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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography	A molecular hybridization approach for the design of potent, highly selective, and brain-penetrant n-myristoyltransferase inhibitors Justin R. Harrison , Stephen Brand , Victoria Smith , David A. Robinson , Stephen Thompson , Alasdair Smith , Kenneth Davies , Ngai Mok , Leah S. Torrie , Iain Collie , Irene Hallyburton , Suzanne Norval , Frederick R. C. Simeons , Laste Stojanovski , Julie A. Frearson , Ruth Brenk , Paul G. Wyatt , Ian H. Gilbert , Kevin D. Read Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.8b00884 Diamond Proposal Number(s): [7705] Show Abstract ▼ Abstract: Crystallography has guided the hybridization of two series of Trypanosoma brucei N-myristoyltransferase (NMT) inhibitors, leading to a novel highly selective series. The effect of combining the selectivity enhancing elements from two pharmacophores is shown to be additive and has led to compounds that have greater than 1000-fold selectivity for TbNMT vs HsNMT. Further optimization of the hybrid series has identified compounds with significant trypanocidal activity capable of crossing the blood–brain barrier. By using CF-1 mdr1a deficient mice, we were able to demonstrate full cures in vivo in a mouse model of stage 2 African sleeping sickness. This and previous work provides very strong validation for NMT as a drug target for human African trypanosomiasis in both the peripheral and central nervous system stages of disease.	Sep 2018
I04-1-Macromolecular Crystallography (fixed wavelength)	Optimization of a “Bump-and-Hole” Approach to Allele-Selective BET Bromodomain Inhibition Andrew Runcie , Michael Zengerle , Kwok-ho Chan , Andrea Testa , Lars Van Beurden , Matthias Baud , Ola Epemolu , Lucy Ellis , Kevin Read , Victoria Coulthard , Alex Brien , Alessio Ciulli Chemical Science DOI: 10.1039/C7SC02536J Diamond Proposal Number(s): [10071] Show Abstract ▼ Abstract: Allele-specific chemical genetics enables selective inhibition within families of highly-conserved proteins. The four BET (bromodomain & extra-terminal domain) proteins – BRD2, BRD3, BRD4 and BRDT bind acetylated chromatin via their bromodomains and regulate processes such as cell proliferation and inflammation. BET bromodomains are of particular interest, as they are attractive therapeutic targets but existing inhibitors are pan-selective. We previously established a bump-&-hole system for the BET bromodomains, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold. Here we optimize upon this system with the introduction of a more conservative and less disruptive leucine/valine mutation. Extensive structure-activity-relationships of diverse benzodiazepine analogues guided the development of potent, mutant-selective inhibitors with desirable physiochemical properties. The active enantiomer of our best compound – 9-ME-1 – shows ~200 nM potency, >100-fold selectivity for the L/V mutant over wild-type and excellent DMPK properties. Through a variety of in vitro and cellular assays we validate the capabilities of our optimized system, and then utilize it to compare the relative importance of the first and second bromodomains to chromatin binding. These experiments confirm the primacy of the first bromodomain in all BET proteins, but also significant variation in the importance of the second bromodomain. We also show that, despite having a minor role in chromatin recognition, BRD4 BD2 is still essential for gene expression , likely through the recruitment of non-histone proteins. The disclosed inhibitor:mutant pair provides a powerful tool for future cellular and in vivo target validation studies.	Jan 2018
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Chemical Validation of Methionyl-tRNA Synthetase as a Druggable Target in Leishmania donovani Leah S. Torrie , Stephen Brand , David A. Robinson , Eun Jung Ko , Laste Stojanovski , Frederick R. C. Simeons , Susan Wyllie , John Thomas , Lucy Ellis , Maria Osuna-cabello , Ola Epemolu , Andrea Nühs , Jennifer Riley , Lorna Maclean , Sujatha Manthri , Kevin D. Read , Ian H. Gilbert , Alan H. Fairlamb , Manu De Rycker Acs Infectious Diseases DOI: 10.1021/acsinfecdis.7b00047 Diamond Proposal Number(s): [8268] Show Abstract ▼ Abstract: Methionyl-tRNA synthetase (MetRS) has been chemically validated as a drug target in the kinetoplastid parasite Trypanosoma brucei. In the present study, we investigate the validity of this target in the related trypanosomatid Leishmania donovani. Following development of a robust high-throughput compatible biochemical assay, a compound screen identified DDD806905 as a highly potent inhibitor of LdMetRS (Ki of 18 nM). Crystallography revealed this compound binds to the methionine pocket of MetRS with enzymatic studies confirming DDD806905 displays competitive inhibition with respect to methionine and mixed inhibition with respect to ATP binding. DDD806905 showed activity, albeit with different levels of potency, in various Leishmania cell-based viability assays, with on-target activity observed in both Leishmania promastigote cell assays and a Leishmania tarentolae in vitro translation assay. Unfortunately, this compound failed to show efficacy in an animal model of leishmaniasis. We investigated the potential causes for the discrepancies in activity observed in different Leishmania cell assays and the lack of efficacy in the animal model and found that high protein binding as well as sequestration of this dibasic compound into acidic compartments may play a role. Despite medicinal chemistry efforts to address the dibasic nature of DDD806905 and analogues, no progress could be achieved with the current chemical series. Although DDD806905 is not a developable antileishmanial compound, MetRS remains an attractive antileishmanial drug target.	Oct 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	Group-based optimization of potent and cell-active inhibitors of the von Hippel-Lindau (VHL) E3 ubiquitin ligase Pedro Soares , Morgan S. Gadd , Julianty Frost , Carles Galdeano , Lucy C. J. Ellis , Ola Epemolu , Sonia Rocha , Kevin D. Read , Alessio Ciulli Journal Of Medicinal Chemistry DOI: 10.1021/acs.jmedchem.7b00675 Diamond Proposal Number(s): [10071] Show Abstract ▼ Abstract: The von Hippel–Lindau tumor suppressor protein is the substrate binding subunit of the VHL E3 ubiquitin ligase, which targets hydroxylated α subunit of hypoxia inducible factors (HIFs) for ubiquitination and subsequent proteasomal degradation. VHL is a potential target for treating anemia and ischemic diseases, motivating the development of inhibitors of the VHL:HIF-α protein-protein interaction. Additionally, bifunctional proteolysis targeting chimeras (PROTACs) containing a VHL ligand can hijack the E3 ligase activity to induce degradation of target proteins. We report the structure-guided design and group-based optimization of a series of VHL inhibitors with low nanomolar potencies and improved cellular permeability. Structure-activity relationships led to the discovery of potent inhibitors 10 and chemical probe VH298, with dissociation constants <100 nM, which induced marked HIF-1α intracellular stabilization. Our study provides new chemical tools to probe the VHL-HIF pathways, and new VHL ligands for next-generation PROTACs.	Aug 2017
I04-1-Macromolecular Crystallography (fixed wavelength)	Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition Julianty Frost , Carles Galdeano , Pedro Soares , Morgan S. Gadd , Katarzyna M. Grzes , Lucy Ellis , Ola Epemolu , Satoko Shimamura , Marcus Bantscheff , Paola Grandi , Kevin D. Read , Doreen A. Cantrell , Sonia Rocha , Alessio Ciulli Nature Communications 7 DOI: 10.1038/ncomms13312 Diamond Proposal Number(s): [10071] Open Access Show Abstract ▼ Abstract: Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein–protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.	Nov 2016
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
I03-Macromolecular Crystallography	Development of Small-Molecule Trypanosoma brucei N-Myristoyltransferase Inhibitors: Discovery and Optimisation of a Novel Binding Mode Daniel Spinks , Victoria Smith , Stephen Thompson , David A. Robinson , Torsten Luksch , Alasdair Smith , Leah S. Torrie , Stuart Mcelroy , Laste Stojanovski , Suzanne Norval , Iain T. Collie , Irene Hallyburton , Bhavya Rao , Stephen Brand , Ruth Brenk , Julie A. Frearson , Kevin D. Read , Paul G. Wyatt , Ian H. Gilbert ChemMedChem 10 (11), 1821 - 1836 DOI: 10.1002/cmdc.201500397 PMID: 26395087 Open Access Show Abstract ▼ Abstract: The front cover picture shows the enzyme N-myristoyltransferase from the pathogen Trypanosoma brucei. Next to this is an image of the parasite. Also shown are an inhibitor of the protein that we have previously reported (bottom structure) and two novel scaffolds (top structures) that are reported in our paper in this issue.	Nov 2015
I03-Macromolecular Crystallography	Development of Small-Molecule Trypanosoma Brucei N-Myristoyltransferase Inhibitors: Discovery and Optimisation of a Novel Binding Mode Daniel Spinks , Victoria Smith , Stephen Thompson , David Robinson , Torsten Luksch , Alasdair Smith , Leah S. Torrie , Stuart Mcelroy , Laste Stojanovski , Suzanne Norval , Iain T. Collie , Irene Hallyburton , Bhavya Rao , Stephen Brand , Ruth Brenk , Julie A. Frearson , Kevin D. Read , Paul G. Wyatt , Ian H. Gilbert ChemMedChem 10, 1821 - 1836 DOI: 10.1002/cmdc.201500301 PMID: 26395087 Open Access Show Abstract ▼ Abstract: The enzyme N-myristoyltransferase (NMT) from Trypanosoma brucei has been validated both chemically and biologically as a potential drug target for human African trypanosomiasis. We previously reported the development of some very potent compounds based around a pyrazole sulfonamide series, derived from a high-throughput screen. Herein we describe work around thiazolidinone and benzomorpholine scaffolds that were also identified in the screen. An X-ray crystal structure of the thiazolidinone hit in Leishmania major NMT showed the compound bound in the previously reported active site, utilising a novel binding mode. This provides potential for further optimisation. The benzomorpholinone was also found to bind in a similar region. Using an X-ray crystallography/structure-based design approach, the benzomorpholinone series was further optimised, increasing activity against T. brucei NMT by >1000-fold. A series of trypanocidal compounds were identified with suitable in vitro DMPK properties, including CNS exposure for further development. Further work is required to increase selectivity over the human NMT isoform and activity against T. brucei.	Nov 2015

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