Cystic fibrosis (CF) is progressive genetic disease that predisposes lungs and other organs to multiple long-lasting microbial infections. Pseudomonas aeruginosa is the most prevalent and deadly pathogen among these microbes. Lung function of CF patients worsens following chronic infections with P. aeruginosa and is associated with increased mortality and morbidity. Emergence of multidrug-resistant, extensively drug-resistant and pandrug-resistant strains of P. aeruginosa due to intrinsic and adaptive antibiotic resistance mechanisms has failed the current anti-pseudomonal antibiotics. Hence new antibacterials are urgently needed to treat P. aeruginosa infections. Structure-guided fragment-based drug discovery (FBDD) is a powerful approach in the field of drug development that has succeeded in delivering six FDA approved drugs over the past 20 years targeting a variety of biological molecules. However, FBDD has not been widely used in the development of anti-pseudomonal molecules. In this review, we first give a brief overview of our structure-guided FBDD pipeline and then give a detailed account of FBDD campaigns to combat P. aeruginosa infections by developing small molecules having either bactericidal or anti-virulence properties. We conclude with a brief overview of the FBDD efforts in our lab at the University of Cambridge towards targeting P. aeruginosa infections.

Mutations in hallmark genes are believed to be the main drivers of cancer progression. These mutations are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC). Structural appreciation of where these mutations appear, in protein-protein interfaces, active sites or deoxyribonucleic acid (DNA) interfaces, and predicting the impacts of these mutations using a variety of computational tools are crucial for successful drug discovery and development. Currently, there are 723 genes presented in the COSMIC Cancer Gene Census. Due to the complexity of the gene products, structures of only 87 genes have been solved experimentally with structural coverage between 90% and 100%. Here, we present a comprehensive, user-friendly, web interface (https://cancer-3d.com/) of 714 modelled cancer-related genes, including homo-oligomers, hetero-oligomers, transmembrane proteins and complexes with DNA, ribonucleic acid, ligands and co-factors. Using SDM and mCSM software, we have predicted the impacts of reported mutations on protein stability, protein-protein interfaces affinity and protein-nucleic acid complexes affinity. Furthermore, we also predicted intrinsically disordered regions using DISOPRED3.

Mycobacterium smegmatisMutT1 (MsMutT1) is a sanitation enzyme made up of an N-terminal Nudix hydrolase domain and a C-terminal domain resembling a histidine phosphatase. It has been established that the action of MutT1 on 8-oxo-dGTP, 8-oxo-GTP and diadenosine polyphosphates is modulated by intermolecular interactions. In order to further explore this and to elucidate the structural basis of its differential action on 8-oxo-NTPs and unsubstituted NTPs, the crystal structures of complexes ofMsMutT1 with 8-oxo-dGTP, GMPPNP and GMPPCP have been determined. Replacement soaking was used in order to ensure that the complexes were isomorphous to one another. Analysis of the structural data led to the elucidation of a relationship between the arrangements of molecules observed in the crystals, molecular plasticity and the action of the enzyme on nucleotides. The dominant mode of arrangement involving a head-to-tail sequence predominantly leads to the generation of NDPs. The other mode of packing arrangement appears to preferentially generate NMPs. This work also provides interesting insights into the dependence of enzyme action on the conformation of the ligand. The possibility of modulating the enzyme action through differences in intermolecular interactions and ligand conformations makesMsMutT1 a versatile enzyme.

Mis-incorporation of modified nucleotides, such as 5-methyl-dCTP or 8-oxo-dGTP, in DNA can be detrimental to genomic integrity. MutT proteins are sanitization enzymes which function by hydrolyzing such nucleotides and regulating the pool of free nucleotides in the cytoplasm. Mycobacterial genomes have a set of four MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4. Mycobacterial MutT2 hydrolyzes 5 m-dCTP and 8-oxo-dGTP to their respective monophosphate products. Additionally, it can hydrolyze canonical nucleotides dCTP and CTP, with a suggested role in sustaining their optimal levels in the nucleotide pool. The structures of M. smegmatis MutT2 and its complexes with cytosine derivatives have been determined at resolutions ranging from 1.10 Å to 1.73 Å. The apo enzyme and its complexes with products (dCMP, CMP and 5 m-dCMP) crystallize in space group P21212, while those involving substrates (dCTP, CTP and 5 m-dCTP) crystallize in space group P21. The molecule takes an α/β/α sandwich fold arrangement, as observed in other MutT homologs. The nucleoside moiety of the ligands is similarly located in all the complexes, while the location of the remaining tail exhibits variability. This is the first report of a MutT2-type protein in complex with ligands. A critical interaction involving Asp116 confers the specificity of the enzyme towards cytosine moieties. A conserved set of enzyme-ligand interactions along with concerted movements of important water molecules provide insights into the mechanism of action.

Incorporation in DNA of oxidized or damaged nucleotides, such as 8-oxo-dGTP or 5-me-dCTP generated during cellular metabolism or as a response to stress in the host cell, is detrimental to genomic integrity.Apart from a plethora of DNA repair proteins that work directly on the damaged DNA, organisms also posses a set of 'sanitization' enzymes.MutT proteins are among such sanitizing enzymes that function by hydrolyzing such nucleotides, thereby preventing their misincorporation in DNA.Mycobacteria posses a set of 4 MutT homologs, namely, MutT1, MutT2, MutT3 and MutT4.Of these, MutT1 has been biochemically and structurally characterized in the laboratory [1,2].Here we report structural studies of MutT2 from Mycobacterium smegmatis and its complexes with substrates 5me-dCTP, dCTP and CTP and the respective products.The native protein and complexes with products crystallize in space group P21212, with one molecule in the asymmetric unit.However, a majority of substrate complexes crystallize in space group P21, with two molecules in the asymmetric unit.At a gross level, MutT1 hydrolyses oxidized guanosine nucleotides efficiently, while MutT2 hydrolyses cytosine nucleotides better.Unlike MutT1, which has an additional domain along with the characteristic Nudix domain, MutT2 is a single Nudix domain enzyme.The structure of MutT2 is very similar to single domain homologs: human MutT homolog (MTH1), E. coli MutT and Orf135 proteins.In MutT2, the binding pocket is well formed on the surface and, the substrate and the product bind to the same pocket, unlike in mycobacterial MutT1, in which the product binds at an inter-molecular interface.While the structure of the Nudix domain of MutT1 and MutT2 is very similar, the active and binding site residues exhibit substantial differences.In particular, the loops at the mouth of binding site in MutT2 are longer than in MutT1, a situation similar to that found in EcOrf135 and EcMutT.Another relevant observation is in relation to the depth of the ligand pocket.While the ligand remains closer to surface presumably due to presence of domain 2 in MutT1, the nitrogenous base is positioned relatively deeper in MutT2, a situation similar to that observed in single domain MutT homologs.The work presented here provides insights into the mechanism of action of this enzyme and also into the difference between the molecular basis of the functioning of mycobacterial MutT1 and MutT2.

Diadenosine polyphosphates (ApnA, n=2-6), particularly Ap4A, are involved in several important physiological processes. The substantial sequence identity of the Nudix hydrolase domain (domain 1) of Mycobacterium smegmatis MutT1 (MsMutT1) with a known Ap4A hydrolase suggested that MsMutT1 could also hydrolyse diadenosine polyphosphates. Biochemical experiments yielded results in conformity with this suggestion, with Ap4A as the best among the substrates. ATP is a product in all experiments; small amounts of ADP were also observed in the experiments involving Ap4A and Ap6A. Hydrolysis was inhibited by fluoride ions in all cases. The mechanism of action and its inhibition in relation to ApnA were explored through the X-ray analysis of the crystals of the MsMutT1 complexes with Ap5A; Ap5A and MnCl2; Ap4A; ATP; and ATP.NaF.MgCl2. The aggregation pattern of molecules in the first four crystals is similar to that found in a majority of MsMutT1-NTP crystals. Substrate molecules occupy the primary binding site and ATP occupies a site at an intermolecular interface, in the first two. ATP occupies both the sites in the third and fourth crystal. The protein-ligand interactions observed in these crystal structures lead to an explanation of the molecular mechanism of hydrolysis of ApnA by MsMutT1. The fifth crystal exhibits a new packing arrangement. The structure of the complex provides an explanation for the fluoride inhibition of the activity of the enzyme. It would thus appear that MutT1 has a major role involving the hydrolysis of diadenosine polyphosphates, which could be elucidated at the molecular level.

Mycobacterium smegmatis MutT1, which is made up of a Nudix domain (domain 1) and a histidine phosphatase domain (domain 2), efficiently hydrolyses 8-oxo-GTP and 8-oxo-dGTP to the corresponding nucleoside diphosphates and phosphate in the presence of magnesium ions. Domain 1 alone hydrolyses nucleoside triphosphates less efficiently. Under high concentrations and over long periods, the full-length enzyme as well as domain 1 catalyses the hydrolysis of the nucleoside triphosphates to the respective nucleoside monophosphates and pyrophosphate. The role of domain 2 appears to be limited to speeding up the reaction. Crystal structures of the apoenzyme and those of ligand-bound enzyme prepared in the presence of 8-oxo-GTP or 8-oxo-dGTP and different concentrations of magnesium were determined. In all of the structures except one, the molecules arrange themselves in a head-to-tail fashion in which domain 1 is brought into contact with domain 2 (trans domain 2) of a neighbouring molecule. The binding site for NTP (site A) is almost exclusively made up of residues from domain 1, while those for NDP (site B) and NMP (site C) are at the interface between domain 1 and trans domain 2 in an unusual instance of intermolecular interactions leading to binding sites. Protein-ligand interactions at site A lead to a proposal for the mechanism of hydrolysis of NTP to NDP and phosphate. A small modification in site A in the crystal which does not exhibit the head-to-tail arrangement appears to facilitate the production of NMP and pyrophosphate from NTP. The two arrangements could be in dynamic equilibrium in the cellular milieu.

17 independent crystal structures of family I uracil-DNA glycosylase fromMycobacterium tuberculosis(MtUng) and its complexes with uracil and its derivatives, distributed among five distinct crystal forms, have been determined. Thermodynamic parameters of binding in the complexes have been measured using isothermal titration calorimetry. The two-domain protein exhibits open and closed conformations, suggesting that the closure of the domain on DNA binding involves conformational selection. Segmental mobility in the enzyme molecule is confined to a 32-residue stretch which plays a major role in DNA binding. Uracil and its derivatives can bind to the protein in two possible orientations. Only one of them is possible when there is a bulky substituent at the 5′ position. The crystal structures of the complexes provide a reasonable rationale for the observed thermodynamic parameters. In addition to providing fresh insights into the structure, plasticity and interactions of the protein molecule, the results of the present investigation provide a platform for structure-based inhibitor design.