Reposicionamiento de fármacos para el tratamiento de infecciones causadas por bacilos gramnegativos multirresistentes

El aumento de bacterias multirresistentes y la dificultad de encontrar un tratamiento eficaz para este tipo de infecciones, supone un problema a nivel mundial. Entre los patógenos más destacados se encuentran los bacilos gramnegativos (BGN), tales como Acinetobacter baumannii, Pseudomonas aeruginosa, y Enterobacterales como Escherichia coli y Klebsiella pneumoniae, para los cuales la investigación y el desarrollo de nuevos antimicrobianos es una prioridad crítica. Debido a que el desarrollo de nuevos antibióticos supone un elevado coste económico, de tiempo empleado y poca rentabilidad a causa de la rápida aparición de resistencias, la búsqueda de nuevas alternativas terapéuticas se encuentra en auge. Esta Tesis Doctoral está basada en el uso de un nuevo enfoque conocido como “Drug repurposing” o reposicionamiento de fármacos, que consiste en la búsqueda de nuevas indicaciones para fármacos que se usan en la práctica clínica y en veterinaria. El objetivo principal fue el estudio de dos fármacos distintos: i) el tamoxifeno, anticancerígeno usado para el tratamiento de cáncer de mama y ii) la rafoxanida, antihelmíntico usado en veterinaria para tratar infecciones por trematodos. Por un lado, se demostró que la monoterapia con tamoxifeno presentó eficacia in vivo en un modelo de sepsis peritoneal murino por A. baumannii, P. aeruginosa y E. coli usando ratones inmunocompetentes e inmunodeprimidos. Esta actividad es debida a la reducción de la liberación de la proteína quimioatrayente de monocitos 1 (MCP-1, del inglés monocyte chemoattractant protein-1), que en última instancia produjo una disminución de la migración de células de la inmunidad innata desde la médula ósea hasta el torrente sanguíneo, reduciendo la inflamación exacerbada producida en este modelo. Además, la modificación de la respuesta inmune del huésped va unida a la actividad antimicrobiana que presentaron los metabolitos del tamoxifeno: n-desmetiltamoxifeno, endoxifeno y 4-hidroxitamoxifeno, frente a las cepas bacterianas de estudio. Por otra parte, se demostró que la rafoxanida potenció el efecto de la colistina en cepas sensibles y resistentes a colistina de A. baumannii, P. aeruginosa y K. pneumoniae. La sinergia observada es debida posiblemente al aumento de la permeabilidad de membrana y al aumento de cargas negativas en la superficie de la misma, potenciando la afinidad de la colistina y restaurando su actividad. Además, la rafoxanida también presentó eficacia terapéutica tanto en monoterapia como en terapia combinada con colistina en un modelo de sepsis peritoneal murino causado por cepas resistentes a colistina de los tres patógenos. En conjunto, estos resultados proporcionan nuevos conocimientos sobre el uso del anticancerígeno tamoxifeno y el antihelmíntico rafoxanida en monoterapia o en combinación con colistina, así como sobre el mecanismo de acción de ambos fármacos. Por ello, esta Tesis Doctoral proporciona dos nuevas alternativas terapéuticas para el tratamiento de infecciones causadas por BGN multirresistentes

Drug Repurposing for the Treatment of Bacterial and Fungal Infections

Multidrug-resistant (MDR) pathogens pose a well-recognized global health threat that demands effective solutions; the situation is deemed a global priority by the World Health Organization and the European Centre for Disease Prevention and Control. Therefore, the development of new antimicrobial therapeutic strategies requires immediate attention to avoid the ten million deaths predicted to occur by 2050 as a result of MDR bacteria. The repurposing of drugs as therapeutic alternatives for infections has recently gained renewed interest. As drugs approved by the United States Food and Drug Administration, information about their pharmacological characteristics in preclinical and clinical trials is available. Therefore, the time and economic costs required to evaluate these drugs for other therapeutic applications, such as the treatment of bacterial and fungal infections, are mitigated. The goal of this review is to provide an overview of the scientific evidence on potential non-antimicrobial drugs targeting bacteria and fungi. In particular, we aim to: (i) list the approved drugs identified in drug screens as potential alternative treatments for infections caused by MDR pathogens; (ii) review their mechanisms of action against bacteria and fungi; and (iii) summarize the outcome of preclinical and clinical trials investigating approved drugs that target these pathogens

Antibacterial activity of colloidal silver against Gram-negative and Gram-positive bacteria.

Motivation: Treatment of multidrug-resistant (MDR) bacteria represent a challenge for clinicians and public health authorities. Due to the emergence of resistance to a wide variety of antibiotics new alternative therapies are needed. Silver has been used to treat bacterial infections since antiquity due to its known antimicrobial properties [1]. The objective of this project was to study in vitro the activity of colloidal silver against Gram-negative and Gram-positive bacteria.Methods: Gram-negative bacteria [Acinetobacter baumannii (n=44), Pseudomonas aeruginosa (n=25) and Escherichia coli (n=79)] and Gram-positive bacteria [Staphylococcus aureus (n=34), Syaphylococcus epidermidis (n=14) and Enterococcus spp. (n=15)] were used. All strains were grown in a Mueller-Hinton Broth (MHB) at 37°C for 20-24 h. Minimal Inhibitory Concentration (MIC) was determined for all strains by using microdilution assay. To monitore the antibacterial activity, time-kill curve assays were performed on MHB at colloidal silver concentrations of 0.5x, 1x and 2x MIC with starting inoculum of 1x10^6 colony-forming units (cfu)/mL. Reactive Oxygen Species (ROS) production was measured at 6, 20 and 24 hours at colloidal silver concentrations of 0.25x, 0.5x and 1x MIC.Results:  Colloidal silver MIC range was from 4-8 mg/L for both Gram-negative and Gram-positive bacteria. Colloidal silver showed bactericidal activity against Gram-negative bacteria. However, it showed bacteriostatic activity against Gram-positive bacteria. For A. baumannii (Ab11 and ATCC 17978 strains), P. aeruginosa (Pa238 and Pa01 strains), and E. coli (mcr-1 positive strain) colloidal silver was bactericidal at 1x, and 2x MIC at 24h. However, at 24h, E. coli (ATCC 25922 strain) showed a regrowth at 0.5x, 1x and 2x MIC. Incubation of bacterial strains with colloidal silver led to a significant increase in ROS production at 24h in Gram-negative bacteria.Conclusions: Colloidal silver showed in vitro activity against these kind of pathogens, especially against Gram-negative bacteria. These results suggest that colloidal silver could be a new alternative for treatment of infections caused by MDR pathogens

Interactions between BRD4S, LOXL2, and MED1 drive cell cycle transcription in triple‐negative breast cancer

Cell cycle; Gene expression; Triple-negative breast cancerCiclo celular; Expresión génica; Cáncer de mama triple negativoCicle cel·lular; Expressió gènica; Càncer de mama triple negatiuTriple‐negative breast cancer (TNBC) often develops resistance to single‐agent treatment, which can be circumvented using targeted combinatorial approaches. Here, we demonstrate that the simultaneous inhibition of LOXL2 and BRD4 synergistically limits TNBC proliferation in vitro and in vivo. Mechanistically, LOXL2 interacts in the nucleus with the short isoform of BRD4 (BRD4S), MED1, and the cell cycle transcriptional regulator B‐MyB. These interactions sustain the formation of BRD4 and MED1 nuclear transcriptional foci and control cell cycle progression at the gene expression level. The pharmacological co‐inhibition of LOXL2 and BRD4 reduces BRD4 nuclear foci, BRD4‐MED1 colocalization, and the transcription of cell cycle genes, thus suppressing TNBC cell proliferation. Targeting the interaction between BRD4S and LOXL2 could be a starting point for the development of new anticancer strategies for the treatment of TNBC.We thank the CRG genomics unit, the CRG‐UPF flow cytometry unit, and the VHIO mouse facility for their contribution. We thank Pharmaxis for the supply of PXS LOXL2 inhibitors. SS is supported by the Plan Estatal de I + D + I (COMBAT PID2019‐110598GA‐I00), and the ERC Starting Grant (ERC‐StG‐852343‐EPICAMENTE). LP‐R is supported by the Juan de la Cierva‐Formación fellowship (FJC2019‐040598‐I) and Fundación Franscico Cobos fellowship. TVT is supported by Plan Estatal de I + D + I (PID2019‐108008RJ‐I00), AECC (INVES20036TIAN), and a Ramón y Cajal investigator contract (RYC2020‐029098‐I). DC is supported by the la Caixa Foundation PhD fellowship (ID 100010434; fellowship code LCF/BQ/DI19/11730061)

Anthelmintic Drugs for Repurposing against Gram-Negative Bacilli Infections

Bacterial infections are among the leading causes of death worldwide. The emergence of antimicrobial resistance factors threatens the efficacy of all current antimicrobial agents, with some already made ineffective, and, as a result, there is an urgent need for new treatment approaches. International organizations, such as the World Health Organization and the European Centre for Diseases Control, have recognized infections caused by multi-drug-resistant (MDR) bacteria as a priority for global health action. Classical antimicrobial drug discovery involves in vitro screening for antimicrobial candidates, Structure-Activity Relationship analysis, followed by in vivo testing for toxicity. Bringing drugs from the bench to the bedside involves huge expenditures in time and resources. This, along with the relatively short window of therapeutic application for antibiotics attributed to the rapid emergence of drug resistance, has, at least until recently, resulted in a waning interest in antibiotic discovery among pharmaceutical companies. In this environment, “repurposing” (defined as investigating new uses for existing approved drugs) has gained renewed interest, as reflected by several recent studies, and may help to speed up the drug development process and save years of expensive research invested in antimicrobial drug development. The goal of this review is to provide an overview of the scientific evidence on potential anthelmintic drugs targeting Gram-negative bacilli (GNB). In particular, we aim to: (i) highlight the potential of anthelmintic drugs for treatments of GNB infections, (ii) review their mechanisms of action against these bacteria, (iii) summarize the outcome of preclinical studies investigating approved anthelmintic drugs that target these bacteria, (iv) provide critical challenges for further anthelmintic repurposing drugs development, and (v) list the specific anthelmintic drugs that may be more likely to be repurposed.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants PI16/01378 and PI19/01453), co-financed by European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014-2020. YS was supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP20/00018

Corrigendum: Drug Repurposing for the Treatment of Bacterial and Fungal Infections.

[This corrects the article DOI: 10.3389/fmicb.2019.00041.]

Antibacterial Activity of Colloidal Silver against Gram-Negative and Gram-Positive Bacteria

This article belongs to the Special Issue Drugs Repurposing for the Treatment of Bacterial Infections.Due to the emergence of antimicrobial resistance, new alternative therapies are needed. Silver was used to treat bacterial infections since antiquity due to its known antimicrobial properties. Here, we aimed to evaluate the in vitro activity of colloidal silver (CS) against multidrug-resistant (MDR) Gram-negative and Gram-positive bacteria. A total of 270 strains (Acinetobacter baumannii (n = 45), Pseudomonas aeruginosa (n = 25), Escherichia coli (n = 79), Klebsiella pneumoniae (n = 58)], Staphylococcus aureus (n = 34), Staphylococcus epidermidis (n = 14), and Enterococcus species (n = 15)) were used. The minimal inhibitory concentration (MIC) of CS was determined for all strains by using microdilution assay, and time–kill curve assays of representative reference and MDR strains of these bacteria were performed. Membrane permeation and bacterial reactive oxygen species (ROS) production were determined in presence of CS. CS MIC90 was 4–8 mg/L for all strains. CS was bactericidal, during 24 h, at 1× and 2× MIC against Gram-negative bacteria, and at 2× MIC against Gram-positive bacteria, and it did not affect their membrane permeabilization. Furthermore, we found that CS significantly increased the ROS production in Gram-negative with respect to Gram-positive bacteria at 24 h of incubation. Altogether, these results suggest that CS could be an effective treatment for infections caused by MDR Gram-negative and Gram-positive bacteria.This work was supported by the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación, y Universidades (PI16/01378, PI16/01306, CP15/000132), and by the Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación, y Universidades, Spanish Network for Research in Infectious Diseases (RD16/0016/0009), co-financed by the European Development Regional Fund “A Way to Achieve Europe”, Operative Program Intelligent Growth 2014-2020. Y.S. is supported by the Subprograma Miguel Servet Tipo I, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación, y Universidades, Spain [CP15/00132]. A.R.V. is supported by the Subprograma Río Hortega, Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación, y Universidades, Spain [CM18/00122]

Repositioning rafoxanide to treat Gram-negative bacilli infections

[Objectives] Repurposing drugs provides a new approach to the fight against MDR Gram-negative bacilli (MDR-GNB). Rafoxanide, a veterinary antihelminthic drug, has shown antibacterial activity in vitro against Gram-positive bacteria. We aimed to analyse the in vitro and in vivo efficacy of rafoxanide in combination with colistin against colistin-susceptible (Col-S) and colistin-resistant (Col-R) GNB.[Methods] A collection of Col-S and Col-R Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae were used. Chequerboard and time–kill curve analyses were performed to determine the synergy between rafoxanide and colistin. Changes in membrane structure and permeability were analysed using transmission electron microscopy and fluorescence assays. A murine peritoneal sepsis model using Col-R strains of these pathogens was performed to study the efficacy of rafoxanide (10 mg/kg/24 h, IV), colistimethate sodium (CMS) (20 mg/kg/8 h, intraperitoneally) and rafoxanide (10 mg/kg/24 h, IV) plus CMS (20 mg/kg/8 h, intraperitoneally) for 72 h.[Results] Rafoxanide showed MICs ≥256 mg/L for all Col-S and Col-R strains. Chequerboard and time–kill curve analyses showed that rafoxanide (1 mg/L) is more synergistic with colistin against Col-R than Col-S strains. Col-R, but not Col-S, strains treated with rafoxanide demonstrated higher membrane permeabilization. Transmission electron microscopy visualization confirmed that Col-R strains suffer morphological changes. In the murine peritoneal sepsis model with Col-R strains, rafoxanide plus CMS, compared with CMS alone, increased mouse survival to 53.8% and 73.3%, and reduced bacterial loads in tissues and blood between 2.34 and 4.99 log10 cfu/g or mL, respectively.[Conclusions] Rafoxanide repurposing, as monotherapy and in combination with CMS, may address the urgent need for new treatments for infections caused by MDR-GNB.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants PI16/01378 and PI16/01306), and by Plan Nacional de I + D+i 2013‐2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009)—co‐financed by European Development Regional Fund ‘A way to achieve Europe’, Operative program Intelligent Growth 2014‐2020. Younes Smani is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/01358)

Tamoxifen and its metabolites antibacterial activities

The development of new strategic therapies for multidrug-resistant bacteria, like the use of non-antimicrobial approaches and/or drugs repurposing to be used as monotherapies or in combination with clinically relevant antibiotics, has become an urgent need. A therapeutic alternative for infections by multidrug-resistant Gram-negative bacilli (MDR-GNB) is immune system modulation to improve the infection clearance. We showed that immunocompetent mice infected by Acinetobacter baumannii, Pseudomonas aeruginosa or Escherichia coli in peritoneal sepsis models and treated with tamoxifen at 80 mg/kg/d for three days reduced the release of MCP-1 and its signalling pathway IL-18 and phosphorylated ERK1/2. This reduction of MCP-1 induced the reduction of migration of inflammatory monocytes and neutrophils from bone marrow to blood. Indeed, the treatment with tamoxifen in murine peritoneal sepsis models reduced the bacterial load in tissues and blood; and increased the mice survival from 0% to 60-100%. Tamoxifen treatment of neutropenic mice infected by these pathogens increased mice survival up to 20-60%. Furthermore, susceptibility and time-kill assays showed that the metabolites of tamoxifen, N-desmethyltamoxifen, hydroxytamoxifen and endoxifen, the three together exhibited MIC90 values of 16 mg/L and were bactericidal against clinical isolates of A. baumannii and E. coli. This antimicrobial activity of tamoxifen metabolites parallels' an increased membrane permeability of A. baumannii and E. coli without affecting their outer membrane proteins profiles. Together, these data showed that tamoxifen present a therapeutic efficacy against MDR A. baumannii, P. aeruginosa and E. coli in experimental models of infections and can be repurposed as new treatment for GNB infections.This study was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud (grants CP15/00132, PI16/01378 and PI19/01453) and by Plan Nacional de I+D+i 2013‐ 2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0009) ‐ co‐ financed by European Development Regional Fund “A way to achieve Europe”, Operative program Intelligent Growth 2014‐2020. Younes Smani is supported by the Subprograma Miguel Servet Tipo I from the Ministerio de Economía y Competitividad of Spain (CP15/00132)

Repurposing of the tamoxifen metabolites to combat infections by multidrug-resistant gram-negative bacilli

The development of new strategic antimicrobial therapeutic approaches, such as drug repurposing, has become an urgent need. Previously, we reported that tamoxifen presents therapeutic efficacy against multidrug-resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli in experimental infection models by modulating innate immune system cell traffic. The main objective of this study was to analyze the activity of N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen, three major metabolites of tamoxifen, against these pathogens. We showed that immunosuppressed mice infected with A. baumannii, P. aeruginosa, or E. coli in peritoneal sepsis models and treated with tamoxifen at 80 mg/kg/d for three days still reduced the bacterial load in tissues and blood. Moreover, it increased mice survival to 66.7% (for A. baumannii and E. coli) and 16.7% (for P. aeruginosa) when compared with immunocompetent mice. Further, susceptibility and time-kill assays showed that N-desmethyltamoxifen, 4-hydroxytamoxifen, and endoxifen exhibited minimum inhibitory concentration of the 90% of the isolates (MIC90) values of 16 mg/L, and were bactericidal against clinical isolates of A. baumannii and E. coli. This antimicrobial activity of tamoxifen metabolites paralleled an increased membrane permeability of A. baumannii and E. coli without affecting their outer membrane proteins profiles. Together, these data showed that tamoxifen metabolites presented antibacterial activity against MDR A. baumannii and E. coli, and may be a potential alternative for the treatment of infections caused by these two pathogens.Instituto de Salud Carlos III, Proyectos de Investigación en Salud (becas PI16 / 01378 y PI19 / 01453)Plan Nacional de I + D + i 2013-2016 y el Instituto de Salud Carlos IIIRed Española de Investigación en Enfermedades Infecciosas (REIPI RD16 / 0016/0009)ounes Smani cuenta con el apoyo del Subprograma Miguel Servet Tipo I del Ministerio de Economía y Competitividad de España (CP15 / 00132)