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Inhibiting glycolysis remains an aspirational approach for the treatment of cancer. We have previously identified a subset of cancers harbouring homozygous deletion of the glycolytic enzyme enolase (ENO1) that have exceptional sensitivity to inhibition of its redundant paralogue, ENO2, through a therapeutic strategy known as collateral lethality. Here, we show that a small-molecule enolase inhibitor, POMHEX, can selectively kill ENO1-deleted glioma cells at low-nanomolar concentrations and eradicate intracranial orthotopic ENO1-deleted tumours in mice at doses well-tolerated in non-human primates. Our data provide an in vivo proof of principle of the power of collateral lethality in precision oncology and demonstrate the utility of POMHEX for glycolysis inhibition with potential use across a range of therapeutic settings.

Transactivation response element (TAR) DNA-binding protein 43 (TDP-43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N- and C-terminus of TDP-43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single-molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP-43 NTD is thermodynamically stable, well-folded and undergoes reversible oligomerization. We propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

LiaR is a 'master regulator' of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structural basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR(D191N) increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. Crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Preventing histone recognition by bromodomains emerges as an attractive therapeutic approach in cancer. Overexpression of ATAD2 (ATPase family AAA domain-containing 2 isoform A) in cancer cells is associated with poor prognosis making the bromodomain of ATAD2 a promising epigenetic therapeutic target. In the development of an in vitro assay and identification of small molecule ligands, we conducted structure-guided studies which revealed a conformationally flexible ATAD2 bromodomain. Structural studies on apo-, peptide-and small molecule-ATAD2 complexes (by co-crystallization) revealed that the bromodomain adopts a 'closed', histone-compatible conformation and a more 'open' ligand-compatible conformation of the binding site respectively. An unexpected conformational change of the conserved asparagine residue plays an important role in driving the peptide-binding conformation remodelling. We also identified dimethylisoxazole-containing ligands as ATAD2 binders which aided in the validation of the in vitro screen and in the analysis of these conformational studies.

Staphylococcus aureus VraR, a vancomycin-resistance-associated response regulator, activates a cell-wall-stress stimulon in response to antibiotics that inhibit cell wall formation. X-ray crystal structures of VraR in both unphosphorylated and beryllofluoride-activated states have been determined, revealing a mechanism of phosphorylation-induced dimerization that features a deep hydrophobic pocket at the center of the receiver domain interface. Unphosphorylated VraR exists in a closed conformation that inhibits dimer formation. Phosphorylation at the active site promotes conformational changes that are propagated throughout the receiver domain, promoting the opening of a hydrophobic pocket that is essential for homodimer formation and enhanced DNA-binding activity. This prominent feature in the VraR dimer can potentially be exploited for the development of novel therapeutics to counteract antibiotic resistance in this important pathogen.

The AgrA transcription factor regulates the quorum-sensing response in Staphylococcus aureus, controlling the production of hemolysins and other virulence factors. AgrA binds to DNA via its C-terminal LytTR domain, a domain not found in humans but common in many pathogenic bacteria, making it a potential target for antimicrobial development. We have determined the crystal structure of the apo AgrA LytTR domain and screened a library of 500 fragment compounds to find inhibitors of AgrA DNA binding activity. Using nuclear magnetic resonance, the binding site for five compounds has been mapped to a common locus at the C-terminal end of the LytTR domain, a site known to be important for DNA binding activity. Three of these compounds inhibit AgrA DNA binding. These results provide the first evidence that LytTR domains can be targeted by small organic compounds.

The histone-like nucleoid structuring (H-NS) protein plays a fundamental role in DNA condensation and is a key regulator of enterobacterial gene expression in response to changes in osmolarity, pH, and temperature. The protein is capable of high-order self-association via interactions of its oligomerization domain. Using crystallography, we have solved the structure of this complete domain in an oligomerized state. The observed superhelical structure establishes a mechanism for the self-association of H-NS via both an N-terminal antiparallel coiled-coil and a second, hitherto unidentified, helix-turn-helix dimerization interface at the C-terminal end of the oligomerization domain. The helical scaffold suggests the formation of a H-NS:plectonemic DNA nucleoprotein complex that is capable of explaining published biophysical and functional data, and establishes a unifying structural basis for coordinating the DNA packaging and transcription repression functions of H-NS.

The nucleoid-associated protein H-NS and its paralogue StpA are global regulators of gene expression and form an integral part of the protein scaffold responsible for DNA condensation in Escherichia coli and Salmonella typhimurium. Although protein oligomerization is a requirement for this function, it is not entirely understood how this is accomplished. We address this by reporting on the self-association of H-NS and its hetero-association with StpA. We identify residues 1-77 of H-NS as being necessary and sufficient for high-order association. A multi-technique-based approach was used to measure the effects of salt concentration on the size distribution of H-NS and the thermal stability of H-NS and StpA dimers. The thermal stability of the StpA homodimer is significantly greater than that of H-NS(1-74). Investigation of the hetero-association of H-NS and StpA proteins suggested that the association of H-NS with StpA is more stable than the self-association of either H-NS or StpA with themselves. This provides a clear understanding of the method of oligomerization of these important proteins in effecting DNA condensation and reveals that the different associative properties of H-NS and StpA allow them to perform distinct, yet complementary roles in the bacterial nucleoid.

The nucleoid-associated protein, StpA, of Escherichia coli binds non-specifically to double-stranded DNA (dsDNA) and apparently forms bridges between adjacent segments of the DNA. Such a coating of protein on the DNA would be expected to hinder the action of nucleases. We demonstrate that StpA binding hinders dsDNA cleavage by both the non-specific endonuclease, DNase I, and by the site-specific type I restriction endonuclease, EcoKI. It requires approximately one StpA molecule per 250-300 bp of supercoiled DNA and approximately one StpA molecule per 60-100 bp on linear DNA for strong inhibition of the nucleases. These results support the role of StpA as a nucleoid-structuring protein which binds DNA segments together. The inhibition of EcoKI, which cleaves DNA at a site remote from its initial target sequence after extensive DNA translocation driven by ATP hydrolysis, suggests that these enzymes would be unable to function on chromosomal DNA even during times of DNA damage when potentially lethal, unmodified target sites occur on the chromosome. This supports a role for nucleoid-associated proteins in restriction alleviation during times of cell stress.