Examinando por Autor "Moreno, Diego M."
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Ítem Acceso Abierto 2-Mercaptomethyl-thiazolidines use conserved aromatic–S interactions to achieve broad-range inhibition of metallo-β-lactamases(Royal Society of Chemistry, 2021-01-05) Rossi, María Agustina; Martínez, Verónica; Hinchliffe, Philip; Mojica, María F.; Castillo, Valerie; Moreno, Diego M.; Smith, Ryan; Spellberg, Brad; Drusano, George L.; Banchio, Claudia; Bonomo, Robert A.; Spencer, James; Vila, Alejandro J.; Mahler, Graciela; https://orcid.org/0000-0003-4720-4070; https://orcid.org/0000-0002-3697-5219; https://orcid.org/0000-0001-8611-4743; https://orcid.org/0000-0002-1380-9824; https://orcid.org/0000-0001-5493-8537; https://orcid.org/0000-0002-4602-0571; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0003-0612-0516Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(II) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than D/L-captopril. Unexpectedly, MMTZ binding features a thioether–π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–π interactions can be exploited for general ligand design in medicinal chemistry.Ítem Acceso Abierto A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases(Nature, 2017-09-14) Lisa, María Natalia; Palacios, Antonela R.; Aitha, Mahesh; González, Mariano M.; Moreno, Diego M.; Crowder, Michael W.; Bonomo, Robert A.; Spencer, James; Tierney, David L.; Llarrull, Leticia Irene; Vila, Alejandro J.Ítem Acceso Abierto A transmembrane Histidine Kinase functions as a pH sensor(MDPI, 2020-08-14) Bortolotti, Ana; Vazquez, Daniela Belén; Almada, Juan Cruz; Inda, María Eugenia; Drusin, Salvador Iván; Villalba, Juan Manuel; Moreno, Diego M.; Ruysschaert, Jean Marie; Cybulski, Larisa EstefaníaÍtem Acceso Abierto Estudio bioinformático estructural del reconocimiento de ARN en el procesamiento de miARNs en plantas(2018) Drusin, Salvador Iván; Moreno, Diego M.; Rasia, Rodolfo M.Los microARN (miARN) son moléculas de ARN pequeñas de 21 nucleótidos de longitud que se sintetizan en el núcleo por la ARN polimerasa II. En plantas, están involucrados en la regulación de procesos como el desarrollo, respuestas a estrés y respuestas a hormonas. La biogénesis de miARN comienza con la transcripción de precursores más largos con forma de hebilla dentro de los cuales está contenida su secuencia. Estos precursores son procesados por un complejo proteico formado por la proteína DICER-LIKE 1 (DCL1) junto a otras proteínas accesorias. Los precursores de plantas son sumamente heterogéneos. Sin embargo, la maquinaria de procesamiento, es capaz de liberar con precisión el miARN. En el presente trabajo se realizó un estudio del mecanismo de procesamiento de los precursores de miARN a través de técnicas de simulación computacional. Para ello, se analizaron distintos agentes participantes del proceso: el precursor de miARN, el dominio de unión a ARN de doble hebra de DCL1 (DCL1-1) y los dominios RIIID de DCL1 en los cuales transcurre la reacción de digestión del ARN. La creación de una estructura de un complejo entre DCL1-1 y un ARN de doble hebra (ARNdh) permitió detectar las diferencias entre este dominio y otros similares de diferentes proteínas. El análisis de las simulaciones de dinámica molecular permitió establecer algunos de los elementos principales que le permiten al dominio DCL1-1 identificar al precursor de miARN. Principalmente, se halló que el residuo Arg8 es un residuo importante en este proceso ya que puede reconocer pares de bases no canónicos en el ARNdh objetivo y anclar el dominio en una posición específica del mismo. Estos hallazgos fueron validados experimentalmente. El modelado de diferentes pares de base en la secuencia de dos precursores de miARN permitió establecer el efecto que las mismas tienen sobre la estructura del ARNdh. Se encontró que los pares de bases no canónicos tienen comportamientos heterogéneos con diferentes grados de estabilidad y de interacción entre las bases. Los resultados hallados permiten explicar las diferencias de procesamiento en experimentos de mutación de precursores de miARN. El funcionamiento de los dominios RIIID se estudió a través de la proteína RNasa III bacteriana. Por medio de técnicas de modelado molecular, se construyó el complejo de este dominio con un ARNdh y se realizaron simulaciones de dinámica molecular dirigida a fin de recrear la reacción de hidrólisis sobre su grupo fosfato. Estos experimentos permitieron obtener información sobre el rol que juegan los diferentes elementos que componen el sitio activo: los iones de Mg2+, el nucleófilo, los residuos y las moléculas de solvente cercanas. Se halló que las moléculas de agua vecinas al sitio activo tienen una participación en la reacción al mediar en las transferencias de protones. Por otro lado, se reveló que el nucleófilo no es una molécula de agua, sino un ion hidroxilo. Finalmente, se encontró que los iones catalíticos tienen la capacidad de facilitar la deprotonación de una de las moléculas de agua coordinadas para generar el ion hidroxilo en una posición apropiada para el ataque nucleofílico.Ítem Acceso Abierto Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues(Public Library of Science (PLOS), 2014-01-02) González, Lisandro Javier; Moreno, Diego M.; Bonomo, Robert A.; Vila, Alejandro J.Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.Ítem Acceso Abierto Identification of key sequence features required for microRNA biogenesis in plants(Nature Research, 2020-10-21) Rojas, Arantxa María Larisa; Drusin, Salvador Iván; Chorostecki, Uciel Pablo; Mateos, Julieta L.; Moro, Belén; Bologna, Nicolás G.; Bresso, Edgardo Gabriel; Schapire, Arnaldo L.; Rasia, Rodolfo M.; Moreno, Diego M.; Palatnik, Javier F.; http://orcid.org/0000-0003-4316-4518; http://orcid.org/0000-0001-5350-514X; http://orcid.org/0000-0003-2229-6853; http://orcid.org/0000-0002-1156-6662; http://orcid.org/0000-0002-2810-3533; http://orcid.org/0000-0002-2161-7910; http://orcid.org/0000-0002-9798-459X; http://orcid.org/0000-0003-3940-067X; http://orcid.org/0000-0001-5493-8537; http://orcid.org/0000-0001-7996-5224MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.Ítem Acceso Abierto Longitudinal evolution of the Pseudomonas-Derived Cephalosporinase (PDC) structure and activity in a cystic fibrosis patient treated with b-Lactams(American Society for Microbiology, 2022-09-08) Colque, Claudia A.; Albarracín Orio, Andrea G.; Tomatis, Pablo E.; Dotta, Gina; Moreno, Diego M.; Hedemann, Laura G.; Hickman, Rachel A.; Sommer, Lea M.; Feliziani, Sofía; Moyano, Alejandro José; Bonomo, Robert A.; Johansen, Helle K.; Molin, Soren; Vila, Alejandro J.; Smania, Andrea M.Ítem Acceso Abierto MoleculARweb: a web site for chemistry and structural biology education through interactive augmented reality out of the box in bommodity devices(American Chemical Society, 2021-07-13) Cortés Rodríguez, Fabio; Frattini, Gianfranco; Krapp, Lucien F.; Martínez-Hung, Hassan; Moreno, Diego M.; Roldán, Mariana; Salomón, Jorge; Stemkoski, Lee; Traeger, Sylvain; Del Peraro, Matteo; Abriata, Luciano AndrésÍtem Acceso Abierto Selective inhibition of the amyloid matrix of Escherichia coli biofilms by a bifunctional microbial metabolite(Nature Research, 2023-10-19) Cordisco, Estefanía; Zanor, María Inés; Moreno, Diego M.; Serra, Diego Omar; https://orcid.org/0000-0002-6574-1702; https://orcid.org/0000-0002-8903-0673; https://orcid.org/0000-0001-5493-8537; https://orcid.org/0000-0002-3926-384XThe propensity of bacteria to grow collectively in communities known as biofilms and their ability to overcome clinical treatments in this condition has become a major medical problem, emphasizing the need for anti-biofilm strategies. Antagonistic microbial interactions have extensively served as searching platforms for antibiotics, but their potential as sources for anti-biofilm compounds has barely been exploited. By screening for microorganisms that in agar-set pairwise interactions could antagonize Escherichia coli’s ability to form macrocolony biofilms, we found that the soil bacterium Bacillus subtilis strongly inhibits the synthesis of amyloid fibers –known as curli-, which are the primary extracellular matrix (ECM) components of E. coli biofilms. We identified bacillaene, a B. subtilis hybrid non-ribosomal peptide/polyketide metabolite, previously described as a bacteriostatic antibiotic, as the effector molecule. We found that bacillaene combines both antibiotic and anti-curli functions in a concentration-dependent order that potentiates the ecological competitiveness of B. subtilis, highlighting bacillaene as a metabolite naturally optimized for microbial inhibition. Our studies revealed that bacillaene inhibits curli by directly impeding the assembly of the CsgB and CsgA curli subunits into amyloid fibers. Moreover, we found that curli inhibition occurs despite E. coli attempts to reinforce its protective ECM by inducing curli genes via a RpoS-mediated competition sensing response trigged by the threatening presence of B. subtilis. Overall, our findings illustrate the relevance of exploring microbial interactions not only for finding compounds with unknown and unique activities, but for uncovering additional functions of compounds previously categorized as antibiotics.