I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14043]
Open Access
Abstract: Inosine-5′-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme involved in nucleotide biosynthesis. Because of its critical role in purine biosynthesis, IMPDH is a drug design target for immunosuppressive, anticancer, antiviral and antimicrobial chemotherapy. In this study, we use mass spectrometry and X-ray crystallography to show that the inhibitor 6-Cl-purine ribotide forms a covalent adduct with the Cys-341 residue of Mycobacterium thermoresistibile IMPDH.
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Jan 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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M. Fleur
Sernee
,
Julie E.
Ralton
,
Tracy L.
Nero
,
Lukasz F.
Sobala
,
Joachim
Kloehn
,
Marcel A.
Vieira-lara
,
Simon A.
Cobbold
,
Lauren
Stanton
,
Douglas E. V.
Pires
,
Eric
Hanssen
,
Alexandra
Males
,
Tom
Ward
,
Laurence M.
Bastidas
,
Phillip L.
Van Der Peet
,
Michael W.
Parker
,
David B.
Ascher
,
Spencer J.
Williams
,
Gideon J.
Davies
,
Malcolm J.
Mcconville
Diamond Proposal Number(s):
[13587, 18598]
Open Access
Abstract: Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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Sep 2019
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I24-Microfocus Macromolecular Crystallography
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Vinayak
Singh
,
Angela
Pacitto
,
Stefano
Donini
,
Davide M.
Ferraris
,
Sándor
Boros
,
Eszter
Illyés
,
Bálint
Szokol
,
Menico
Rizzi
,
Tom L.
Blundell
,
David B.
Ascher
,
Janos
Pato
,
Valerie
Mizrahi
Abstract: Tuberculosis (TB) is a major infectious disease associated increasingly with drug resistance. Thus, new anti-tubercular agents with novel mechanisms of action are urgently required for the treatment of drug-resistant TB. In prior work, we identified compound 1 (cyclohexyl(4-(isoquinolin-5-ylsulfonyl)piperazin-1-yl)methanone) and showed that its anti-tubercular activity is attributable to inhibition of inosine-5′-monophosphate dehydrogenase (IMPDH) in Mycobacterium tuberculosis. In the present study, we explored the structure–activity relationship around compound 1 by synthesizing and evaluating the inhibitory activity of analogues against M. tuberculosis IMPDH in biochemical and whole-cell assays. X-ray crystallography was performed to elucidate the mode of binding of selected analogues to IMPDH. We establish the importance of the cyclohexyl, piperazine and isoquinoline rings for activity, and report the identification of an analogue with IMPDH-selective activity against a mutant of M. tuberculosis that is highly resistant to compound 1. We also show that the nitrogen in urea analogues is required for anti-tubercular activity and identify benzylurea derivatives as promising inhibitors that warrant further investigation.
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Apr 2019
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I02-Macromolecular Crystallography
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Palika
Abayakoon
,
Yi
Jin
,
James P.
Lingford
,
Marija
Petricevic
,
Alan
John
,
Eileen
Ryan
,
Janice
Wai-ying Mui
,
Douglas E. V.
Pires
,
David B.
Ascher
,
Gideon J.
Davies
,
Ethan D.
Goddard-borger
,
Spencer J.
Williams
Diamond Proposal Number(s):
[9948]
Abstract: An estimated 10 billion tonnes of sulfoquinovose (SQ) are produced and degraded each year. Prokaryotic sulfoglycolytic pathways catabolize sulfoquinovose (SQ) liberated from plant sulfolipid, or its delipidated form α-d-sulfoquinovosyl glycerol (SQGro), through the action of a sulfoquinovosidase (SQase), but little is known about the capacity of SQ glycosides to support growth. Structural studies of the first reported SQase (Escherichia coli YihQ) have identified three conserved residues that are essential for substrate recognition, but crossover mutations exploring active-site residues of predicted SQases from other organisms have yielded inactive mutants casting doubt on bioinformatic functional assignment. Here, we show that SQGro can support the growth of E. coli on par with d-glucose, and that the E. coli SQase prefers the naturally occurring diastereomer of SQGro. A predicted, but divergent, SQase from Agrobacterium tumefaciens proved to have highly specific activity toward SQ glycosides, and structural, mutagenic, and bioinformatic analyses revealed the molecular coevolution of catalytically important amino acid pairs directly involved in substrate recognition, as well as structurally important pairs distal to the active site. Understanding the defining features of SQases empowers bioinformatic approaches for mapping sulfur metabolism in diverse microbial communities and sheds light on this poorly understood arm of the biosulfur cycle.
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Sep 2018
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Abstract: Tuberculosis (TB) remains a major cause of mortality worldwide, and improved treatments are needed to combat emergence of drug resistance. Inosine 5´-monophosphate dehydrogenase (IMPDH), a crucial enzyme required for de novo synthesis of guanine nucleotides, is an attractive TB drug target. Herein, we describe the identification of potent IMPDH inhibitors using fragment-based screening and structure-based design techniques. Screening of a fragment library for Mycobacterium thermoresistible (Mth) IMPDH(ΔCBS) inhibitors identified a low affinity phenylimidazole derivative. X-ray crystallography of the Mth IMPDH ΔCBS −IMP−inhibitor complex revealed that two molecules of the fragment were bound in the NAD binding pocket of IMPDH. The linking the two molecules of the fragment afforded compounds with more than 1000-fold improvement in IMPDH affinity over the initial fragment hit.
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Mar 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9007, 9537]
Abstract: DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a central component of nonhomologous end joining (NHEJ), repairing DNA double-strand breaks that would otherwise lead to apoptosis or cancer. We have solved its structure in complex with the C-terminal peptide of Ku80 at 4.3 angstrom resolution using x-ray crystallography. We show that the 4128–amino acid structure comprises three large structural units: the N-terminal unit, the Circular Cradle, and the Head. Conformational differences between the two molecules in the asymmetric unit are correlated with changes in accessibility of the kinase active site, which are consistent with an allosteric mechanism to bring about kinase activation. The location of KU80ct194 in the vicinity of the breast cancer 1 (BRCA1) binding site suggests competition with BRCA1, leading to pathway selection between NHEJ and homologous recombination.
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Feb 2017
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[9537]
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.
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Oct 2016
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I24-Microfocus Macromolecular Crystallography
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Vinayak
Singh
,
Stefano
Donini
,
Angela
Pacitto
,
Claudia
Sala
,
Ruben C.
Hartkoorn
,
Neeraj
Dhar
,
Gyorgy
Keri
,
David B.
Ascher
,
Guillaume
Mondésert
,
Anthony
Vocat
,
Andréanne
Lupien
,
Raphael
Sommer
,
Hélène
Vermet
,
Sophie
Lagrange
,
Joe
Buechler
,
Digby F.
Warner
,
John D.
Mckinney
,
Janos
Pato
,
Stewart T.
Cole
,
Tom L.
Blundell
,
Menico
Rizzi
,
Valerie
Mizrahi
Diamond Proposal Number(s):
[9537]
Abstract: VCC234718, a molecule with growth inhibitory activity against Mycobacterium tuberculosis (Mtb), was identified by phenotypic screening of a 15344-compound library. Sequencing of a VCC234718-resistant mutant identified a Y487C substitution in the inosine monophosphate dehydrogenase, GuaB2, which was subsequently validated to be the primary molecular target of VCC234718 in Mtb. VCC234718 inhibits Mtb GuaB2 with a Ki of 100 nM and is uncompetitive with respect to IMP and NAD+. This compound binds at the NAD+ site, after IMP has bound, and makes direct interactions with IMP; therefore, the inhibitor is by definition uncompetitive. VCC234718 forms strong pi interactions with the Y487 residue side chain from the adjacent protomer in the tetramer, explaining the resistance-conferring mutation. In addition to sensitizing Mtb to VCC234718, depletion of GuaB2 was bactericidal in Mtb in vitro and in macrophages. When supplied at a high concentration (≥125 μM), guanine alleviated the toxicity of VCC234718 treatment or GuaB2 depletion via purine salvage. However, transcriptional silencing of guaB2 prevented Mtb from establishing an infection in mice, confirming that Mtb has limited access to guanine in this animal model. Together, these data provide compelling validation of GuaB2 as a new tuberculosis drug target.
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Sep 2016
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I02-Macromolecular Crystallography
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Open Access
Abstract: The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt–Hogg–Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.
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Dec 2015
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I03-Macromolecular Crystallography
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Open Access
Abstract: The growth/motility factor hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, the tyrosine kinase MET, constitute a signalling system essential for embryogenesis and for tissue/organ regeneration in post-natal life. HGF/SF-MET signalling, however, also plays a key role in the onset of metastasis of a large number of human tumours. Both HGF/SF and MET are high molecular weight proteins that bury an extensive interface upon complex formation and thus constitute a challenging target for the development of low molecular weight inhibitors. Here we have used surface plasmon resonance (SPR), nuclear magnetic resonance (NMR) and X-ray crystallography to screen a diverse fragment library of 1338 members as well as a range of piperazine-like compounds. Several small molecules were found to bind in the lysine-binding pocket of the kringle 1 domain of HGF/SF and its truncated splice variant NK1. We have defined the binding mode of these compounds, explored their biological activity and we show that selected fragments inhibit MET downstream signalling. Thus we demonstrate that targeting the lysine-binding pocket of NK1 is an effective strategy to generate MET receptor antagonists and we offer proof of concept that the HGF/SF-MET interface may be successfully targeted with small molecules. These studies have broad implications for the development of HGF/SF-MET therapeutics and cancer treatment.
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Jul 2015
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