Professor Peter Lewis

We report the discovery of the first bacterial ribosomal RNA (rRNA) synthesis inhibitor that has specific antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). A pharmacophore model was constructed on the basis of the protein-protein interaction between essential bacterial rRNA transcription factors NusB and NusE and employed for an in silico screen to identify potential leads. One compound, (E)-2-{[(3-ethynylphenyl)imino]methyl}-4-nitrophenol (MC4), demonstrated antimicrobial activity against a panel of S. aureus strains, including MRSA, without significant toxicity to mammalian cells. MC4 resulted in a decrease in the rRNA level in bacteria, and the target specificity of MC4 was confirmed at the molecular level. Results obtained from this work validated the bacterial rRNA transcription machinery as a novel antimicrobial target. This approach may be extended to other factors in rRNA transcription, and MC4 could be applied as a chemical probe to dissect the relationship among MRSA infection, MRSA growth rate, and rRNA synthesis, in addition to its therapeutic potential.

Bacterial transcription is a proven target for antibacterial research. However, most of the known inhibitors targeting transcription are from natural extracts or are hits from screens where the binding site remains unidentified. Using an RNA polymerase holoenzyme homology structure from the model Gram-positive organism Bacillus subtilis, we created a pharmacophore model and used it for in silico screening of a publicly available library for compounds able to inhibit holoenzyme formation. The hits demonstrated specific affinity to bacterial RNA polymerase and excellent activity using in vitro assays and showed no binding to the equivalent structure from human RNA polymerase II. The target specificity in live cells and antibacterial activity was demonstrated in microscopy and growth inhibition experiments. This is the first example of targeted inhibitor development for a bacterial RNA polymerase, outlining a complete discovery process from virtual screening to biochemical validation. This approach could serve as an appropriate platform for the future identification of inhibitors of bacterial transcription.

Our ongoing research focused on targeting transcription initiation in bacteria has resulted in synthesis of several classes of mono-indole and mono-benzofuran inhibitors that targeted the essential protein-protein interaction between RNA polymerase core and s 70/s A factors in bacteria. In this study, the reaction of indole-2-, indole-3-, indole-7- and benzofuran-2-glyoxyloyl chlorides with amines and hydrazines afforded a variety of glyoxyloylamides and glyoxyloylhydrazides. Similarly, condensation of 2- and 7-trichloroacetylindoles with amines and hydrazines delivered amides and hydrazides. The novel molecules were found to inhibit the RNA polymerase-s 70/s A interaction as measured by ELISA, and also inhibited the growth of both Gram-positive and Gram-negative bacteria in culture. Structure-activity relationship (SAR) studies of the mono-indole and mono-benzofuran inhibitors suggested that the hydrophilic-hydrophobic balance is an important determinant of biological activity.

Abstract Nitric oxide (NO) and its auto-oxidation products are known to disrupt normal bacterial function and NO releasing molecules have the potential to be developed as antibacterial leads in drug discovery. We have designed and synthesized a series of novel nitrated compounds by combining NO releasing groups with ocotillol-type triterpenoids, which have previously demonstrated activity only against Gram-positive bacteria. The in vitro NO release capacity and antibacterial activity were sequentially evaluated and the data showed that most of the synthesized compounds could release nitric oxide. Compound 16a, 17a and 17c, with nitrated aliphatic esters at C-3 position, displayed higher NO release than other analogues, correlating to their good antibacterial activity, in which 17c demonstrated broad-spectrum activity against both Gram positive and -negative bacteria, as well as excellent synergism at sub-minimum inhibitory concentration when using with kanamycin and chloramphenicol. Furthermore, the epifluorescent microscopic study indicated that the ocotillol-type triterpenoid core may induce NO release on the bacterial membrane. Our results demonstrate that nitrated substitutions at C-3 of ocotillol-type derivatives could provide an approach to expand their antibacterial spectrum, and that ocotillol-type triterpenoids may also be developed as appropriate carriers for NO donors in antibacterial agent discovery with low cytotoxicity.

Very few clinically available antibiotics target bacterial RNA polymerase (RNAP) suggesting it is an underutilized target. The advent of detailed structural information of RNAP holoenzyme (HE) has allowed the design and in silico screening of novel transcription inhibitors. Here, we describe our approach for the design and testing of small molecule transcription inhibitors that work by preventing the interaction between the essential transcription initiation factor s and RNAP. With the appropriate structural information this approach can be easily modified to other essential protein-protein interactions.

Triterpenoid saponins are involved in plant defense systems to inhibit bacterial invasion. A new series of hydrophilic ocotillol-type triterpenoid derivatives 5-26 have been synthesized with antibacterial activity against Gram-positive bacteria, including a community associated methicillin-resistant Staphylococcus aureus (CA-MRSA; strain USA300). From this series, compounds 6 and 15 were found to be the most active, both with MIC values of 2 µg/mL against B. subtilis 168 and 8 µg/mL against S. aureus USA300, respectively. Furthermore, subsequent assays showed that compounds 6 and 15 displayed strong synergistic effects at sub-MIC levels against both S. aureus USA300 and B. subtilis 168 when combined with two commercial antibiotics, kanamycin and chloramphenicol. Preliminary structure-activity relationship studies were also performed.

The increasing resistance of bacteria against clinically approved antibiotics is resulting in an alarming decrease in therapeutic options for today's clinicians. We have targeted the essential interaction between bacterial RNA polymerase and s70/sA for the development of lead molecules exhibiting a novel mechanism of antibacterial activity. Several classes of structurally related bis-indole inhibitors of bacterial transcription initiation complex formation were synthesized and their antimicrobial activities were evaluated. Condensation of indole-7- and indole-2-carbohydrazides with 7- and 2-trichloroacetylindoles or indole-7- and indole-2-glyoxyloyl chlorides resulted in the successful synthesis of 7,7'-, 2,2'-, 2,7'- and 3,2'-linked bis-indole derivatives with -CO-NH-NH-CO- and -CO-CO-NH-NH-CO- linkers. Indole-7-glyoxyloyl chlorides were reacted with hydrazine hydrate in different ratios to afford respective -CO-CO-NH-NH-CO-CO- bis-indole or hydrazide derivatives. The resulting compounds were found to be active against the ß'-CH- s70/sA2.2 interaction in ELISA assays and inhibited the growth of both Gram-positive and Gram-negative bacteria. Structure-activity relationship (SAR) studies were performed in order to identify the structural features of the synthesized inhibitors required for biological activity. This journal is © the Partner Organisations 2014.

Acinetobacter species are widely distributed bacteria in the environment, and have recently gained notoriety as opportunistic nosocomial pathogens. Here we characterize a novel RNA polymerase-interacting protein named acidic transcription factor A, AtfA. It is small and highly acidic, and is widely distributed throughout the ¿ proteobacteria, including other significant pathogens in the genera Moraxella, Pseudomonas, Legionella and Vibrio. In the model species A. baylyiADP1, deletion of atfA significantly affects expression of over 500 genes, resulting in a large cell phenotype, reduced cell fitness, impaired biofilm formation and twitching motility, and increased sensitivity to antibiotics. Deletion of atfA also causes dramatically enhanced sensitivity to ethanol, which is an important growth promoter and virulence factor in Acinetobacter spp. The results suggest that auxiliary factors of RNA polymerase with important biological roles remain to be discovered.

Diffuse intrinsic pontine glioma (DIPG) is a fatal brain cancer that arises in the brainstem of children, with no effective treatment and near 100% fatality. The failure of most therapies can be attributed to the delicate location of these tumors and to the selection of therapies on the basis of assumptions that DIPGs are molecularly similar to adult disease. Recent studies have unraveled the unique genetic makeup of this brain cancer, with nearly 80% found to harbor a p.Lys27Met histone H3.3 or p.Lys27Met histone H3.1 alteration. However, DIPGs are still thought of as one disease, with limited understanding of the genetic drivers of these tumors. To understand what drives DIPGs, we integrated whole-genome sequencing with methylation, expression and copy number profiling, discovering that DIPGs comprise three molecularly distinct subgroups (H3-K27M, silent and MYCN) and uncovering a new recurrent activating mutation affecting the activin receptor gene ACVR1 in 20% of DIPGs. Mutations in ACVR1 were constitutively activating, leading to SMAD phosphorylation and increased expression of the downstream activin signaling targets ID1 and ID2. Our results highlight distinct molecular subgroups and novel therapeutic targets for this incurable pediatric cancer. © 2014 Nature America, Inc. All rights reserved.

A range of novel hydrazine bridged bis-indoles was prepared from readily available indole-7-glyoxyloylchlorides and 7-trichloroacetylindoles and underwent cyclodehydration to produce 2,5-di(7-indolyl)-1,3,4-oxadiazoles and a 2,2'-bi-1,3,4-oxadiazolyl with phosphoryl chloride in ethyl acetate. This efficient protocol was subsequently used for the synthesis of 2- and 7-indolyl 2-(1,3,4-thiadiazolyl)ketones from related indolyl-hydrazine carbothioamides. The synthesised bis-indoles were evaluated for their antimicrobial properties, particularly the inhibition of protein-protein complex formation between RNA polymerase and s factor and their bactericidal effect on Gram positive Bacillus subtilis and Gram negative Escherichia coli. © 2014 Elsevier Ltd. All rights reserved.

We have examined the localization of DNA replication of the Bacillus subtilis phage f29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.

Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

We report the development of a series of plasmid vectors for the construction of fusions to mutants of the intrinsically fluorescent green fluorescent protein, GFPmut1 (Cormack et al., 1996. Gene 173, 33-38) and GFPuv (Crameri et al., 1996. Nature Biotechnology 14, 315-319). Both N- and C-terminal fusions can be produced, and their expression can be finely controlled from the inducible Pxyl promoter following double crossover integration into the amyE locus of the Bacillus subtilis chromosome. Other vectors designed for single crossover insertion into the chromosome allow downstream genes to be placed under inducible control. We also show that fusions to GFPmut1 and GFPuv can be co-localized within the cell by virtue of their different excitation spectra.

Immunofluorescence microscopy was used to study the establishment of compartment-specific transcription during sporulation in Bacillus subtilis. Analysis of the distribution of the anti-anti-sigma factor, SpoIIAA, in a variety of mutant backgrounds supports a model in which the SpoIIE phosphatase, which activates SpoIIAA by dephosphorylation, is sequestered onto the prespore face of the asymmetric septum. Thus, prespore-specific gene expression apparently arises as a result of the compartmentalization of SpoIIE protein. The results also suggest the existence of at least two compartment-specific programs of proteolysis, one dependent on the mother cell-specific sigma factor s(E) and the other dependent on the prespore- specific sigma factor s(F).