I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775, 18566]
Open Access
Abstract: Alanine racemase (Alr) is a pyridoxal 5′-phosphate-dependent enzyme that catalyzes the racemization of l-alanine to d-alanine. Alr is one of the two targets of the broad-spectrum antibiotic d-cycloserine (DCS), a structural analogue of d-alanine. Despite being an essential component of regimens used to treat multi- and extensively drug-resistant tuberculosis for almost seven decades, resistance to DCS has not been observed in patients. We previously demonstrated that DCS evades resistance due to an ultralow rate of emergence of mutations. Yet, we identified a single polymorphism (converting Asp322 to Asn) in the alr gene, which arose in 8 out of 11 independent variants identified and that confers resistance. Here, we present the crystal structure of the Alr variant D322N in both the free and DCS-inactivated forms and the characterization of its DCS inactivation mechanism by UV–visible and fluorescence spectroscopy. Comparison of these results with those obtained with wild-type Alr reveals the structural basis of the 240-fold reduced inhibition observed in Alr D322N.
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Mar 2023
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Martyn
Frederickson
,
Irwin R.
Selvam
,
Dimitrios
Evangelopoulos
,
Kirsty J.
Mclean
,
Mona M.
Katariya
,
Richard B.
Tunnicliffe
,
Bethany
Campbell
,
Madeline E.
Kavanagh
,
Sitthivut
Charoensutthivarakul
,
Richard T.
Blankley
,
Colin W.
Levy
,
Luiz Pedro S.
De Carvalho
,
David
Leys
,
Andrew W.
Munro
,
Anthony G.
Coyne
,
Chris
Abell
Diamond Proposal Number(s):
[17773, 24447]
Abstract: There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 μM), a novel hit compound suitable as a starting point for a more involved lead-to-clinical candidate medicinal chemistry campaign.
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Jan 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[13775]
Abstract: The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5′-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.
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Mar 2020
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I04-Macromolecular Crystallography
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Safaa M.
Kishk
,
Kirsty J.
Mclean
,
Sakshi
Sood
,
Darren
Smith
,
Jack W. D.
Evans
,
Mohamed A.
Helal
,
Mohamed S.
Gomaa
,
Ismail
Salama
,
Samia M.
Mostafa
,
Luiz Pedro S.
De carvalho
,
Colin W.
Levy
,
Andrew W.
Munro
,
Claire
Simons
Diamond Proposal Number(s):
[17773]
Open Access
Abstract: The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (KD). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, KD 0.22 μM (10 f) and 4.81 μM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H‐bond acceptors/H‐bond donors 4/0, number of rotatable bonds 5–6, molecular volume >340 Å3, topological polar surface area <40 Å2.
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Jul 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13775]
Open Access
Abstract: The biosynthetic pathway of peptidoglycan is essential for Mycobacterium tuberculosis. We report here the acetyltransferase substrate specificity and catalytic mechanism of the bifunctional N-acetyltransferase/uridyltransferase from M. tuberculosis (GlmU). This enzyme is responsible for the final two steps of the synthesis of UDP-N-acetylglucosamine, which is an essential precursor of peptidoglycan, from glucosamine-1-phosphate, acetyl coenzyme A and uridine-5'-triphosphate. GlmU utilizes requires ternary complex formation to transfer an acetyl from acetyl coenzyme A to glucosamine-1-phosphate to form N-acetylglucosmaine-1-phosphate. Steady-state kinetic studies and equilibrium binding experiments indicate that GlmU follows a steady-state ordered kinetic mechanism, with acetyl coenzyme A binding first, which triggers a conformational change on GlmU, followed by glucosamine-1-phosphate binding. Coenzyme A is the last product to dissociate. Chemistry is partially rate-limiting as indicated by pH-rate studies and solvent kinetic isotope effects. A novel crystal structure of a mimic of the Michaelis complex, with glucose-1-phosphate and acetyl-coenzyme A, helps us to propose the residues involved in deprotonation of glucosamine-1-phosphate and the loop movement that likely generates the active site required for glucosamine-1-phosphate to bind. Together, these results pave the way for the rational discovery of improved inhibitors against M. tuberculosis GlmU, some of which might become candidates for antibiotic discovery programs.
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Apr 2018
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I04-Macromolecular Crystallography
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Sarah
Batson
,
Cesira
De Chiara
,
Vita
Majce
,
Adrian J.
Lloyd
,
Stanislav
Gobec
,
Dean
Rea
,
Vilmos
Fulop
,
Christopher
Thoroughgood
,
Katie J.
Simmons
,
Christopher G.
Dowson
,
Colin W. G.
Fishwick
,
Luiz Pedro S.
De Carvalho
,
David I.
Roper
Open Access
Abstract: D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.
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Dec 2017
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I02-Macromolecular Crystallography
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Ismail M.
Taban
,
Hosam E. A. E.
Elshihawy
,
Beyza
Touran
,
Benedetta
Zucchini
,
Clare J.
Williamson
,
Dania
Altuwairigi
,
Adeline S. T.
Ngu
,
Kirsty J.
Mclean
,
Colin W.
Levy
,
Sakshi
Sood
,
Leonardo B.
Marino
,
Andrew W.
Munro
,
Luiz Pedro S.
De Carvalho
,
Claire
Simons
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: Three series of biarylpyrazole imidazole and triazoles are described, which vary in the linker between the biaryl pyrazole and imidazole/triazole group. The imidazole and triazole series with the short –CH2- linker displayed promising antimycobacterial activity, with the imidazole–CH2- series (7) showing low MIC values (6.25-25 μg/mL), which was also influenced by lipophilicity. Extending the linker to –C(O)NH(CH2)2- resulted in loss of antimycobacterial activity. Binding affinity of the compounds with CYP121A1 was determined by UV-visible optical titrations with KD values of 2.63, 35.6 and 290 μM, respectively, for the tightest binding compounds 7e, 8b and 13d from their respective series. Both binding affinity assays and docking studies of the CYP121A1 inhibitors suggests type II indirect binding through interstitial water molecules, with key binding residues Thr77, Val78, Val82, Val83, Met86, Ser237, Gln385 and Arg386, comparable with the binding interactions observed with fluconazole and the natural substrate dicyclotyrosine.
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Nov 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7707, 9826]
Open Access
Abstract: ATP-phosphoribosyltransferase (ATP-PRT) is a hexameric enzyme in conformational equilibrium between an open and seemingly active state and a closed and presumably inhibited form. The structure-function relationship of allosteric regulation in this system is still not fully understood. Here, we develop a screening strategy for modulators of ATP-PRT and identify 3-(2-thienyl)-l-alanine (TIH) as an allosteric activator of this enzyme. Kinetic analysis reveals co-occupancy of the allosteric sites by TIH and l-histidine. Crystallographic and native ion-mobility mass spectrometry data show that the TIH-bound activated form of the enzyme closely resembles the inhibited l-histidine-bound closed conformation, revealing the uncoupling between ATP-PRT open and closed conformations and its functional state. These findings suggest that dynamic processes are responsible for ATP-PRT allosteric regulation and that similar mechanisms might also be found in other enzymes bearing a ferredoxin-like allosteric domain.
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Aug 2017
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Madeline E.
Kavanagh
,
Anthony G.
Coyne
,
Kirsty J.
Mclean
,
Guy G.
James
,
Colin W.
Levy
,
Leonardo B.
Marino
,
Luiz Pedro S.
De Carvalho
,
Daniel S. H.
Chan
,
Sean A.
Hudson
,
Sachin
Surade
,
David
Leys
,
Andrew W.
Munro
,
Chris
Abell
Diamond Proposal Number(s):
[8997]
Open Access
Abstract: The essential enzyme CYP121 is a target for
drug development against antibiotic resistant strains of
Mycobacterium tuberculosis. A triazol-1-yl phenol fragment 1
was identified to bind to CYP121 using a cascade of
biophysical assays. Synthetic merging and optimization of 1
produced a 100-fold improvement in binding affinity, yielding
lead compound 2 (KD = 15 μM). Deconstruction of 2 into its
component retrofragments allowed the group efficiency of
structural motifs to be assessed, the identification of more LE
scaffolds for optimization and highlighted binding affinity
hotspots. Structure-guided addition of a metal-binding
pharmacophore onto LE retrofragment scaffolds produced
low nanomolar (KD = 15 nM) CYP121 ligands. Elaboration of
these compounds to target binding hotspots in the distal active site afforded compounds with excellent selectivity against human
drug-metabolizing P450s. Analysis of the factors governing ligand potency and selectivity using X-ray crystallography, UV−vis
spectroscopy, and native mass spectrometry provides insight for subsequent drug development.
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Mar 2016
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I04-Macromolecular Crystallography
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David C.
Goldstone
,
Valerie
Ennis-Adeniran
,
Joseph J.
Hedden
,
Harriet C. T.
Groom
,
Gillian I.
Rice
,
Evangelos
Papagrigoriou
,
Phil A.
Walker
,
Geoff
Kelly
,
Lesley F.
Haire
,
Melvyn W.
Yap
,
Luiz Pedro S.
De Carvalho
,
Jonathan P.
Stoye
,
Yanick J.
Crow
,
Ian A.
Taylor
,
Michelle
Webb
Abstract: SAMHD1, an analogue of the murine interferon (IFN)-gamma-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells(2,3) and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction(4-7). SAMHD1 is also associated with Aicardi-Goutieres syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-alpha(8). The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.
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Nov 2011
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