I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Simon R.
Stockwell
,
Duncan E.
Scott
,
Gerhard
Fischer
,
Estrella
Guarino
,
Timothy P. C.
Rooney
,
Tzu-Shean
Feng
,
Tommaso
Moschetti
,
Rajavel
Srinivasan
,
Esther
Alza
,
Alice
Asteian
,
Claudio
Dagostin
,
Anna
Alcaide
,
Mathieu
Rocaboy
,
Beata
Blaszczyk
,
Alicia
Higueruelo
,
Xuelu
Wang
,
Maxim
Rossmann
,
Trevor R.
Perrior
,
Tom L.
Blundell
,
David R.
Spring
,
Grahame
Mckenzie
,
Chris
Abell
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Marko
Hyvonen
Diamond Proposal Number(s):
[9537, 14043]
Open Access
Abstract: Aurora A kinase, a cell division regulator, is frequently overexpressed in various cancers, provoking genome instability and resistance to antimitotic chemotherapy. Localization and enzymatic activity of Aurora A are regulated by its interaction with the spindle assembly factor TPX2. We have used fragment-based, structure-guided lead discovery to develop small molecule inhibitors of the Aurora A-TPX2 protein–protein interaction (PPI). Our lead compound, CAM2602, inhibits Aurora A:TPX2 interaction, binding Aurora A with 19 nM affinity. CAM2602 exhibits oral bioavailability, causes pharmacodynamic biomarker modulation, and arrests the growth of tumor xenografts. CAM2602 acts by a novel mechanism compared to ATP-competitive inhibitors and is highly specific to Aurora A over Aurora B. Consistent with our finding that Aurora A overexpression drives taxane resistance, these inhibitors synergize with paclitaxel to suppress the outgrowth of pancreatic cancer cells. Our results provide a blueprint for targeting the Aurora A-TPX2 PPI for cancer therapy and suggest a promising clinical utility for this mode of action.
|
Aug 2024
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18548, 25402]
Open Access
Abstract: Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment-based competitive 19F Ligand Based-NMR (LB-NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand-binding hotspots. As a proof of concept, the method was applied to 4’-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X-ray crystallographic characterisation of the hits from a 960-member fragment screen identified three ligand-binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19F LB-NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X-ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment-based competitive 19F LB-NMR screening platform for hotspot-directed ligand profiling.
|
Jun 2024
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Mona M.
Katariya
,
Matthew
Snee
,
Richard B.
Tunnicliffe
,
Madeline E.
Kavanagh
,
Helena I. M.
Boshoff
,
Cecilia N.
Amadi
,
Colin W.
Levy
,
Andrew W.
Munro
,
Chris
Abell
,
David
Leys
,
Anthony G.
Coyne
,
Kirsty J.
Mclean
Diamond Proposal Number(s):
[17773, 24447]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb) was responsible for approximately 1.6 million deaths in 2021. With the emergence of extensive drug resistance, novel therapeutic agents are urgently needed, and continued drug discovery efforts required. Host-derived lipids such as cholesterol support Mtb growth, and are also suspected to function in immunomodulation, with links to persistence and immune evasion. Mtb cytochrome P450 (CYP) enzymes facilitate key steps in lipid catabolism and thus present potential targets for inhibition. Here we present a series of compounds based on an ethyl 5-(pyridin-4-yl)-1H-indole-2-carboxylate pharmacophore which bind strongly to both Mtb cholesterol oxidases CYP125 and CYP142. Using a structure-guided approach, combined with biophysical characterization, compounds with micromolar range in-cell activity against clinically relevant drug-resistant isolates were obtained. These will support further development of much-needed additional treatment options and provide routes to probe the role of CYP125 and CYP142 in Mtb pathogenesis.
|
Mar 2023
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Sherine E.
Thomas
,
William J.
Mccarthy
,
Jamal
El Bakali
,
Karen P.
Brown
,
So Yeon
Kim
,
Michal
Blaszczyk
,
Vitor
Mendes
,
Chris
Abell
,
R. Andres
Floto
,
Anthony G.
Coyne
,
Tom L.
Blundell
Diamond Proposal Number(s):
[9537, 14043, 18548]
Open Access
Abstract: Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (Kd 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.
|
May 2022
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
|
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]
Open Access
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.
|
Jan 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Sitthivut
Charoensutthivarakul
,
Sherine E.
Thomas
,
Amy
Curran
,
Karen P.
Brown
,
Juan M.
Belardinelli
,
Andrew J.
Whitehouse
,
Marta
Acebron-Garcia-De-Eulate
,
Jaspar
Sangan
,
Subramanian G.
Gramani
,
Mary
Jackson
,
Vitor
Mendes
,
R. Andres
Floto
,
Tom L.
Blundell
,
Anthony G.
Coyne
,
Chris
Abell
Diamond Proposal Number(s):
[9537, 14043, 18548]
Abstract: Mycobacterium abscessus (Mab) has emerged as a challenging threat to individuals with cystic fibrosis. Infections caused by this pathogen are often impossible to treat due to the intrinsic antibiotic resistance leading to lung malfunction and eventually death. Therefore, there is an urgent need to develop new drugs against novel targets in Mab to overcome drug resistance and subsequent treatment failure. In this study, SAICAR synthetase (PurC) from Mab was identified as a promising target for novel antibiotics. An in-house fragment library screen and a high-throughput X-ray crystallographic screen of diverse fragment libraries were explored to provide crucial starting points for fragment elaboration. A series of compounds developed from fragment growing and merging strategies, guided by crystallographic information and careful hit-to-lead optimization, have achieved potent nanomolar binding affinity against the enzyme. Some compounds also show a promising inhibitory effect against Mab and Mtb. This work utilizes a fragment-based design and demonstrates for the first time the potential to develop inhibitors against PurC from Mab.
|
Jan 2022
|
|
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Marta
Acebron-Garcia-De-Eulate
,
Joan
Mayol-Llinàs
,
Matthew T. O.
Holland
,
So Yeon
Kim
,
Karen P.
Brown
,
Chiara
Marchetti
,
Jeannine
Hess
,
Ornella
Di Pietro
,
Vitor
Mendes
,
Chris
Abell
,
R. Andres
Floto
,
Anthony G.
Coyne
,
Tom L.
Blundell
Diamond Proposal Number(s):
[18548]
Abstract: Pseudomonas aeruginosa is of major concern for cystic fibrosis patients where this infection can be fatal. With the emergence of drug-resistant strains, there is an urgent need to develop novel antibiotics against P. aeruginosa. MurB is a promising target for novel antibiotic development as it is involved in the cell wall biosynthesis. MurB has been shown to be essential in P. aeruginosa, and importantly, no MurB homologue exists in eukaryotic cells. A fragment-based drug discovery approach was used to target Pa MurB. This led to the identification of a number of fragments, which were shown to bind to MurB. One fragment, a phenylpyrazole scaffold, was shown by ITC to bind with an affinity of Kd = 2.88 mM (LE 0.23). Using a structure guided approach, different substitutions were synthesized and the initial fragment was optimized to obtain a small molecule with Kd = 3.57 μM (LE 0.35).
|
Jan 2022
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Pooja
Gupta
,
Sherine E.
Thomas
,
Shaymaa A.
Zaidan
,
Maria A.
Pasillas
,
James
Cory-Wright
,
Víctor
Sebastián-Pérez
,
Ailidh
Burgess
,
Emma
Cattermole
,
Clio
Meghir
,
Chris
Abell
,
Anthony G.
Coyne
,
William R.
Jacobs
,
Tom L.
Blundell
,
Sangeeta
Tiwari
,
Vitor
Mendes
Diamond Proposal Number(s):
[14043, 18548]
Open Access
Abstract: The L-arginine biosynthesis pathway consists of eight enzymes that catalyse the conversion of L-glutamate to L-arginine. Arginine auxotrophs (argB/argF deletion mutants) of Mycobacterium tuberculosis are rapidly sterilised in mice, while inhibition of ArgJ with Pranlukast was found to clear chronic M. tuberculosis infection in a mouse model. Enzymes in the arginine biosynthetic pathway have therefore emerged as promising targets for anti-tuberculosis drug discovery. In this work, the ligandability of four enzymes of the pathway ArgB, ArgC, ArgD and ArgF is assessed using a fragment-based approach. We identify several hits against these enzymes validated with biochemical and biophysical assays, as well as X-ray crystallographic data, which in the case of ArgB were further confirmed to have on-target activity against M. tuberculosis. These results demonstrate the potential for more enzymes in this pathway to be targeted with dedicated drug discovery programmes.
|
Jun 2021
|
|
I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Duncan E.
Scott
,
Nicola J.
Francis-Newton
,
May E.
Marsh
,
Anthony G.
Coyne
,
Gerhard
Fischer
,
Tommaso
Moschetti
,
Andrew R.
Bayly
,
Timothy D.
Sharpe
,
Kalina T.
Haas
,
Lorraine
Barber
,
Chiara R.
Valenzano
,
Rajavel
Srinivasan
,
David J.
Huggins
,
Miyoung
Lee
,
Amy
Emery
,
Bryn
Hardwick
,
Matthias
Ehebauer
,
Claudio
Dagostin
,
Alessandro
Esposito
,
Luca
Pellegrini
,
Trevor
Perrior
,
Grahame
Mckenzie
,
Tom L.
Blundell
,
Marko
Hyvonen
,
John
Skidmore
,
Ashok R.
Venkitaraman
,
Chris
Abell
Diamond Proposal Number(s):
[315, 7141]
Open Access
Abstract: BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
|
Mar 2021
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
|
Vitor
Mendes
,
Simon R.
Green
,
Joanna C.
Evans
,
Jeannine
Hess
,
Michael
Blaszczyk
,
Christina
Spry
,
Owain
Bryant
,
James
Cory-Wright
,
Daniel S-H.
Chan
,
Pedro H. M.
Torres
,
Zhe
Wang
,
Navid
Nahiyaan
,
Sandra
O’neill
,
Sebastian
Damerow
,
John
Post
,
Tracy
Bayliss
,
Sasha L.
Lynch
,
Anthony G.
Coyne
,
Peter C.
Ray
,
Chris
Abell
,
Kyu Y.
Rhee
,
Helena I. M.
Boshoff
,
Clifton E.
Barry
,
Valerie
Mizrahi
,
Paul G.
Wyatt
,
Tom L.
Blundell
Diamond Proposal Number(s):
[9537, 14043, 18548]
Open Access
Abstract: Coenzyme A (CoA) is a fundamental co-factor for all life, involved in numerous metabolic pathways and cellular processes, and its biosynthetic pathway has raised substantial interest as a drug target against multiple pathogens including Mycobacterium tuberculosis. The biosynthesis of CoA is performed in five steps, with the second and third steps being catalysed in the vast majority of prokaryotes, including M. tuberculosis, by a single bifunctional protein, CoaBC. Depletion of CoaBC was found to be bactericidal in M. tuberculosis. Here we report the first structure of a full-length CoaBC, from the model organism Mycobacterium smegmatis, describe how it is organised as a dodecamer and regulated by CoA thioesters. A high-throughput biochemical screen focusing on CoaB identified two inhibitors with different chemical scaffolds. Hit expansion led to the discovery of potent and selective inhibitors of M. tuberculosis CoaB, which we show to bind to a cryptic allosteric site within CoaB.
|
Jan 2021
|
|
