An ever-increasing demand for novel antimicrobials to treat life-threatening infections caused by the global spread of multidrug-resistant bacterial pathogens stands in stark contrast to the current level of investment in their development, particularly in the fields of natural-product-derived and synthetic small molecules. New agents displaying innovative chemistry and modes of action are desperately needed worldwide to tackle the public health menace posed by antimicrobial resistance. Here, our consortium presents a strategic blueprint to substantially improve our ability to discover and develop new antibiotics. We propose both short-term and long-term solutions to overcome the most urgent limitations in the various sectors of research and funding, aiming to bridge the gap between academic, industrial and political stakeholders, and to unite interdisciplinary expertise in order to efficiently fuel the translational pipeline for the benefit of future generations.
Flaviviral NS3 is a multifunctional protein displaying N-terminal protease activity in addition to C-terminal helicase, nucleoside 5'-transferase (NTPase), and 5'-terminal RNA triphosphatase (RTPase) activities. NS3 is held to support the separation of RNA daughter and template strands during viral replication.We solved the three-dimensional structure (at 3.1 Å resolution) of the NS3 helicase domain (residues 186-619; NS3:186-619) from Kunjin virus, an Australian variant of the West Nile virus (1). We showed that NS3:186-619 displays both ATPase and RTPase activity, that it can unwind a double-stranded RNA substrate, being however inactive on a double-stranded DNA substrate. Analysis of different constructs shows that full length NS3 displays increased helicase activity, suggesting that the protease domain plays an assisting role in the RNA unwinding process. The structural interaction between the helicase and protease domain has been assessed using small angle X-ray scattering on full length NS3, disclosing that the protease and helicase domains build a rather elongated molecular assembly differing from that observed in the NS3 protein from hepatitis C virus. S.A.X.S. data were collected on the X33 camera of the EMBL on the storage ring DORIS III (DESY, Hamburg, Germany). The data were recorded using a MAR345 two-dimensional image plate detector at a sample-detector distance of 2.7 m and a wavelength of λ = 1.5 Å, covering the range of momentum transfer 0.012 < s < 0.45 Å -1 (s = 4π sinθ/λ, where 2θ is the scattering angle). The solutions containing NS3FL at 3.0, 1.5 and 0.75 mg/ml in Hepes 10 mM, NaCl 300 mM and protease inhibitors cocktail (Roche), with the addition of 2 mM DTT to reduce radiation damage, were placed in a 70 µl cell with polystyrene windows and exposed to X-ray beam. To check for radiation damage, the data were collected in two successive 2-minute exposures, and no changes were observed in the scattering patterns with time, i.e., there was no measurable radiation damage. All measurements were performed at 15°C. The data were averaged after normalization to the intensity of the incident beam and the scattering of the buffer was subtracted. The difference curves were scaled for the solute concentration and extrapolated to infinite dilution. All data manipulations were performed using the program package PRIMUS (2).The forward scattering I(0) and the radius of gyration (R g ) were evaluated using the Guinier approximation (3); the maximum diameter D max of the particle were computed from the entire scattering patterns using program GNOM (4), and the excluded volume V p of the particle was computed from the Porod invariant (5). The molecular masses of the solutes were evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (66 kDa). The ab initio shape reconstruction was performed using the program GASBOR (6), using the total number of the residues in NS3FL (619).The estimated molecular mass of the NS3FL sample, 65±5 kDa, is compatible with mon...
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, 7MeG5′-ppp5′-A2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and 7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppACn and 7MeGpppACn (1 ≤ n ≤ 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 ≤ n ≤ 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
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