I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Madeline E.
Kavanagh
,
Kirsty J.
Mclean
,
Sophie H.
Gilbert
,
Cecilia N.
Amadi
,
Matthew
Snee
,
Richard B.
Tunnicliffe
,
Kriti
Arora
,
Helena I. M.
Boshoff
,
Alexander
Fanourakis
,
Maria Jose
Rebollo-Lopez
,
Fatima
Ortega
,
Colin W.
Levy
,
Andrew W.
Munro
,
David
Leys
,
Chris
Abell
,
Anthony G.
Coyne
Diamond Proposal Number(s):
[8997, 17773, 24447]
Open Access
Abstract: Tuberculosis is the deadliest infectious disease in history and new drugs are urgently required to combat multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (Mtb). Here, we exploit the relience of Mtb on host-derived cholesterol to develop a novel class of antitubercular compounds that target Mtb CYP125 and CYP142; the enzymes that catalyze the first step of cholesterol metabolism. A combination of fragment screening and structure-based drug design was used to identify a hit compound and guide synthetic optimization of a dual CYP125/142 ligand 5m (KD 40–160 nM), which potently inhibits enzyme activity in vitro (KI < 100 nM), and the growth of Mtb in extracellular (MIC99 0.4–1.5 μM) and intracellular assays (IC50 1.7 μM). The structural data and lead compounds reported here will help study Mtb cholesterol metabolism and guide the development of novel antibiotics to combat MDR Mtb.
|
Jul 2025
|
|
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
|
|
B23-Circular Dichroism
|
Cédric
Couturier
,
Quentin
Ronzon
,
Giulia
Lattanzi
,
Iain
Lingard
,
Sebastien
Coyne
,
Veronique
Cazals
,
Nelly
Dubarry
,
Stephane
Yvon
,
Corinne
Leroi-Geissler
,
Obdulia Rabal
Gracia
,
Joanne
Teague
,
Sylvie
Sordello
,
David
Corbett
,
Caroline
Bauch
,
Chantal
Monlong
,
Lloyd
Payne
,
Thomas
Taillier
,
Hazel
Fuchs
,
Mark
Broenstrup
,
Peter H.
Harrison
,
Lucile
Moynie
,
Abirami
Lakshminarayanan
,
Tiberiu-Marius
Gianga
,
Rohanah
Hussain
,
James H.
Naismith
,
Michael
Mourez
,
Eric
Bacqué
,
Fredrik
Björkling
,
Jean-Francois
Sabuco
,
Henrik
Franzyk
Diamond Proposal Number(s):
[26447]
Abstract: Tridecaptins comprise a class of linear cationic lipopeptides with an N-terminal fatty acyl moiety. These 13-mer antimicrobial peptides consist of a combination of d- and l-amino acids, conferring increased proteolytic stability. Intriguingly, they are biosynthesized by non-ribosomal peptide synthetases in the same bacterial species that also produce the cyclic polymyxins displaying similar fatty acid tails. Previously, the des-acyl analog of TriA1 (termed H-TriA1) was found to possess very weak antibacterial activity, albeit it potentiated the effect of several antibiotics. In the present study, two series of des-acyl tridecaptins were explored with the aim of improving the direct antibacterial effect. At the same time, overall physico-chemical properties were modulated by amino acid substitution(s) to diminish the risk of undesired levels of hemolysis and to avoid an impairment of mammalian cell viability, since these properties are typically associated with highly hydrophobic cationic peptides. Microbiology and biophysics tools were used to determine bacterial uptake, while circular dichroism and isothermal calorimetry were used to probe the mode of action. Several analogs had improved antibacterial activity (as compared to that of H-TriA1) against Enterobacteriaceae. Optimization enabled identification of the lead compound 29 that showed a good ADMET profile as well as in vivo efficacy in a variety of mouse models of infection.
|
Feb 2024
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Dihydrofolate reductase (DHFR) is a key enzyme involved in the folate pathway that has been heavily targeted for the development of therapeutics against cancer and bacterial and protozoa infections amongst others. Despite being an essential enzyme for Mycobacterium tuberculosis (Mtb) viability, DHFR remains an underexploited target for tuberculosis (TB) treatment. Herein, we report the preparation and evaluation of a series of compounds against Mtb DHFR (MtbDHFR). The compounds have been designed using a merging strategy of traditional pyrimidine-based antifolates with a previously discovered unique fragment hit against MtbDHFR. In this series, four compounds displayed a high affinity against MtbDHFR, with sub-micromolar affinities. Additionally, we determined the binding mode of six of the best compounds using protein crystallography, which revealed occupation of an underutilised region of the active site.
|
May 2023
|
|
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
|
|
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
|
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
|
|
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
|
|
