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Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both in vitro and ex vivo. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in Mycobacterium tuberculosis whole-cell assays

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.

Small-molecule inhibitors of the mycobacterial transcriptional repressor EthR have previously been shown to act as boosters of the second-line antituberculosis drug ethionamide. Fragment-based drug discovery approaches have been used in the past to make highly potent EthR inhibitors with ethionamide boosting activity both $\textit{in vitro}$ and $\textit{ex vivo}$. Herein, we report the development of fragment-sized EthR ligands with nanomolar minimum effective concentration values for boosting the ethionamide activity in $\textit{Mycobacterium tuberculosis}$ whole-cell assays.

The important plant hormone salicylic acid (SA; 2-hydroxybenzoic acid) regulates several key plant responses including, most notably, defence against pathogens. A key enzyme for SA biosynthesis is isochorismate synthase (ICS), which converts chorismate into isochorismate, and for which there are two genes in $\textit{Arabidopsis thaliana}$ One ($\textit{AtICS1}$) has been shown to be required for increased SA biosynthesis in response to pathogens and its expression can be stimulated throughout the leaf by virus infection and exogenous SA. The other ($\textit{AtICS2}$) appears to be expressed constitutively, predominantly in the plant vasculature. Here, we characterise the enzymatic activity of both isozymes expressed as hexahistidine fusion proteins in $\textit{Escherichia coli}$. We show for the first time that recombinant AtICS2 is enzymatically active. Both isozymes are Mg$^{2+}$-dependent with similar temperature optima (ca. 33°C) and similar K$_{m}$ values for chorismate of 34.3 ± 3.7 and 28.8 ± 6.9 µM for ICS1 and ICS2, respectively, but reaction rates were greater for ICS1 than for ICS2, with respective values for V$_{max}$ of 63.5 ± 2.4 and 28.3 ± 2.0 nM s$^{-1}$ and for k$_{cat}$ of 38.1 ± 1.5 and 17.0 ± 1.2 min$^{-1}$ However, neither enzyme displayed isochorismate pyruvate lyase (IPL) activity, which would enable these proteins to act as bifunctional SA synthases, i.e. to convert chorismate into SA. These results show that although $\textit{Arabidopsis}$ has two functional ICS enzymes, it must possess one or more IPL enzymes to complete biosynthesis of SA starting from chorismate.

Similarity in the ligand binding profile of two enzymes may provide insight for functional characterization and be of greater relevance to inhibitor development than sequence similarity or structural homology. Fragment screening is an efficient approach to characterizing the ligand profile of an enzyme and has been applied here to study the family of cytochrome P450 enzymes (P450s) expressed by Mycobacterium tuberculosis (Mtb). The Mtb P450s have important roles in bacterial virulence, survival and pathogenicity. Comparing the fragment profiles of seven of these enzymes revealed that P450s which share a similar biological function have significantly similar fragment profiles, while functionally unrelated or orphan P450s exhibit distinct ligand binding properties, despite overall high structural homology. Chemical structures that exhibit promiscuous binding between enzymes have been identified, as have selective fragments that could provide leads for inhibitor development. The similarity in the fragment binding profile of the orphan enzyme CYP144A1 to CYP121A1, an enzyme important for Mtb viability, provides a case study illustrating the subsequent identification of novel CYP144A1 ligands. The different binding modes of these compounds to CYP144A1 provide insight into structural and dynamic aspects of the enzyme, suggest a hypothesis into biological function and provide opportunity for inhibitor development. Expanding this fragment profiling approach to include a greater number of functionally characterized and orphan proteins may provide a valuable resource for understanding enzyme-ligand interactions.

There is an emerging trend towards the fabrication of microcapsules at liquid interfaces. In order to control the parameters of such capsules, the interfacial processes governing their formation must be understood. Here, poly(vinyl alcohol) films are assembled at the interface of water-in-oil microfluidic droplets. The polymer is cross-linked using cucurbit[8]uril ternary supramolecular complexes. It is shown that compression-induced phase change causes the onset of buckling in the interfacial film. On evaporative compression, the interfacial film both increases in density and thickens, until it reaches a critical density and a phase change occurs. We show that this increase in density can be simply related to the film Poisson ratio and area compression.This description captures fundamentals of many compressive interfacial phase changes and can also explain the observation of a fixed thickness-to-radius ratio at buckling, $\left(\frac TR\right)$$_{buck}$.

The macrocyclic host, cucurbit[8]uril, is used to facilitate cross-linking of colloidal particles and polymers in microdroplets resulting in thermo- and photo-responsive supramolecular colloidal microcapsules. Methyl viologen-bearing colloidal particles were prepared using template polymerisation and combined with cucurbit[8]uril and an azobenzene-functionalised polymer within microfluidic droplets. The colloidal particles self-assembled at the droplet interface, whereupon polymeric cross-links formed $\textit{via}$ ternary host-guest complexation with cucurbit[8]uril. The resultant supramolecular colloidal microcapsules were uniform in size and were able to retain a macromolecular cargo. It is shown that the capsule skin porosity, and consequently the rate of release of encapsulated cargo, can be remotely controlled $\textit{via}$ either temperature or light triggers. This simple and versatile method could be extended to other polymer or colloidal derivatives for the fabrication of nano- and microcapsules with dual stimuli response for controlled release.

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site.

RAD51 is a recombinase involved in the homologous recombination of double-strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an ‘FxxA’ motif, and the same recognition sequence is similarly utilised to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X-ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered.

Here we report a new class of hydrogels formed by polymers that are cross-linked through subcomponent self-assembled metal–organic cages. Selective encapsulation of guest molecules within the cages creates two distinct internal phases within the hydrogel, which allows for contrasting release profiles of related molecules depending on their aptitude for encapsulation within the cages. The hydrogels were fabricated into microparticles via a droplet-based microfluidic approach and proved responsive to a variety of stimuli, including acid and competing amine or aldehyde subcomponents, allowing for the triggered release of cargo.

Protein-protein interactions are difficult therapeutic targets, and inhibiting pathologically relevant interactions without disrupting other essential ones presents an additional challenge. Herein we report how this might be achieved for the potential anticancer target, the TPX2-importin-α interaction. Importin-α is a nuclear transport protein that regulates the spindle assembly protein TPX2. It has two binding sites--major and minor-to which partners bind. Most nuclear transport cargoes use the major site, whereas TPX2 binds principally to the minor site. Fragment-based approaches were used to identify small molecules that bind importin-α, and crystallographic studies identified a lead series that was observed to bind specifically to the minor site, representing the first ligands specific for this site. Structure-guided synthesis informed the elaboration of these fragments to explore the source of ligand selectivity between the minor and major sites. These ligands are starting points for the development of inhibitors of this protein-protein interaction.

We have isolated a Lotus japonicus cDNA for pantothenate (vitamin B(5)) synthetase (PS) by functional complementation of an Escherichia coli panC mutant (AT1371). A rice (Oryza sativum) expressed sequence tag, identified by sequence similarity to PS, was also able to complement the E. coli auxotroph, as was an open reading frame from Saccharomyces cerevisiae (baker's yeast). The Lotus and rice cDNAs encode proteins of approx. 34 kDa, which are 65% similar at the amino acid level and do not appear to encode N-terminal extensions by comparison with PS sequences from other organisms. Furthermore, analysis of genomic sequence flanking the coding sequence for PS in Lotus suggests the original cDNA is full-length. The Lotus and rice PSs are therefore likely to be cytosolic. Southern analysis of Lotus genomic DNA indicates that there is a single gene for PS. Recombinant PS from Lotus, overexpressed in E. coli AT1371, is a dimer. The enzyme requires d-pantoate, beta-alanine and ATP for activity and has a higher affinity for pantoate (K(m) 45 microM) than for beta-alanine (K(m) 990 microM). Uncompetitive substrate inhibition becomes significant at pantoate concentrations above 1 mM. The enzyme displays optimal activity at about 0.5 mM pantoate (k(cat) 0.63 s(-1)) and at pH 7.8. Neither oxopantoate nor pantoyl-lactone can replace pantoate as substrate. Antibodies raised against recombinant PS detected a band of 34 kDa in Western blots of Lotus proteins from both roots and leaves. The implications of these findings for pantothenate biosynthesis in plants are discussed.

Complex hierarchical architectures are ubiquitous in nature. By designing and controlling the interaction between elementary building blocks, nature is able to optimize a large variety of materials with multiple functionalities. Such control is, however, extremely challenging in man-made materials, due to the difficulties in controlling their interaction at different length scales simultaneously. Here, hierarchical cholesteric architectures are obtained by the self-assembly of cellulose nanocrystals within shrinking, micron-sized aqueous droplets. This confined, spherical geometry drastically affects the colloidal self-assembly process, resulting in concentric ordering within the droplet, as confirmed by simulation. This provides a quantitative tool to study the interactions of cellulose nanocrystals beyond what has been achieved in a planar geometry. Our developed methodology allows us to fabricate truly hierarchical solid-state architectures from the nanometer to the macroscopic scale using a renewable and sustainable biopolymer.

EthR is a transcriptional repressor that increases Mycobacterium tuberculosis resistance to ethionamide. In this study, the EthR-DNA interaction has been investigated by native electrospray-ionization mass spectrometry for the first time. The results show that up to six subunits of EthR are able to bind to its operator., D.S.-H. Chan acknowledges the support of the Croucher Foundation and the Cambridge Commonwealth, European and International Trust for receipt of a Croucher Cambridge International Scholarship. W.-G. Seetoh was supported by the Agency for Science, Technology and Research (A*STAR) Singapore (PhD sponsorship) and the Wellcome Trust Strategic Award (090340/Z/09/Z). B.N. McConnell acknowledges Cambridge Australia Scholarships for the award of a Poynton Scholarship, the Cambridge Philosophical Society and the Access to Learning Fund. S.E. Thomas is supported by the Cystic Fibrosis Trust. V. Mendes and M. Blaszczyk acknowledge the Bill & Melinda Gates Foundation (subcontract by the Foundation for the National Institutes of Health - NIH) (OPP1024021).

We have studied the effect of dialysates from lung cancer homogenates to alter both the expression of complement (C3b) receptors per se and also to inhibit leucoattractant-induced enhancement of complement rosettes on monocytes from healthy individuals. Enhancement and enhancement-inhibition by tumour extracts were compared with material derived from normal lung excised from distance from the tumour. There was no significant difference between tumour homogenate (TH) and normal lung homogenate (NLH) in terms of enhancement of complement rosettes per se. In contrast, TH produced a dose- and time-dependent inhibition of leucoattractant-induced enhancement of C3b rosettes which was significantly different from that obtained with NLH. This enhancement-inhibition was observed with four undifferentiated, four squamous and three adenocarcinomas of lung. The degree of enhancement-inhibition was not related to the type of tumour or varying accompanying histological features such as necrosis and the degree of infiltration with inflammatory cells. Following gel filtration on Sephadex G-50 each type of cancer gave a major peak of inhibitory activity which eluted with molecules having an apparent molecular size of approximately 3,000 daltons. A second larger peak (8,000-10,000 daltons) was also detected with extracts from the undifferentiated and adenocarcinomas. These results support previous findings, mainly from experimental animals, indicating that 'anti-macrophage/monocyte principles' are elaborated from certain tumour types.

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands "moonlight" as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity.

The alarming increase of pathogenic bacteria that are resistant to multiple antibiotics is now recognized as a major health issue fuelling demand for new drugs. Bacterial resistance is often caused by molecular changes at the bacterial surface, which alter the nature of specific drug-target interactions. Here, we identify a novel mechanism by which drug-target interactions in resistant bacteria can be enhanced. We examined the surface forces generated by four antibiotics; vancomycin, ristomycin, chloroeremomycin and oritavancin against drug-susceptible and drug-resistant targets on a cantilever and demonstrated significant differences in mechanical response when drug-resistant targets are challenged with different antibiotics although no significant differences were observed when using susceptible targets. Remarkably, the binding affinity for oritavancin against drug-resistant targets (70 nM) was found to be 11,000 times stronger than for vancomycin (800 μM), a powerful antibiotic used as the last resort treatment for streptococcal and staphylococcal bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Using an exactly solvable model, which takes into account the solvent and membrane effects, we demonstrate that drug-target interactions are strengthened by pronounced polyvalent interactions catalyzed by the surface itself. These findings further enhance our understanding of antibiotic mode of action and will enable development of more effective therapies.