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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1 Adrian Richter , Ines Rudolph , Ute Möllmann , Kerstin Voigt , Chun-Wa Chung , Onkar M. P. Singh , Michael Rees , Alfonso Mendoza-Losana , Robert Bates , Lluís Ballell , Sarah Batt , Natacha Veerapen , Klaus Fütterer , Gurdyal Besra , Peter Imming , Argyrides Argyrou Scientific Reports 8 DOI: 10.1038/s41598-018-31316-6 Diamond Proposal Number(s): [12279] Open Access Show Abstract ▼ Abstract: Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.	Sep 2018
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Target identification of Mycobacterium tuberculosis phenotypic hits using a concerted chemogenomic, biophysical, and structural approach Grace Mugumbate , Vitor Mendes , Michal Blaszczyk , Mohamad Sabbah , George Papadatos , Joel Lelievre , Lluis Ballell , David Barros , Chris Abell , Tom L. Blundell , John P. Overington Frontiers In Pharmacology 8 DOI: 10.3389/fphar.2017.00681 Diamond Proposal Number(s): [9537, 14043] Show Abstract ▼ Abstract: Mycobacterium phenotypic hits are a good reservoir for new chemotypes for the treatment of tuberculosis. However, the absence of defined molecular targets and modes of action could lead to failure in drug development. Therefore, a combination of ligand-based and structure-based chemogenomic approaches followed by biophysical and biochemical validation have been used to identify targets for Mycobacterium tuberculosis phenotypic hits. Our approach identified EthR and InhA as targets for several hits, with some showing dual activity against these proteins. From the 35 predicted EthR inhibitors, eight exhibited an IC50 below 50 μM against M. tuberculosis EthR and three were confirmed to be also simultaneously active against InhA. Further hit validation was performed using X-ray crystallography yielding eight new crystal structures of EthR inhibitors. Although the EthR inhibitors attain their activity against M. tuberculosis by hitting yet undefined targets, these results provide new lead compounds that could be further developed to be used to potentiate the effect of EthA activated pro-drugs, such as ethionamide, thus enhancing their bactericidal effect.	Sep 2017
I04-Macromolecular Crystallography	Inhibiting mycobacterial tryptophan synthase by targeting the inter-subunit interface Katherine A. Abrahams , Jonathan A. G. Cox , Klaus Futterer , Joaquín Rullas , Fátima Ortega-Muro , Nicholas J. Loman , Patrick J. Moynihan , Esther Pérez-Herrán , Elena Jiménez , Jorge Esquivias , David Barros , Lluís Ballell , Carlos Alemparte , Gurdyal S. Besra Scientific Reports 7 DOI: 10.1038/s41598-017-09642-y Open Access Show Abstract ▼ Abstract: Drug discovery efforts against the pathogen Mycobacterium tuberculosis (Mtb) have been advanced through phenotypic screens of extensive compound libraries. Such a screen revealed sulfolane 1 and indoline-5-sulfonamides 2 and 3 as potent inhibitors of mycobacterial growth. Optimization in the sulfolane series led to compound 4, which has proven activity in an in vivo murine model of Mtb infection. Here we identify the target and mode of inhibition of these compounds based on whole genome sequencing of spontaneous resistant mutants, which identified mutations locating to the essential α- and β-subunits of tryptophan synthase. Over-expression studies confirmed tryptophan synthase as the biological target. Biochemical techniques probed the mechanism of inhibition, revealing the mutant enzyme complex incurs a fitness cost but does not prevent inhibitor binding. Mapping of the resistance conferring mutations onto a low-resolution crystal structure of Mtb tryptophan synthase showed they locate to the interface between the α- and β-subunits. The discovery of anti-tubercular agents inhibiting tryptophan synthase highlights the therapeutic potential of this enzyme and draws attention to the prospect of other amino acid biosynthetic pathways as future Mtb drug targets.	Aug 2017
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Essential but not vulnerable: indazole sulfonamides targeting inosine monophosphate dehydrogenase as potential leads against Mycobacterium tuberculosis Yumi Park , Angela Pacitto , Tracy Bayliss , Laura A. T. Cleghorn , Zhe Wang , Travis Hartman , Kriti Arora , Thomas R. Ioerger , Jim Sacchettini , Menico Rizzi , Stefano Donini , Tom L. Blundell , David B. Ascher , Kyu Rhee , Ardala Breda , Nian Zhou , Veronique Dartois , Surendranadha Reddy Jonnala , Laura E. Via , Valerie Mizrahi , Ola Epemolu , Laste Stojanovski , Fred Simeons , Maria Osuna-Cabello , Lucy Ellis , Claire J. Mackenzie , Alasdair R. C. Smith , Susan H. Davis , Dinakaran Murugesan , Kirsteen I. Buchanan , Penelope A. Turner , Margaret Huggett , Fabio Zuccotto , Maria Jose Rebollo-Lopez , Maria Jose Lafuente-Monasterio , Olalla Sanz , Gracia Santos Diaz , Joël Lelièvre , Lluis Ballell , Carolyn Selenski , Matthew Axtman , Sonja Ghidelli-Disse , Hannah Pflaumer , Markus Bösche , Gerard Drewes , Gail M. Freiberg , Matthew D. Kurnick , Myron Srikumaran , Dale J. Kempf , Simon R. Green , Peter C. Ray , Kevin Read , Paul Wyatt , Clifton E. Barry , Helena I. Boshoff Acs Infectious Diseases DOI: 10.1021/acsinfecdis.6b00103 Diamond Proposal Number(s): [9537] Show Abstract ▼ Abstract: A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH–IMP–inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi–pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time–kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5–2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.	Oct 2016
I04-Macromolecular Crystallography	Identification of KasA as the cellular target of an anti-tubercular scaffold Katherine A. Abrahams , Chun-Wa Chung , Sonja Ghidelli-Disse , Joaquín Rullas , María José Rebollo-López , Sudagar S. Gurcha , Jonathan A. G. Cox , Alfonso Mendoza , Elena Jiménez-Navarro , María Santos Martínez-Martínez , Margarete Neu , Anthony Shillings , Paul Homes , Argyrides Argyrou , Ruth Casanueva , Nicholas J. Loman , Patrick J. Moynihan , Joël Lelièvre , Carolyn Selenski , Matthew Axtman , Laurent Kremer , Marcus Bantscheff , Iñigo Angulo-Barturen , Mónica Cacho Izquierdo , Nicholas C. Cammack , Gerard Drewes , Lluis Ballell , David Barros , Gurdyal S. Besra , Robert H. Bates Nature Communications 7 DOI: 10.1038/ncomms12581 Diamond Proposal Number(s): [12279] Open Access Show Abstract ▼ Abstract: 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.	Sep 2016
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	THPP target assignment reveals EchA6 as an essential fatty acid shuttle in mycobacteria Jonathan A. G. Cox , Katherine A. Abrahams , Carlos Alemparte , Sonja Ghidelli-Disse , Joaquín Rullas , Iñigo Angulo-Barturen , Albel Singh , Sudagar S. Gurcha , Vijayashankar Nataraj , Stephen Bethell , Modesto J. Remuiñán , Lourdes Encinas , Peter J. Jervis , Nicholas C. Cammack , Apoorva Bhatt , Ulrich Kruse , Marcus Bantscheff , Klaus Fütterer , David Barros , Lluis Ballell , Gerard Drewes , Gurdyal S. Besra Nature Microbiology 1 DOI: 10.1038/nmicrobiol.2015.6 Diamond Proposal Number(s): [8359, 10369] Show Abstract ▼ Abstract: Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.	Feb 2016
I04-Macromolecular Crystallography	Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target Sudagar S. Gurcha , Veeraraghavan Usha , Jonathan A. G. Cox , Klaus Futterer , K Abrahams , Apoorva Bhatt , Luke Alderwick , Robert C. Reynolds , Nicholas J. Loman , Vijayashankar Nataraj , Carlos Alemparte , David Barros , Adrian J. Lloyd , Lluis Ballell , Judith V. Hobrath , Gurdyal S. Besra Plos One 9 (11) DOI: 10.1371/journal.pone.0113568 PMID: 25409504 Diamond Proposal Number(s): [10369] Open Access Show Abstract ▼ Abstract: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.	Nov 2014

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