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Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors., Graphical Abstract, Vink and colleagues describe a novel method to generate stable producer cell lines for lentiviral vector manufacturing using a bacterial artificial chromosome that encodes all vector components.

Through an internal virtual screen at GlaxoSmithKline a distinct class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors were discovered. Careful evaluation of crystal structures and SAR led to a novel, potent, and orally active imidazopyridine inhibitor of H-PGDS, 20b. Herein, describes the identification of 2 classes of inhibitors, their syntheses, and their challenges.

A high-throughput RapidFire mass spectrometry assay is described for the JMJD2 family of Fe(2+), O(2), and α-ketoglutarate-dependent histone lysine demethylases. The assay employs a short amino acid peptide substrate, corresponding to the first 15 amino acid residues of histone H3, but mutated at two positions to increase assay sensitivity. The assay monitors the direct formation of the dimethylated-Lys9 product from the trimethylated-Lys9 peptide substrate. Monitoring the formation of the monomethylated and des-methylated peptide products is also possible. The assay was validated using known inhibitors of the histone lysine demethylases, including 2,4-pyridinedicarboxylic acid and an α-ketoglutarate analogue. With a sampling rate of 7 s per well, the RapidFire technology permitted the single-concentration screening of 101 226 compounds against JMJD2C in 10 days using two instruments, typically giving Z' values of 0.75 to 0.85. Several compounds were identified of the 8-hydroxyquinoline chemotype, a known series of inhibitors of the Lys9-specific histone demethylases. The peptide also functions as a substrate for JMJD2A, JMJD2D, and JMJD2E, thus enabling the development of assays for all 3 enzymes to monitor progress in compound selectivity. The assay represents the first report of a RapidFire mass spectrometry assay for an epigenetics target.

New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a ‘pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested., Type IIA topoisomerases (topo2As) create transient double-strand DNA breaks. Here, the authors report structures showing how QPT-1 binds in the DNA/topo2A complex at the same site as the fluoroquinolone moxifloxacin, and discuss the potential for developing new classes of antibiotics.

Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis β-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related β-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis., Screens for bactericidal compounds have resulted in promising anti-tubercular hits. Here, the authors analyse in detail the target of an indazole sulfonamide (GSK3011724A), and find that it has a different mode of inhibition compared to other Kas inhibitors of fatty acid biosynthesis in bacteria.

Fluoroquinolone drugs such as moxifloxacin kill bacteria by stabilizing the normally transient double-stranded DNA breaks created by bacterial type IIA topoisomerases. Previous crystal structures of Staphylococcus aureus DNA gyrase with asymmetric DNAs have had static disorder (with the DNA duplex observed in two orientations related by the pseudo-twofold axis of the complex). Here, 20-base-pair DNA homoduplexes were used to obtain crystals of covalent DNA-cleavage complexes of S. aureus DNA gyrase. Crystals with QPT-1, moxifloxacin or etoposide diffracted to between 2.45 and 3.15 Å resolution. A G/T mismatch introduced at the ends of the DNA duplexes facilitated the crystallization of slightly asymmetric complexes of the inherently flexible DNA-cleavage complexes.

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.

Type IIA topoisomerases resolve topological problems in DNA by making a double-stranded break in one DNA segment, passing another DNA duplex through this break, and then resealing the break. Drugs (such as the widely used fluoroquinolone antibacterials and anti-cancer compounds such as etoposide) that stabilize double-strandedly cleaved DNA complexes with type IIA topoisomerases are cytotoxic. In GlaxoSmithKline a new class of novel bacterial topoisomerase inhibitors (NBTIs) have been developed. A 2.1Å crystal structure of a complex of GSK299423 with DNA and S. aureus DNA gyrase showed how the NBTI inhibits the enzyme by interacting with both the DNA and the protein. A pocket occupied by the compound in the protein (at the dimer interface) is absent in the apo structure, while the pocket occupied by the compound in the DNA has been formed by the enzyme stretching and untwisting the DNA between the two active sites. The NBTI structure has trapped a pre-cleavage complex of the enzyme, before the four base-pair double stranded break has occurred, and the structure gives insights into the role of metal ions in the cleavage mechanism of type IIA topoisomerases. Stuctures suggest how relatively small movements at the active sites (for example an ~3Å movement of a magnesium ion) can cause the cleavage of phosphate ester bonds and are coupled to the large domain movements involved in the catalytic cycle of these conformationally flexible enzymes. The binding site for the NBTI is close to but distinct from those for fluoroquinolones. Structures shows how the fluoroquinolone interacts with both the protein and the DNA by binding a non-catalytic magnesium ion and four associated waters. This provides a structural explanation for both fluoroquinolone resistance mutations and SAR (structure-activity relationships). Mechanistic implications of recent structural studies will be discussed.