Attenuation of SARS‐CoV‐2 replication and associated inflammation by concomitant targeting of viral and host cap 2'‐O‐ribose methyltransferases

The SARS‐CoV‐2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2''‐O‐ribose cap needed for viral immune escape. We find that the host cap 2''‐O‐ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS‐CoV‐2 replication. Using in silico target‐based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti‐SARS‐CoV‐2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co‐substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID‐19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection‐induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID‐19.     

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

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Orotidine-5′-monophosphate decarboxylase fromPseudomonas aeruginosa PAO1: Cloning,overexpression, and enzyme characterization

Orotidine-5-monophosphate decarboxylase (OMPdecase) catalyzes the final step in pyrimidine biosynthesis, the conversion of orotidine-5-monophosphate (OMP) to uridine-5-monophosphate. ThepyrF gene, encoding OMPdecase, was isolated from a chromosomal library ofPseudomonas aeruginosa PAO1 by screening for complementation of anEscherichia coli and aP. aeruginosa pyrF mutant. The nucleotide sequence of a 2510-bp chromosomal DNA fragment, complementing both strains, was determined (EMBL accession number X65613). On this a 696-bp open reading frame capable of encoding the 24 kDa OMPdecase was identified. Despite a generally good correspondence to other OMPdecase sequences, theP. aeruginosa gene was unique in that it did not constitute part of an operon. ThepyrF gene was amplified by polymerase chain reaction, overexpressed in the pT7-7/E. coli BL21(DE3) system and purified to near electrophoretic homogeneity by anion exchange chromatography. Characterization of the purified enzyme revealed the following data, aK _m value for OMP of 9.91 M and an isoelectric point of 6.65. No major decrease in enzyme activity was observed in a pH range between 7.8 and 10.2. Gel electrophoresis under nondenaturing conditions suggested that the native form of OMPdecase is the dimer.     

Novel cyclohexyl-amides as potent antibacterials targeting bacterial type IIA topoisomerases

As part of our wider efforts to exploit novel mode of action antibacterials, we have discovered a series of cyclohexyl-amide compounds that has good Gram positive and Gram negative potency. The mechanism of action is via inhibition of bacterial topoisomerases II and IV. We have investigated various subunits in this series and report advanced studies on compound 7 which demonstrates good PK and in vivo efficacy properties.     

Compensatory Hemagglutinin Mutations Alter Antigenic Properties of Influenza Viruses

Influenza viruses routinely acquire mutations in antigenic sites on the globular head of the hemagglutinin (HA) protein. Since these antigenic sites are near the receptor binding pocket of HA, many antigenic mutations simultaneously alter the receptor binding properties of HA. We previously reported that a K165E mutation in the Sa antigenic site of A/Puerto Rico/8/34 (PR8) HA is associated with secondary neuraminidase (NA) mutations that decrease NA activity. Here, using reverse genetics, we show that the K165E HA mutation dramatically decreases HA binding to sialic acid receptors on cell surfaces. We sequentially passaged reverse-genetics-derived PR8 viruses with the K165E antigenic HA mutation in fertilized chicken eggs, and to our surprise, viruses with secondary NA mutations did not emerge. Instead, viruses with secondary HA mutations emerged in 3 independent passaging experiments, and each of these mutations increased HA binding to sialic acid receptors. Importantly, these compensatory HA mutations were located in the Ca antigenic site and prevented binding of Ca-specific monoclonal antibodies. Taken together, these data indicate that HA antigenic mutations that alter receptor binding avidity can be compensated for by secondary HA or NA mutations. Antigenic diversification of influenza viruses can therefore occur irrespective of direct antibody pressure, since compensatory HA mutations can be located in distinct antibody binding sites.     

High Sequence Variability of the ppE18 Gene of Clinical Mycobacterium tuberculosis Complex Strains Potentially Impacts Effectivity of Vaccine Candidate M72/AS01E

The development of an effective vaccine is urgently needed to fight tuberculosis (TB) which is still the leading cause of death from a single infectious agent worldwide. One of the promising vaccine candidates M72/AS01E consists of two proteins subunits PepA and PPE18 coded by Rv0125 and Rv1196. However, preliminary data indicate a high level of sequence variability among clinical Mycobacterium tuberculosis complex (MTBC) strains that might have an impact on the vaccine efficacy. To further investigate this finding, we determined ppE18 sequence variability in a well-characterized reference collection of 71 MTBC strains from 23 phylogenetic lineages representing the global MTBC diversity. In total, 100 sequence variations consisting of 96 single nucleotide polymorphisms (SNPs), three insertions and one deletion were detected resulting in 141 variable positions distributed over the entire gene. The majority of SNPs detected were non-synonymous (n = 68 vs. n = 28 synonymous). Strains from animal adapted lineages, e.g., M. bovis, showed a significant higher diversity than the human pathogens such as M. tuberculosis Haarlem. SNP patterns specific for different lineages as well as for deeper branches in the phylogeny could be identified. The results of our study demonstrate a high variability of the ppE18 gene even in the N-terminal domains that is normally highly conserved in ppe genes. As the N-terminal region interacts with TLR2 receptor inducing a protective anti-inflammatory immune response, genetic heterogeneity has a potential impact on the vaccine efficiency, however, this has to be investigated in future studies.     

Conservation of plasmids among plant-pathogenic Pseudomonas syringae isolates of diverse origins

Thirty isolates of Pseudomonas syringae pv. tabaci, pv. angulata (pathogens on tobacco), pv. coronafaciens, and pv. striafaciens (pathogens on oats) were examined for plasmid DNAs. The strains were obtained from plants throughout the world, some over 50 years ago. Of the 22 tobacco pathogens, 16 contain predominantly one type of plasmid, the pJP27.00 type. The remaining six tobacco-specific strains do not harbor detectable plasmids. The oat pathogens contain one, two, or three plasmids. DNA homology studies indicate that the plasmid DNAs are highly conserved. More importantly, the plasmids harbored by strains isolated from one host plant are conserved most stringently; e.g., the plasmids from the tobacco pathogens are, with one exception, indistinguishable by restriction endonuclease digestion and Southern hybridization. There is also extensive homology among plasmids indigenous to the oat-specific P. syringae pv. coronafaciens and pv. striafaciens strains.