than ASCE (80% inhibition at five µM, IC50=3.176 µM). Molecular modelling studies showed different orientation of testosterone in ASWT and ASCE crystal structures. Conclusions: The inhibitory effect of testosterone on ASWT or ASCE enzymes is a novel non-genomic testosterone action, suggesting that further clinical studies are needed to assess the role of testosterone in the screening and diagnosis of primary aldosteronism.
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    Usefulness and Pitfalls in Sodium Intake Estimation : Comparison of Dietary Assessment and Urinary Excretion in Chilean Children and Adults
    (2016) Campino Johnson, María del Carmen; Hill, Caroline; Baudrand Biggs, René; Martínez Aguayo, Alejandro Gregorio; Aglony Imbarack, Marlene Elizabeth; Carrasco, Carmen A.; Ferrada, Clarita; Loureiro Pérez, Carolina Andrea; Vecchiola Cárdenas, Andrea Paola; Bancalari, Rodrigo; Grob Lunecke, Francisca Andrea; Carvajal, Cristian A.; Lagos, Carlos F.; Valdivia, Carolina; Tapia-Castillo, Alejandra; Fuentes Zúñiga, Cristóbal Andrés; Mendoza, Carolina; García Bruce, Hernán; Uauy, Ricardo; Fardella B., Carlos; Campino Johnson, María del Carmen; Hill, Caroline; Baudrand Biggs, René; Martínez Aguayo, Alejandro Gregorio; Aglony Imbarack, Marlene Elizabeth; Carrasco, Carmen A.; Ferrada, Clarita; Loureiro Pérez, Carolina Andrea; Vecchiola Cárdenas, Andrea Paola; Bancalari, Rodrigo; Grob Lunecke, Francisca Andrea; Carvajal, Cristian A.; Lagos, Carlos F.; Valdivia, Carolina; Tapia-Castillo, Alejandra; Fuentes, Cristóbal A.; Mendoza, Carolina; García Bruce, Hernán; Uauy, Ricardo; Fardella B., Carlos