I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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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.
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Jul 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31440]
Open Access
Abstract: Antimicrobial resistance has emerged as a critical global public health threat, impacting human, animal and environmental health. An important mechanism of resistance is the production of β-lactamases, enzymes that hydrolyze the β-lactam ring, rendering β-lactam antibiotics ineffective. Metallo-β-lactamases (MBLs), which contain zinc ions in their active sites, are particularly challenging to counter as there are currently no inhibitors targeting these enzymes available on the market. Therefore, there is an urgent need for innovative drug discovery strategies to develop MBL-targeted therapies. New Delhi Metallo-β-Lactamase 1 (NDM-1) is the most widely disseminated MBL, with a global distribution in Enterobacterales. In this study, we used our library of fragment-sized chloroacetamides as a starting point to synthesize mercaptoacetamides as potential NDM-1 inhibitors. This resulted in a compound (14a) with an IC50 of 20 μM, which crystallography shows binds to NDM-1 in two different poses. Using this structure as a starting point for in silico design, we developed a series of larger thiol-based compounds designed to occupy more space in the active site and to utilize other novel zinc-binding groups. Although some showed minimal inhibition (which makes them valuable as decoys for metalloenzyme studies) one compound exhibited an IC50 of 14 μM, with crystallography indicating that an additional aromatic group, compared to 14a, interacts with hydrophobic residues on an NDM-1 active site loop. These data identify promising scaffolds for the further development of potent MBL inhibitors and show the utility of repurposing chemical libraries to target clinically important enzymes.
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Jun 2025
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I03-Macromolecular Crystallography
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Anthony K.
Edmonds
,
Dimitrios-Ilias
Balourdas
,
Graham P.
Marsh
,
Robert
Felix
,
Bradley
Brasher
,
Jeff
Cooper
,
Cari
Graber-Feesl
,
Madhu
Kollareddy
,
Karim
Malik
,
Helen
Stewart
,
Timothy J. T.
Chevassut
,
Ella
Lineham
,
Simon
Morley
,
Oleg
Fedorov
,
James
Bennett
,
Mohan B.
Rajasekaran
,
Samuel
Ojeda
,
Drew A.
Harrison
,
Christopher J.
Ott
,
Andreas C.
Joerger
,
Hannah J.
Maple
,
John
Spencer
Open Access
Abstract: Degraders with dual activity against BRD4 and CBP/EP300 were designed. A structure-guided design approach was taken to assess and test potential exit vectors on the dual BRD4 and CBP/EP300 inhibitor, ISOX-DUAL. Candidate degrader panels revealed that VHL-recruiting moieties could mediate dose-responsive ubiquitination of BRD4. A panel of CRBN-recruiting thalidomide-based degraders was unable to induce ubiquitination or degradation of target proteins. High-resolution protein cocrystal structures revealed an unexpected interaction between the thalidomide moiety and Trp81 on the first bromodomain of BRD4. The inability to form a ternary complex provides a potential rationale for the lack of degrader activity with these compounds, some of which have remarkable affinities close to those of (+)-JQ1, as low as 65 nM in a biochemical assay, vs 1.5 μM for their POI ligand, ISOX-DUAL. Such a “degrader collapse” may represent an under-reported mechanism by which some putative degrader molecules are inactive with respect to target protein degradation.
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Apr 2025
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I03-Macromolecular Crystallography
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Dóra
Laczi
,
Sofia Schönbauer
Huamán
,
Taylah
Andrews-Clark
,
Stephen M.
Laidlaw
,
Eidarus
Salah
,
Leo
Dumjahn
,
Petra
Lukacik
,
Hani
Choudhry
,
Martin A.
Walsh
,
Miles W.
Carroll
,
Christopher J.
Schofield
,
Lennart
Brewitz
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Nirmatrelvir is a substrate-related inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) that is clinically used in combination with ritonavir to treat COVID-19. Derivatives of nirmatrelvir, modified at the substrate P2-equivalent position, have been developed to fine-tune inhibitor properties and are now in clinical use. We report the synthesis of nirmatrelvir derivatives with a (R)-4,4-dimethyl-4-silaproline (silaproline) group at the P2-equivalent position. Mass spectrometry (MS)-based assays demonstrate that silaproline-bearing nirmatrelvir derivatives efficiently inhibit isolated recombinant Mpro, albeit with reduced potency compared to nirmatrelvir. Investigations with SARS-CoV-2 infected VeroE6 cells reveal that the silaproline-bearing inhibitors with a CF3 group at the P4-equivalent position inhibit viral progression, implying that incorporating silicon atoms into Mpro inhibitors can yield in vivo active inhibitors with appropriate optimization. MS and crystallographic studies show that the nucleophilic active site cysteine residue of Mpro (Cys145) reacts with the nitrile group of the silaproline-bearing inhibitors. Substituting the electrophilic nitrile group for a non-activated terminal alkyne shifts the inhibition mode from reversible covalent inhibition to irreversible covalent inhibition. One of the two prochiral silaproline methyl groups occupies space in the S2 pocket that is unoccupied in Mpro:nirmatrelvir complex structures, highlighting the value of sila-derivatives in structure-activity-relationship (SAR) studies. The combined results highlight the potential of silicon-containing molecules for inhibition of Mpro and, by implication, other nucleophilic cysteine enzymes.
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Apr 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[17212, 23269]
Open Access
Abstract: Dual angiotensin-converting enzyme (ACE) and neprilysin (NEP) inhibitors such as omapatrilat showed promise as potent treatments for hypertension but produced adverse effects due to their high affinity for both domains of ACE (nACE and cACE). This led to the search for compounds that retained NEP potency but selectively inhibit cACE, leaving nACE active to degrade other peptides such as bradykinin. Lisinopril-tryptophan (LisW) has previously been reported to have cACE selectivity. Three mercapto-3-phenylpropanoyl inhibitors were synthesized, combining features of omapatrilat and LisW to probe structural characteristics required for potent dual cACE/NEP inhibition. We report the synthesis of these inhibitors, enzyme inhibition data, and high-resolution crystal structures in complex with nACE and cACE. This provides valuable insight into factors driving potency and selectivity and shows that the mercapto-3-phenylpropanoyl backbone is significantly better for NEP potency than a P1 carboxylate. Future chemistry efforts could be directed at identifying alternative chemotypes for optimization of cACE/NEP inhibitors.
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Apr 2025
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[21773]
Open Access
Abstract: There is an urgent need for new antibiotics. FabF (3-oxoacyl-[acyl-carrier-protein] synthase 2), which catalyses the rate limiting condensation reaction in the fatty acid synthesis II pathway, is an attractive target. Very few inhibitors of FabF are known and most are derived from natural products. In an effort to further explore the chemical space of FabF ligands, we have carried out fragment screening by X-ray crystallography against an intermediated state-mimicking variant of P. aeruginosa FabF (PaFabF C164Q). This screen has resulted in 48 hits out of which 16 bind in or close to the malonyl-CoA or fatty acid binding site or an adjacent dimer interface. None of the closer investigated fragments were active in a binding assay, but the same was the case for fragments derived from a potent FabF inhibitor. For hit optimization, we focused on the two fragments binding close to the catalytic residues of FabF. Different strategies were followed in the optimization process: exploration of commercially available analogues, fragment merging, virtual screening of a combinatorial make-on-demand space, and design and in-house synthesis of analogues. In total, more than 90 analogues of the hit compounds were explored, and for 10 of those co-crystal structures could be determined. The most potent ligand was discovered using manual structure-based design and has a binding affinity of 65 μM. This data package forms a strong foundation for the development of more potent and diverse FabF inhibitors.
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Apr 2025
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[34182]
Open Access
Abstract: Human diphtheria toxin-like ADP-ribosyltransferases, PARPs and tankyrases, transfer ADP-ribosyl groups to other macromolecules, thereby controlling various signaling events in cells. They are considered promising drug targets, especially in oncology, and a vast number of inhibitors have already been successfully developed. These inhibitors typically occupy the nicotinamide binding site and extend along the NAD+ binding groove of the catalytic domain. Quinazolin-4-ones have been explored as compelling scaffolds for such inhibitors and we have identified a new position within the catalytic domain that has not been extensively studied yet. In this study, we investigate larger substituents at the C-8 position and, using X-ray crystallography, we demonstrate that nitro- and diol-substituents engage in new interactions with TNKS2, improving both affinity and selectivity. Both diol- and nitro-substituents exhibit intriguing inhibition of TNKS2, with the diol-based compound EXQ-1e displaying a pIC50 of 7.19, while the nitro-based compound EXQ-2d's pIC50 value is 7.86. Both analogues impact and attenuate the tankyrase-controlled WNT/β-catenin signaling with sub-micromolar IC50. When tested against a wider panel of enzymes, the nitro-based compound EXQ-2d displayed high selectivity towards tankyrases, whereas the diol-based compound EXQ-1e also inhibited other PARPs. Compound EXQ-2d displays in vitro cell growth inhibition of the colon cancer cell line COLO 320DM, while compound EXQ-1e displays nonspecific cell toxicity. Collectively, the results offer new insights for inhibitor development targeting tankyrases and PARPs by focusing on the subsite between a mobile active site loop and the canonical nicotinamide binding site.
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Apr 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[31353]
Open Access
Abstract: The 2-oxoglutarate (2OG)/Fe(II)-dependent γ-butyrobetaine hydroxylase (BBOX) catalyzes the final step in l-carnitine biosynthesis, i.e., stereoselective C-3 oxidation of γ-butyrobetaine (GBB). BBOX inhibition is a validated clinical strategy to modulate l-carnitine levels and to enhance cardiovascular efficiency. Reported BBOX inhibitors, including the clinically used cardioprotective agent Mildronate, manifest moderate inhibitory activity in vitro, limited selectivity, and/or unfavorable physicochemical properties, indicating a need for improved BBOX inhibitors. We report that the clinically used hypoxia-inducible factor-α prolyl residue hydroxylase (PHD) inhibitors Desidustat, Enarodustat, and Vadadustat efficiently inhibit isolated recombinant BBOX, suggesting that BBOX inhibition by clinically used PHD inhibitors should be considered as a possible off-target effect. Structure–activity relationship studies on the Desidustat scaffold enabled development of potent BBOX inhibitors that manifest high levels of selectivity for BBOX inhibition over representative human 2OG oxygenases, including PHD2. The Desidustat derivatives will help to enable investigations into the biological roles of l-carnitine and the therapeutic potential of BBOX inhibition.
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Apr 2025
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I03-Macromolecular Crystallography
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Kevin M.
Cottrell
,
Kimberly J.
Briggs
,
Alice
Tsai
,
Matthew R.
Tonini
,
Douglas A.
Whittington
,
Shanzhong
Gong
,
Colin
Liang
,
Patrick
Mccarren
,
Minjie
Zhang
,
Wenhai
Zhang
,
Alan
Huang
,
John P.
Maxwell
Open Access
Abstract: The gene encoding for MTAP is one of the most commonly deleted genes in cancer, occurring in approximately 10–15% of all human cancer. We have previously described the discovery of TNG908, a brain-penetrant clinical-stage compound that selectively targets MTAP-deleted cancer cells by binding to and inhibiting PRMT5 cooperatively with MTA, which is present in elevated concentrations in MTAP-deleted cells. Herein we describe the discovery of TNG462, a more potent and selective MTA-cooperative PRMT5 inhibitor with improved DMPK properties that is selective for MTAP-deleted cancers and is currently in Phase I/II clinical trials.
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Mar 2025
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Charles W.
Parry
,
Francesca
Pellicano
,
Alexander W.
Schuettelkopf
,
Kim S.
Beyer
,
Justin
Bower
,
Amy
Bryson
,
Kenneth
Cameron
,
Nichole M.
Cerutti
,
Jonathan P.
Clark
,
Stuart C.
Davidson
,
Keneth
Davies
,
Martin J.
Drysdale
,
Jeffrey
Engelman
,
Anna
Estevan-Barber
,
Andrea
Gohlke
,
Christopher H.
Gray
,
Daniel A.
Guthy
,
Min
Hong
,
Alana
Hopkins
,
Luke D.
Hutchinson
,
Jennifer
Konczal
,
Michel
Maira
,
Duncan
Mcarthur
,
Mokdad
Mezna
,
Heather
Mckinnon
,
Ridvan
Nepravishta
,
Nils
Ostermann
,
Camila C.
Pasquali
,
Katie
Pollock
,
Angelo
Pugliese
,
Nicholas
Rooney
,
Niko
Schmiedeberg
,
Paul
Shaw
,
Camilo
Velez-Vega
,
Christopher
West
,
Ryan
West
,
Frederic
Zecri
,
John B.
Taylor
Diamond Proposal Number(s):
[8659, 11651, 16258, 26339]
Open Access
Abstract: Activating mutations of Ras are one of the most prevalent drivers of cancer and are often associated with poor clinical outcomes. Despite FDA approval for two irreversible inhibitors that target the inactive state of KRasG12C, significant unmet clinical need still exists, and the susceptibility of non-G12C mutants to inactive-state inhibition remains unclear. Here we report the discovery of a novel series of reversible inhibitors that bind in an enlarged version of the switch I–II pocket with nanomolar affinities. Dependent on chemotype these can either preferentially bind to the inactive or active state or bind both with similar affinity. The active-state binders inhibit the Raf interaction for wild-type Ras, and a broad range of oncogenic KRas mutants with nanomolar potency. A subseries of these molecules displays cellular inhibition of Ras–Raf binding, as well as decreased phosphorylation of the downstream protein ERK, demonstrating that potent multivariant Ras inhibitors can be accessed from this novel pocket.
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Mar 2025
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