I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
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Simon R.
Green
,
Susan H.
Davis
,
Sebastian
Damerow
,
Curtis A.
Engelhart
,
Michael
Mathieson
,
Beatriz
Baragaña
,
David A.
Robinson
,
Jevgenia
Tamjar
,
Alice
Dawson
,
Fabio K.
Tamaki
,
Kirsteen I.
Buchanan
,
John
Post
,
Karen
Dowers
,
Sharon M.
Shepherd
,
Chimed
Jansen
,
Fabio
Zuccotto
,
Ian H.
Gilbert
,
Ola
Epemolu
,
Jennifer
Riley
,
Laste
Stojanovski
,
Maria
Osuna-Cabello
,
Esther
Pérez-Herrán
,
María José
Rebollo
,
Laura
Guijarro López
,
Patricia
Casado Castro
,
Isabel
Camino
,
Heather C.
Kim
,
James M.
Bean
,
Navid
Nahiyaan
,
Kyu Y.
Rhee
,
Qinglan
Wang
,
Vee Y.
Tan
,
Helena I. M.
Boshoff
,
Paul J.
Converse
,
Si-Yang
Li
,
Yong S.
Chang
,
Nader
Fotouhi
,
Anna M.
Upton
,
Eric L.
Nuermberger
,
Dirk
Schnappinger
,
Kevin D.
Read
,
Lourdes
Encinas
,
Robert H.
Bates
,
Paul G.
Wyatt
,
Laura A. T.
Cleghorn
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
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Oct 2022
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I03-Macromolecular Crystallography
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Rachel
Milne
,
Natalie
Wiedemar
,
Victoriano
Corpas-Lopez
,
Eoin
Moynihan
,
Richard J.
Wall
,
Alice
Dawson
,
David A.
Robinson
,
Sharon M.
Shepherd
,
Robert J.
Smith
,
Irene
Hallyburton
,
John M.
Post
,
Karen
Dowers
,
Leah S.
Torrie
,
Ian H.
Gilbert
,
Beatriz
Baragaña
,
Stephen
Patterson
,
Susan
Wyllie
Diamond Proposal Number(s):
[19844]
Open Access
Abstract: There is a pressing need for new medicines to prevent and treat malaria. Most antimalarial drug discovery is reliant upon phenotypic screening. However, with the development of improved target validation strategies, target-focused approaches are now being utilized. Here, we describe the development of a toolkit to support the therapeutic exploitation of a promising target, lysyl tRNA synthetase (PfKRS). The toolkit includes resistant mutants to probe resistance mechanisms and on-target engagement for specific chemotypes; a hybrid KRS protein capable of producing crystals suitable for ligand soaking, thus providing high-resolution structural information to guide compound optimization; chemical probes to facilitate pulldown studies aimed at revealing the full range of specifically interacting proteins and thermal proteome profiling (TPP); as well as streamlined isothermal TPP methods to provide unbiased confirmation of on-target engagement within a biologically relevant milieu. This combination of tools and methodologies acts as a template for the development of future target-enabling packages.
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Aug 2022
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment, including host defense responses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. Because this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the Δgna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective sub-micromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.
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Apr 2020
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I03-Macromolecular Crystallography
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Leah S.
Torrie
,
David A.
Robinson
,
Michael G.
Thomas
,
Judith V.
Hobrath
,
Sharon M
Shepherd
,
John M.
Post
,
Eun-Jung
Ko
,
Rafael Augusto
Alves Ferreira
,
Claire J.
Mackenzie
,
Karolina
Wrobel
,
Darren
Edwards
,
Ian H.
Gilbert
,
David W.
Gray
,
Alan H.
Fairlamb
,
Manu
De Rycker
Diamond Proposal Number(s):
[14980]
Open Access
Abstract: Methionyl-tRNA synthetase (MetRS) is a chemically validated drug target in the kinetoplastid parasites Trypanosoma brucei and Leishmania donovani. To date, all kinetoplastid MetRS inhibitors described bind in a similar way to an expanded methionine pocket and an adjacent, auxiliary pocket. In the current study we have identified a structurally novel class of inhibitors containing a 4,6-diamino substituted pyrazolopyrimidine core (‘MetRS02’ series). Crystallographic studies revealed that ‘MetRS02’ compounds bind to an allosteric pocket in L. major MetRS not previously described and enzymatic studies demonstrated a non-competitive mode of inhibition. Homology modelling of the Trypanosoma cruzi MetRS enzyme revealed key differences in the allosteric pocket between the T. cruzi and Leishmania enzymes. These provide a likely explanation for the lower ‘MetRS02’ potencies that we observed for the T. cruzi enzyme compared to the Leishmania enzyme. The identification of a new series of MetRS inhibitors and the discovery of a new binding site in kinetoplastid MetRS enzymes provides a novel strategy in the search for new therapeutics for kinetoplastid diseases.
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Apr 2020
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I24-Microfocus Macromolecular Crystallography
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Beatriz
Baragaña
,
Barbara
Forte
,
Ryan
Choi
,
Stephen
Nakazawa Hewitt
,
Juan A.
Bueren-Calabuig
,
Joao Pedro
Pisco
,
Caroline
Peet
,
David M.
Dranow
,
David A.
Robinson
,
Chimed
Jansen
,
Neil R.
Norcross
,
Sumiti
Vinayak
,
Mark
Anderson
,
Carrie F.
Brooks
,
Caitlin A.
Cooper
,
Sebastian
Damerow
,
Michael
Delves
,
Karen
Dowers
,
James
Duffy
,
Thomas E.
Edwards
,
Irene
Hallyburton
,
Benjamin G.
Horst
,
Matthew A.
Hulverson
,
Liam
Ferguson
,
María Belén
Jiménez-Díaz
,
Rajiv S.
Jumani
,
Donald D.
Lorimer
,
Melissa S.
Love
,
Steven
Maher
,
Holly
Matthews
,
Case W.
Mcnamara
,
Peter
Miller
,
Sandra
O’neill
,
Kayode K.
Ojo
,
Maria
Osuna-Cabello
,
Erika
Pinto
,
John
Post
,
Jennifer
Riley
,
Matthias
Rottmann
,
Laura M.
Sanz
,
Paul
Scullion
,
Arvind
Sharma
,
Sharon M.
Shepherd
,
Yoko
Shishikura
,
Frederick R. C.
Simeons
,
Erin E.
Stebbins
,
Laste
Stojanovski
,
Ursula
Straschil
,
Fabio K.
Tamaki
,
Jevgenia
Tamjar
,
Leah S.
Torrie
,
Amélie
Vantaux
,
Benoît
Witkowski
,
Sergio
Wittlin
,
Manickam
Yogavel
,
Fabio
Zuccotto
,
Iñigo
Angulo-Barturen
,
Robert
Sinden
,
Jake
Baum
,
Francisco-Javier
Gamo
,
Pascal
Mäser
,
Dennis E.
Kyle
,
Elizabeth A.
Winzeler
,
Peter J.
Myler
,
Paul G.
Wyatt
,
David
Floyd
,
David
Matthews
,
Amit
Sharma
,
Boris
Striepen
,
Christopher D.
Huston
,
David W.
Gray
,
Alan H.
Fairlamb
,
Andrei V.
Pisliakov
,
Chris
Walpole
,
Kevin D.
Read
,
Wesley C.
Van Voorhis
,
Ian H.
Gilbert
Diamond Proposal Number(s):
[10071]
Open Access
Abstract: Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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Apr 2019
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I03-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[7705]
Open Access
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.
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Sep 2018
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[8268]
Open Access
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.
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Oct 2017
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I03-Macromolecular Crystallography
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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
Open Access
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.
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Nov 2015
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis. Pyrazole sulfonamide (DDD85646), a potent inhibitor of TbNMT, has been identified in previous studies; however, poor central nervous system exposure restricts its use to the haemolymphatic form (stage 1) of the disease. In order to identify new chemical matter, a fragment screen was carried out by ligand-observed NMR spectroscopy, identifying hits that occupy the DDD85646 binding site. Crystal structures of hits from this assay have been obtained in complex with the closely related NMT from Leishmania major, providing a structural starting point for the evolution of novel chemical matter.
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May 2015
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8268]
Open Access
Abstract: Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.
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Feb 2014
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