RESUMEN
Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.
Asunto(s)
Antibacterianos , Descubrimiento de Drogas , Microbioma Gastrointestinal , Bacterias Gramnegativas , Infecciones por Bacterias Gramnegativas , Simbiosis , Animales , Femenino , Humanos , Masculino , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Línea Celular , Clostridioides difficile/efectos de los fármacos , Infecciones por Clostridium/microbiología , Infecciones por Clostridium/tratamiento farmacológico , Modelos Animales de Enfermedad , Diseño de Fármacos , Farmacorresistencia Bacteriana Múltiple , Microbioma Gastrointestinal/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Infecciones por Bacterias Gramnegativas/microbiología , Lipoproteínas/metabolismo , Ratones Endogámicos C57BL , Transporte de Proteínas/efectos de los fármacos , Sepsis/microbiología , Sepsis/tratamiento farmacológico , Especificidad por Sustrato , Simbiosis/efectos de los fármacosRESUMEN
Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open-closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF ß-barrel. Mutation of key residues important for the loop's conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated â¼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.
Asunto(s)
Antibacterianos/metabolismo , Farmacorresistencia Bacteriana/fisiología , Porinas/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Bacterias Gramnegativas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Teóricos , Simulación de Dinámica Molecular , Permeabilidad , Porinas/fisiología , Porinas/ultraestructuraRESUMEN
Cytochrome P450 2D6 (CYP2D6) is primarily expressed in the liver and in the central nervous system. It is known to be highly polymorphic in nature. It metabolizes several endogenous substrates such as anandamide (AEA). Concomitantly, it is involved in phase 1 metabolism of several antidepressants, antipsychotics, and other drugs. Research in the field of phytocannabinoids (pCBs) has recently accelerated owing to their legalization and increasing medicinal use for pain and inflammation. The primary component of cannabis is THC, which is well-known for its psychotropic effects. Since CYP2D6 is an important brain and liver P450 and is known to be inhibited by CBD, we investigated the interactions of four important highly prevalent CYP2D6 polymorphisms with selected phytocannabinoids (CBD, THC, CBDV, THCV, CBN, CBG, CBC, ß-carophyllene) that are rapidly gaining popularity. We show that there is differential binding of CYP2D6*17 to pCBs as compared to WT CYP2D6. We also perform a more detailed comparison of WT and *17 CYP2D6, which reveals the possible regulation of AEA metabolism by CBD. Furthermore, we use molecular dynamics to delineate the mechanism of this binding, inhibition, and regulation. Taken together, we have found that the interactions of CYP2D6 with pCBs vary by polymorphism and by specific pCB class.
Asunto(s)
Cannabinoides/metabolismo , Cannabinoides/farmacología , Citocromo P-450 CYP2D6/genética , Cannabidiol/metabolismo , Cannabidiol/farmacología , Cannabinol/metabolismo , Cannabinol/farmacología , Cannabis/química , Cannabis/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Dronabinol/metabolismo , Dronabinol/farmacología , Humanos , Simulación de Dinámica Molecular , Fitoquímicos/metabolismo , Polimorfismo Genético/efectos de los fármacosRESUMEN
Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and form favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.