Small molecule inhibitors of the response regulator ArsR exhibit bactericidal activity against Helicobacter pylori

Helicobacter pylori is considered the most prevalent bacterial pathogen in humans. The increasing antibiotic resistance evolved by this microorganism has raised alarm bells worldwide due to the significant reduction in the eradication rates of traditional standard therapies. A major challenge in this antibiotic resistance crisis is the identification of novel microbial targets whose inhibitors can overcome the currently circulating resistome. In the present study, we have validated the use of the essential response regulator ArsR as a novel and promising therapeutic target against H. pylori infections. A high-throughput screening of a repurposing chemical library using a fluorescence-based thermal shift assay identified several ArsR binders. At least four of these low-molecular weight compounds noticeably inhibited the DNA binding activity of ArsR and showed bactericidal effects against antibiotic-resistant strains of H. pylori. Among the ArsR inhibitors, a human secondary bile acid, lithocholic acid, quickly destroyed H. pylori cells and exhibited partial synergistic action in combination with clarithromycin or levofloxacin, while the antimicrobial effect of this compound against representative members of the normal human microbiota such as Escherichia coli and Staphylococcus epidermidis appeared irrelevant. Our results enhance the battery of novel therapeutic tools against refractory infections caused by multidrug-resistant H. pylori strains

Galleria mellonella como modelo animal de la infección por Helicobacter pylori y para estudios de eficacia preclínica de nuevos antimicrobianos frente a este patógeno.

H. pylori es una bacteria Gram-negativa que coloniza el estómago de los humanos. La prevalencia de la infección por H. pylori en el mundo varía en función de numerosos factores como la edad, el origen étnico, el estado geográfico y socioeconómico. Más del 50 % de la población mundial está infectada con H. pylori y se considera un agente etiológico importante en el desarrollo de enfermedades como gastritis, úlcera péptica, linfoma gástrico asociado a mucosa (MALT) y cáncer gástrico. El dinamismo genético del organismo, unido a diversos factores ambientales y del propio huésped determina el daño epitelial y la capacidad colonizadora de H. pylori, lo que explica la diversidad de enfermedades causadas por la bacteria. Existen varios factores de virulencia que se han relacionado con la agresividad de la bacteria y por tanto, están implicados en la colonización y daño en la mucosa. Los regímenes farmacológicos más utilizados para tratar la infección por H. pylori han variado durante los últimos años y no existe un tratamiento único. La creciente prevalencia de cepas de H. pylori resistentes a antibióticos es una de las principales causas de la dificultad actual de erradicar la infección. Además, la Organización Mundial de la Salud en 2017 catalogó a H. pylori como una de las infecciones con resistencia a antibióticos alta. Por ello, hoy en día en necesario encontrar una nueva terapia efectiva contra H. pylori. Como modelo animal para el estudio de la eficacia de tratamientos en la infección por H. pylori se han utilizado modelos animales como ratones y jerbos, pero debido al gran coste que suponen y a los impedimentos éticos que supone el uso de animales actualmente se están buscando alternativas que permitan reducir el número de los mismos. Por ello, se están reemplazando con modelos de animales invertebrados como es el caso de G. mellonella. Se ha demostrado que este modelo es susceptible a la infección por H. pylori y se puede utilizar para estudiar mecanismos patogénicos y factores de virulencia de la bacteria. <br /

Inhibición del Transporte de Protones como estrategia terapéutica del Adenocarcinoma de Esófago: estudio in vitro

La alteración del gradiente de pH intra/extracelular consecuencia del aumento de expresión de transportadores de H+ de membrana es una característica de las células neoplásicas que tiene un enorme impacto en el metabolismo energético de la célula y se ha visto implicado tanto en la transformación neoplásica como en la progresión y metástasis de los tumores. OBJETIVO Evaluar el efecto que la inhibición farmacológica de diferentes transportadores de protones, en concreto la bomba de protones V-H+ATPasa y el transportador MCT1, ejerce sobre los fenómenos de proliferación y apoptosis en una línea celular de adenocarcinoma de esófago. METODOS Se utilizó una línea de células de adenocarcinoma de esófago humano (OE33) que expresa V-H+ATPasa a nivel de membrana. Se evaluó la expresión de MCT1 (inmunocitoquímica), concentración intracelular de lactato, apoptosis y proliferación (citometría de flujo). Se analizó el efecto de Esomeprazol (para inhibir la V-H+ATPasa y de AZD3965 (inhibidor selectivo de MCT1) en dos condiciones experimentales (con/sin sobrecarga de glucosa). RESULTADOS La línea OE33 expresa el transportador MCT1. El tratamiento con Esomeprazol a concentraciones altas indujo la apoptosis e inhibió la proliferación celular. La presencia de mayor concentración de glucosa en el medio (30 mM) aumentó la concentración de lactato intracelular, aumentó el efecto pro-apoptótico de Esomeprazol pero revirtió el efecto anti-proliferativo del mismo. El tratamiento de las células con el inhibidor de MCT1, AZD3965, a pesar de que indujo un aumento del lactato intracelular, no produjo ningún efecto en la proliferación ni en la apoptosis de las células OE33. CONCLUSIÓN Esomeprazol a alta concentración ejerce efectos antineoplásicos en células de adenocarcinoma de esófago humano, induciendo la apoptosis e inhibiendo la proliferación. La presencia de altas concentraciones de glucosa en el medio de cultivo potencia el efecto proapoptótico de Esomeprazol pero revierte el efecto antiproliferativo del mismo. A pesar de que la línea celular empleada expresa MCT1 y que su inhibición con AZD3965 aumenta la concentración de lactato intracelular, no ejerce ningún efecto citotóxico en las células OE33

Inhibición de la actividad biológica del regulador transcripcional HsrA de Helicobacter pylori mediante el empleo de ligandos de bajo peso molecular y estudio de la capacidad antimicrobiana de estos compuestos

Helicobacter pylori es el patógeno bacteriano de mayor prevalencia mundial, infectando a más de la mitad de la población y estando relacionado con más del 90% de casos de cáncer gástrico. El incremento y acumulación de resistencias frente a la mayoría de los antibióticos convencionales ha determinado una disminución apreciable de la eficacia de los regímenes de erradicación empleados para este patógeno. En la actualidad resulta imperativa la búsqueda de nuevas dianas terapéuticas para la investigación y desarrollo de nuevas familias de antimicrobianos efectivas, que logren evadir los mecanismos de resistencia actuales. Nuestro grupo de investigación ha sido el primero en validar el regulador de respuesta esencial HsrA de H. pylori como diana molecular para el descubrimiento de nuevos fármacos con actividad bactericida frente a este microorganismo. Mediante cribados masivos de quimiotecas han sido identificados varios ligandos de HsrA que se unen al regulador, formando un complejo con mayor estabilidad térmica que la proteína. En el presente trabajo se evaluó la capacidad inhibitoria de 20 ligandos de bajo peso molecular y naturaleza química diversa sobre la actividad in vitro del HsrA mediante estudios de retardo en gel. Se estudió la interacción molecular proteína-inhibidores mediante acoplamiento (docking) molecular y se analizó la actividad antimicrobiana de los inhibidores de HsrA frente a diferentes cepas de H. pylori mediante la determinación de las concentraciones mínimas inhibitorias (CMI) y las concentraciones mínimas bactericidas (CMB). Seis de los ligandos estudiados inhibieron efectivamente la actividad biológica in vitro de la proteína HsrA a concentraciones iguales o inferiores a 2 mM. Todos los inhibidores de HsrA se unieron preferiblemente al dominio efector C-terminal de la proteína, interaccionando con residuos directamente implicados en la estructura del motivo HTH de unión al DNA o con residuos aparentemente imprescindibles para la estabilidad del dominio. Al menos 2 de estos inhibidores mostraron una alta capacidad antimicrobiana frente a H. pylori, con valores de CMI y CMB ≤ 8 mg/L. Los resultados obtenidos avalan el uso del regulador transcripcional HsrA como diana terapéutica e incrementan la batería de potenciales nuevas herramientas para el tratamiento de infecciones refractarias causadas por cepas de H. pylori resistentes a los antibióticos convencionales. ABSTRACTHelicobacter pylori is the most worldwide prevalent bacterial pathogen, infecting more than half of the world’s population and it has been associated with more than 90% of gastric cancer cases. The increase and accumulation in the resistance against most of the conventional antibiotics has determined a greatly and noticeable reduction in the efficacy of eradication therapies used for the treatment of infections caused by this pathogen. Currently, it is imperative to search for new therapeutic targets for the research and development of novel and effective antimicrobial families which can surpass current resistance mechanisms. Our research group has been the first one to validate the essential response regulator HsrA from H. pylori as a molecular target for the discovery of new drugs with bactericidal activity against this microorganism. Through high-throughput screening of some chemical libraries several HsrA ligands that bind to the response regulator were found, forming a complex with a higher thermal stability compared to the free protein. In the present work we studied the inhibitory capacity of 20 low-weight and structurally diverse ligands over the in vitro activity of HsrA by electrophoretic mobility shift assays. The molecular interaction between the protein and its inhibitors was studied by molecular docking and antimicrobial activity of HsrA inhibitors was analysed against different H. pylori strains by determining the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). Six of the studied ligands effectively inhibited the in vitro biological activity of HsrA at concentration equal to or lower than 2 mM. All HsrA inhibitors bound preferably to the C-terminal effector domain in the protein, interacting with residues directly involved in the structure of the HTH DNA-binding motif or with residues apparently essential for the domain stability. At least two of these inhibitors showed a high antimicrobial activity against H. pylori, with MIC and MBC values ≤ 8 mg/L. The results obtained in this study support the use of response regulator HsrA as a therapeutic target and increase the battery of potential novel tools for the treatment of refractory infection caused by H. pylori strains resistant to conventional antibiotics.<br /

Antitumor effects of lactate transport inhibition on esophageal adenocarcinoma cells

As a consequence of altered glucose metabolism, cancer cell intake is increased, producing large amounts of lactate which is pumped out the cytosol by monocarboxylate transporters (MCTs). MCT 1 and MCT4 are frequently overexpressed in tumors, and recently, MCT inhibition has been reported to exert antineoplastic effects. In the present study, MCT1 and MCT4 levels were assessed in esophageal adenocarcinoma (EAC) cells and the effects of the MCT-1 selective inhibitor AZD3965, hypoxia, and a glucose overload were evaluated in vitro. Two EAC cell lines (OE33 and OACM5.1C) were treated with AZD3965 (10–100 nM) under different conditions (normoxia/hypoxia) and also different glucose concentrations, and parameters of cytotoxicity, oxidative stress, intracellular pH (pHi), and lactate levels were evaluated. MCT1 was present in both cell lines whereas MCT4 was expressed in OE33 cells and only in a small proportion of OACM5.1C cells. Glucose addition did not have any effect on apoptosis nor cell proliferation. AZD3965 increased apoptosis and reduced proliferation of OACM5.1C cells, effects which were abrogated when cells were growing in hypoxia. MCT1 inhibition increased intracellular lactate levels in all the cells evaluated, but this increase was higher in cells expressing only MCT1 and did not affect oxidative stress. AZD3965 induced a decrease in pHi of cells displaying low levels of MCT4 and also increased the sodium/hydrogen exchanger 1 (NHE-1) expression on these cells. These data provide in vitro evidence supporting the potential of MCT inhibitors as novel antineoplastic drugs for EAC and highlight the importance of achieving a complete MCT inhibition

Repurposing dihydropyridines for treatment of helicobacter pylori infection

Antibiotic resistance is a major cause of the increasing failures in the current eradication therapies against Helicobacter pylori. In this scenario, repurposing drugs could be a valuable strategy to fast-track novel antimicrobial agents. In the present study, we analyzed the inhibitory capability of 1, 4-dihydropyridine (DHP) antihypertensive drugs on the essential function of the H. pylori response regulator HsrA and investigated both the in vitro antimicrobial activities and the in vivo efficacy of DHP treatments against H. pylori. Six different commercially available and highly prescribed DHP drugs—namely, Nifedipine, Nicardipine, Nisoldipine, Nimodipine, Nitrendipine, and Lercanidipine—noticeably inhibited the DNA binding activity of HsrA and exhibited potent bactericidal activities against both metronidazole-and clarithromycin-resistant strains of H. pylori, with minimal inhibitory concentration (MIC) values in the range of 4 to 32 mg/L. The dynamics of the decline in the bacterial counts at 2 × MIC appeared to be correlated with the lipophilicity of the drugs, suggesting different translocation efficiencies of DHPs across the bacterial membrane. Oral treatments with 100 mg/kg/day of marketed formulations of Nimodipine or Nitrendipine in combination with omeprazole significantly reduced the H. pylori gastric colonization in mice. The results presented here support a novel therapeutic solution for treatment of antibiotic-resistant H. pylori infections

CD24 Expression Is Increased in 5-Fluorouracil-Treated Esophageal Adenocarcinoma Cells

The cancer stem cell (CSC) model suggests that there are subsets of cells within a tumor with increased proliferation and self-renewal capacity, which play a key role in therapeutic resistance. The importance of cyclooxygenase-2 (COX-2) in carcinogenesis has been previously established and the use of COX-2 inhibitors as celecoxib has been shown to exert antitumor effects. The present study investigated whether treatment of esophageal adenocarcinoma (EAC) cells with 5-fluorouracil (5-FU) or the growth of tumor spheres increased the proportion of CSCs and also if treatment with celecoxib was able to reduce the putative CSC markers in this tumor. OE19 and OE33 EAC cells surviving 5-FU exposure exhibited an increase in CSC markers CD24 and ABCG2 and also an increased resistance to apoptosis. EAC cell lines had the capacity to form multiple spheres displaying typical CSC functionalities such as self-renewal and increased CD24 levels. In addition, after the induction of differentiation, cancer cells reached levels of CD24 similar to those observed in the parental cells. Treatment with celecoxib alone or in combination with 5-FU also resulted in a reduction of CD24 expression. Moreover, celecoxib inhibited the growth of tumor spheres. These findings showing a reduction in CSC markers induced by celecoxib suggest that the COX-2 inhibitor might be a candidate for combined chemotherapy in the treatment of EAC. However, additional clinical and experimental studies are needed

La fosfoenolpiruvato carboxilasa (PEPC): enzima clave de los metabolismos fotosintéticos C4 y CAM

http://digital.csic.es/bitstream/10261/29768/13/echevarria.pdfLa fosfoenolpiruvato carboxilasa (PEPC; EC 4.1.1.31) cataliza la β-carboxilación del fosfoenolpiruvato (PEP) en presencia de HCO3 - y Mg2+, para producir oxaloacetato (OAA) y Pi (Chollet et al., 1996). La PEPC está ampliamente distribuida en plantas, algas verdes y microorganismos pero ausente en levaduras y animales (Chollet et al., 1996). En plantas vasculares su papel estelar está relacionado con la fotosíntesis C4 y CAM («Crassulacean acid metabolism»), sin embargo desempeña otras funciones como la anaplerótica, en relación a la síntesis de proteínas, homeostasis del pH citosólico, electroneutralidad y osmolaridad. Está formada por una pequeña familia multigénica algunos de cuyos representantes están regulados a nivel transcripcional por factores como luz, hormonas y metabolitos (Chollet et al., 1996; Vidal y Chollet, 1997). La naturaleza alostérica de la enzima permite una regulación fina en relación a diferentes ambientes metabólicos. La PEPC está regulada por fosforilación reversible, proceso ligado a una cascada de transducción de señales de alta complejidad. En la actualidad es uno de los mejores modelos de señalización descritos en plantas. Este capítulo se centra en los eventos relacionados con este proceso en plantas C4 y CAM, los dos sistemas mejor estudiados en la actualidad (Chollet et al., 1996; Echevarría y Vidal, 2003; Izui et al., 2004; Nimmo, 2000; Vidal y Chollet, 1997).Phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) catalyzes the b-carboxylation of phosphoenolpyruvate (PEP) by HCO3 - in the presence of Mg2+ to yiel oxaloacetate and Pi (Chollet et al., 1996). PEPC is a widely distributed enzyme in plants, green algae and micro-organisms but absent in yeast and animals (Chollet et al., 1996). In higher plants, it catalyses a pivotal reaction related to such important processes as C4 and Crassulacean acid metabolism (CAM) photosynthesis, the anaplerotic pathway linked to amino acid synthesis, homeostasis of cytosolic pH, electroneutrality and osmolarity. PEPC belongs to a small multigenic family (Chollet et al., 1996; Vidal y Chollet, 1997). At the transcriptional level, some PEPC genes respond to external and internal factors (light, hormones and metabolites), while at the protein level, the allosteric nature of the enzyme allows its activity to be fine-tuned in relation to a varying metabolic environment. PEPC undergoes a posttranslational control by a phosphorylation process linked to a highly complex signal transduction cascade. Today, it is one of the best-described models of plant signaling. This chapter will focus on what is known about these processes in leaves of C4 and CAM plants, the two systems that have been studied in detail so far (Chollet et al., 1996; Echevarría y Vidal, 2003; Izui et al., 2004; Nimmo, 2000; Vidal y Chollet, 1997)

Decreased cardiotrophin-1 levels are associated with a lower risk of developing the metabolic syndrome in overweight/obese children after a weight loss program

Objective: Cardiotrophin-1 (CT-1) shares some similarities with other cytokines, and participates in the control of energy metabolism. Higher circulating levels are observed in obese humans, but little information is gathered in weight loss (WL) programs. Therefore, we aimed to investigate the association of serum CT-1 levels with metabolic variables and the risk of developing metabolic syndrome (MetS) after a WL program in overweight/obese children. Subjects and Methods: Forty-four overweight/obese children (mean age 11.5 yr; 50% males) undergoing a 10-week WL program were enrolled. Subjects were dichotomized at the median of Body Mass Index-Standard Deviation Score (BMI-SDS) change, as high and low responders after intervention. Results: CT-1 levels were significantly reduced (-48 fmol/mL, p=0.043) in the high responder group after the WL program. They had significantly lower body weight (-3.7 kg, p<0.001), body fat mass (-8%, p<0.001), BMI-SDS (-0.78, p<0.001) and waist circumference (-5.4 cm, p<0.001), and a significant improvement in lipid and glucose profiles (p<0.05). Interestingly, decreased CT-1 levels significantly predicted changes in total cholesterol (41%) and LDL-cholesterol (28%). Moreover, in our participants the lower the CT-1 levels, the higher the reduction in MetS risk components, after the 10- week intervention, (p-ANCOVA=0.040, p-trend=0.024). Conclusion: We showed, for the first time, a reduction in serum CT-1 levels after a WL program and this decrease in CT-1 was strongly associated with a reduction in cholesterol levels and in MetS risk factors in overweight/obese children. Our findings may suggest that CT-1 could be an indirect marker for the diagnosis of MetS in this population

Spatiotemporal Characteristics of the Largest HIV-1 CRF02_AG Outbreak in Spain: Evidence for Onward Transmissions

Background and Aim: The circulating recombinant form 02_AG (CRF02_AG) is the predominant clade among the human immunodeficiency virus type-1 (HIV-1) non-Bs with a prevalence of 5.97% (95% Confidence Interval-CI: 5.41–6.57%) across Spain. Our aim was to estimate the levels of regional clustering for CRF02_AG and the spatiotemporal characteristics of the largest CRF02_AG subepidemic in Spain.Methods: We studied 396 CRF02_AG sequences obtained from HIV-1 diagnosed patients during 2000–2014 from 10 autonomous communities of Spain. Phylogenetic analysis was performed on the 391 CRF02_AG sequences along with all globally sampled CRF02_AG sequences (N = 3,302) as references. Phylodynamic and phylogeographic analysis was performed to the largest CRF02_AG monophyletic cluster by a Bayesian method in BEAST v1.8.0 and by reconstructing ancestral states using the criterion of parsimony in Mesquite v3.4, respectively.Results: The HIV-1 CRF02_AG prevalence differed across Spanish autonomous communities we sampled from (p &lt; 0.001). Phylogenetic analysis revealed that 52.7% of the CRF02_AG sequences formed 56 monophyletic clusters, with a range of 2–79 sequences. The CRF02_AG regional dispersal differed across Spain (p = 0.003), as suggested by monophyletic clustering. For the largest monophyletic cluster (subepidemic) (N = 79), 49.4% of the clustered sequences originated from Madrid, while most sequences (51.9%) had been obtained from men having sex with men (MSM). Molecular clock analysis suggested that the origin (tMRCA) of the CRF02_AG subepidemic was in 2002 (median estimate; 95% Highest Posterior Density-HPD interval: 1999–2004). Additionally, we found significant clustering within the CRF02_AG subepidemic according to the ethnic origin.Conclusion: CRF02_AG has been introduced as a result of multiple introductions in Spain, following regional dispersal in several cases. We showed that CRF02_AG transmissions were mostly due to regional dispersal in Spain. The hot-spot for the largest CRF02_AG regional subepidemic in Spain was in Madrid associated with MSM transmission risk group. The existence of subepidemics suggest that several spillovers occurred from Madrid to other areas. CRF02_AG sequences from Hispanics were clustered in a separate subclade suggesting no linkage between the local and Hispanic subepidemics