Identification of new antibacterial targets in RNA polymerase of Mycobacterium tuberculosis by detecting positive selection sites

Bacterial RNA polymerase (RNAP) is an effective target for antibacterial treatment. In order to search new potential targets in RNAP of Mycobacterium, we detected adaptive selections of RNAP related genes in 13 strains of Mycobacterium by phylogenetic analysis. We first collected sequences of 17 genes including rpoA, rpoB, rpoC, rpoZ, and sigma factor A-M. Then maximum likelihood trees were constructed, followed by positive selection detection. We found that sigG shows positive selection along the clade (M. tuberculosis, M. bovis), suggesting its important evolutionary role and its potential to be a new antibacterial target. Moreover, the regions near 933Cys and 935His on the rpoB subunit of M. tuberculosis showed significant positive selection, which could also be a new attractive target for anti-tuberculosis drugs

Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided

Antimicrobial activity and mode of action of bacteriocin AS-48 against Mycobacterium tuberculosis

La creciente incidencia de cepas multirresistentes de Mycobacteriumtuberculosis y los pocos medicamentos disponibles para su tratamiento estánpromoviendo el desarrollo de nuevos medicamentos que tratan de resolver estosproblemas. Siguiendo esta idea, este trabajo ha explorado el péptido antibacterianoAS-48, producido por Enterococcus faecalis, cuya diana es la membrana bacterianay, además, es activo contra varias bacterias grampositivas. Se ha demostrado queAS-48 tiene una acción bactericida contra M. tuberculosis, incluyendo H37Rv yotras cepas clínicas y de referencia, también contra algunas especiesmicobacterianas clínicas no tuberculosas. Cabe destacar el efecto sinérgico de lacombinación de AS-48 con lisozima o etambutol (comúnmente utilizado en eltratamiento de la tuberculosis), dos compuestos que aumentan la acciónantimicrobiana de AS-48. En estas condiciones, AS-48 mata a M. tuberculosis a unadosis más baja y muestra una CIM (Concentración Inhibitoria Mínima) cercana aalgunos de los agentes anti-TB de primera línea.Además, se ha ensayado la citotoxicidad de AS-48 contra las líneas celularesde macrófagos THP-1, MHS y J774, y se ha encontrado que a concentracionescercanas a la CIM de AS-48 no se pudo detectar ningún efecto citotóxico. Laactividad de AS-48 para inhibir el crecimiento de M. tuberculosis también seobservó dentro de los macrófagos infectados; en este modelo, también se observósinergia para las combinaciones de AS-48 y etambutol.Se ha explorado el mecanismo de acción de la bacteriocina AS-48 contra M.tuberculosis que muestra que AS-48 está afectando la membrana, que es una de lasdianas menos estudiadas, y que reduciría la aparición de nuevas cepas resistentes.Además, algunos mecanismos relacionados con la aparición de cepas persistentesparecen estar alterados después de tratar M. tuberculosis con AS-48. Se ha realizadoun análisis transcriptómico para comprender mejor el modo de acción AS-48.Curiosamente, se ha demostrado que AS-48 también tiene como diana lamovilidad del ADN, probablemente afectando a otros procesos moleculares. Porlo tanto, podemos considerar que AS-48 es un fármaco multidiana, y como tal, suuso reduciría las probabilidades de desarrollar cepas mutantes resistentes.Se han realizado estudios de toxicidad in vivo que muestran que la cepaBALB/c es menos sensible a AS-48 que C57B/6. Los ensayos de toxicidadsubcrónica muestran que los ratones se mantienen saludables en tratamientos dediez días con dosis de 2-1 mg/Kg de peso corporal de AS-48.Sin embargo, en el ensayo de eficacia, AS-48 no muestra la actividadantituberculosis observada en los ensayos in vitro; incluso junto con etambutol, nodemostró reducir la carga bacteriana en ratones infectados con M. tuberculosis.En resumen, este trabajo revela algunos aspectos positivos de AS-48(actividad antimicrobiana, baja toxicidad, sinergismo con etambutol) pero tambiénotros aspectos que tienen que mejorarse (ausencia de actividad in vivo o más bienalta toxicidad). Así más trabajos y refinamientos son necesarios para poderpresentar a AS-48 como un candidato terapéutico contra tuberculosis.<br /

Identification and Evaluation of Bacterial RNA Polymerase Inhibitors Using a Novel Plasmid-based Transcription Assay

Tuberculosis (TB) is a global health problem caused by Mycobacterium tuberculosis with 8-10 million new cases each year according to the World Health Organization. The current treatment includes a 6-9 month treatment with four different drugs. Due to the long treatment time and lack of adherence to the treatment regimen, there is a rise in the number of multi-drug resistant strains of Mycobacterium tuberculosis (MDR-TB). Rifampin, a long-time staple in TB treatment, is an effective drug against TB which acts by inhibiting the bacterial enzyme RNA polymerase (RNAP). However, there are problems of resistance to rifampin due to mutations in the gene encoding for RNAP and rifampin is a very effective inducer of CYP450s. It is of interest to develop potent inhibitors against bacterial RNAP that are effective against both the wild-type M. tuberculosis RNAP and common mutant RNAPs and do not induce CYP450s. Nucleic acid aptamers are very useful oligonucleotides that bind specifically to a target molecule based on the nucleotide sequence. An in vitro plasmid-based RNAP transcription assay was developed and adapted to high-throughput screening, in which a malachite green aptamer (MGA) is used as the detection method for RNA transcription. Compounds identified were evaluated for activity against a panel of 10 bacterial RNAPs (E. coli and MTB - WT and RifR RNAPs), and active scaffolds were further studied resulting in identification of a cyanopyrimidine scaffold for bacterial RNAP inhibition. Previous studies have shown benzoxazinorifamycins to have improved activity compared to rifampin in vitro against RifR RNAP enzymes. Additionally, benzoxazinorifamycins have decreased induction of the human pregnane X receptor (hPXR), which leads to expression of CYP3A4 and drug-drug interactions for patients taking rifampin concurrently with HIV medications. Novel benzoxazinorifamycins were evaluated for in vitro activity against WT and RifR bacterial RNAPs (E. coli and MTB) using the plasmid-based transcription assay. We hypothesize that identification of rifamycins that do not activate hPXR and are still active against M. tuberculosis RNAP and M. tuberculosis in culture will allow for the development of anti-TB drugs that can be taken concurrently with HIV medications, as TB-HIV coinfection is a global problem.PHDMedicinal ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140941/1/scharfn_1.pd

Substandard antimicrobial drugs: detection methods and their contributions to antibiotic resistance

Substandard and counterfeit medicines are major obstacles to the treatment of infectious diseases. Substandard medicines vary from standard drugs in terms of dose, bioavailability, or the presence of impurities. Current methods to identify substandard and counterfeit antimicrobial drugs are either resource intensive or have poor specificity. This dissertation examined two issues related to poor quality antimicrobial medicines: 1) Methods to detect and prevent the consumption of substandard drugs. 2) The relationship between substandard medicines and the evolution of rifampicin resistance. This dissertation advanced two technologies that may aid in the detection of substandard medicines: aptamers and biosensors. Oligonucleotide aptamers may be adapted for drug detection by coupling binding events to changes in fluorescence, luminescence or colorimetric signals. A computational model was developed to discover experimental factors that increase the probability of selecting a high affinity aptamer. Among them are: micromolar drug target concentration, high affinity substrate to partition aptamers, and high aptamer library affinity distribution. Random losses of aptamers due to experimental noise greatly decreased the probability of selecting an aptamer. Experimental parameters to optimize the process of aptamer discovery for small molecules are discussed. Bacterial biosensors are an alternative strategy for the detection of active pharmaceutical ingredients. Here, luciferase-expressing Escherichia coli were used to create profiles of drug interactions for anti-mycobacterial drugs. Drug interactions were tested by the Loewe additivity model. A novel method to differentiate rifamycin drugs from the drug degradation product rifampicin quinone was developed by analyzing each drug’s unique interactions. While subinhibitory drug doses are known to select for antimicrobial resistance in vitro, the role of substandard anti-mycobacterial medicines in the development of rifampicin resistance remains poorly understood. The role of the drug degradation product rifampicin quinone on rifamycin resistance was assessed through in vitro studies of bacteria. Wild type Escherichia coli and Mycobacterium smegmatis cultured in the presence of rifampicin quinone acquired high levels of resistance to rifamycin drugs. Resistance was associated with genetic mutations in the rifampicin resistance cluster of the rpoB gene. The studies presented here demonstrate that substandard medicines can contribute towards rifamycin resistance, and offer methodologies to identify substandard medicines.2020-10-24T00:00:00

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived from a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. As the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets

Deciphering the Details of RNA Aminoglycoside Interactions: From Atomistic Models to Biotechnological Applications

Aminoglycosides are a class of antibiotics functioning through binding to 16S rRNA A-site and inhibiting the bacterial translation. However, the continuous emergence of drug-resistant strains makes the development of new and more potent antibiotics necessary. Aminoglycosides are also known to interact with various biologically crucial RNA molecules other than 16S rRNA A-site and inhibit their functions. As a result, they are considered as the single most important model to understand the principles of RNA small molecule recognition. The detailed understanding of these interactions is necessary for the development of novel antibacterial, antiviral or even anti-oncogenic agents. In our studies, we have studied both the natural aminoglycoside targets like Rev responsive element (RRE), trans-activating region (TAR) of HIV-1 and thymidylate synthase mRNA 5\u27 untranslated (UTR) region as well as the in vitro selected neomycin, tobramycin and kanamycin RNA aptamers. By this way, we think we have covered a variety of binding pockets to figure out the critical nucleic acid residues playing essential role in aminoglycoside recognition. Along with all these RNAs, we studied more than 10 aminoglycoside ligands to pinpoint the chemical groups in close contact with RNAs. To determine thermodynamic parameters for these interactions, we utilized isothermal titration calorimetry (ITC) assay by which we found that the majority of these interactions are enthalpy driven. More specifically, RNA aminoglycoside interactions are mainly derived by electrostatic and hydrogen binding interactions. Our studies indicated that the amino groups on the first ring of the aminoglycosides are essential for high affinity binding whereas having bulky groups on ring II sterically eliminate their interactions with RNAs. RNA binding trend of aminoglycosides are as follows: neomycin-B \u3e ribostamycin \u3e kanamycin-B \u3e tobramycin \u3e paromomycin \u3e sisomicin \u3e gentamicin \u3e kanamycin-A \u3e geneticin \u3e amikacin \u3e netilmicin. Aminoglycoside binding to the aptamer was shown highly buffer dependent. This phenomenon was analyzed in five different buffers and found that cacodylate-based buffer changes the specificity of the aptamer. In addition to ITC, we have used molecular docking to specifically find out the chemical groups in these interactions. We have specified the nucleic acid residues interacting with aminoglycosides. In parallel, molecular dynamics (MD) simulations of neomycin RNA aptamer with neomycin-B in an all-atom platform in GROMACS were carried out. The results showed a mobile structure consistent with the ability of this aptamer to interact with a wide range of ligands. From molecular docking and MD simulations, we identified the neomycin-B aptamer residues that might contribute to its ligand selectivity and designed a series of new aptamers accordingly. Also, A16 was found to be flexible, which was confirmed by 2AP fluorescence studies. In this analysis, the buffer dependence was also confirmed against neomycin-B, ribostamycin and paromomycin. One of the challenges in therapeutics is the emergence of resistant cells. They become reistant to the drugs via changing the target site, or enzymatically modifying the drug, or producing drug pumps to export the drugs. To overcome the very last challenge, we are utilizing RNA-aminoglycoside partners to keep high intracellular drug concentration and increase the efficacy of aminoglycosides against bacteria. We called the system as DRAGINs (Drug binding aptamers for growing intracellular numbers). We express these RNAs in bacteria and detect their growth rate in order to evaluate their response to different concentration of aminoglycosides. In this study, we found that we could successfully decrease the IC50 values by 2 to 5 fold with the help of aminoglycoside-binding RNA aptamers. Finally, we are mathematically modeling the effect of aptamers on IC50 values of drugs with the use of four-compartment model. In our research group, we are utilizing these RNA-aminoglycoside partners to develop tags for detecting RNA in vivo and in real time. We called this system as intracellular multiaptamer genetic tags (IMAGEtags)