I03-Macromolecular Crystallography
|
Barbara
Forte
,
Fiona
Bellany
,
Peter S.
Campbell
,
Giulia
Chemi
,
Alice
Dawson
,
Mark
Anderson
,
Yaw
Aniweh
,
Anna Y.
Burkhard
,
Anna Caroline Campos
Aguiar
,
Alisje
Churchyard
,
Caitlin A.
Cooper
,
Amália
Dos Santos Ferreira
,
Mufuliat Toyin
Famodimu
,
Francis G.
Fang
,
Xiao
Hu
,
Tonnie
Huijs
,
Delphine
Baud
,
Chimed
Jansen
,
María Belén
Jiménez Díaz
,
Roger
Bonnert
,
Susan
Boyd
,
Benigno
Crespo-Fernández
,
Branko
Mitasev
,
Simone
Montagna
,
Sachel
Mok
,
Dinakaran
Murugesan
,
Sunil K.
Narwal
,
Neil R.
Norcross
,
John
Okombo
,
Heekuk
Park
,
Caroline
Peet
,
Dhelio B.
Pereira
,
John M.
Post
,
Janette
Reader
,
Jennifer
Riley
,
David A.
Robinson
,
Raku
Shinkyo
,
Frederick R. C.
Simeons
,
Laura
Simpson
,
Alasdair
Smith
,
Dennis
Smith
,
Josefine
Striepen
,
Carolina B. G.
Teles
,
Rianne
Van Der Laak
,
Anne-Catrin
Uhlemann
,
Amélie
Vantaux
,
Caroline
Wilson
,
Benoît
Witkowski
,
Gavin
Wood
,
Tomas
Yeo
,
Fabio
Zuccotto
,
Iñigo
Angulo-Barturen
,
Jake
Baum
,
Judith M.
Bolscher
,
Rafael Victorio Carvalho
Guido
,
Lyn-Marié
Birkholtz
,
Michael J.
Delves
,
Laurent
Dembele
,
David A.
Fidock
,
Francisco Javier
Gamo
,
Dennis E.
Kyle
,
Steven P.
Maher
,
Jean
Popovici
,
Chris
Walpole
,
Fabian
Gusovsky
,
Paul A.
Willis
,
Kevin D.
Read
,
Ian H.
Gilbert
,
Beatriz
Baragaña
Diamond Proposal Number(s):
[10071, 26793]
Open Access
Abstract: A fused dihydropyrrolidino-pyrimidine hit with low lipophilicity and excellent ligand efficiency was identified in a biochemical screen of the Global Health Chemical Diversity Library (GHCDL) against Plasmodium lysyl-tRNA synthetase (KRS). Structure-guided lead optimization delivered analogues with potent parasite growth inhibition, excellent biochemical and cellular selectivity (>1000-fold), and oral efficacy in the malaria NOD-scid-IL2Rγnull (SCID) mouse model. Structural information and computational methods were deployed to identify a potent and selective basic KRS inhibitor (30) with an extended half-life to reduce the dose regimen to a single-dose cure. Compound 30 displayed a long half-life across preclinical species, favorable safety, and activity across Plasmodium species as well as against drug-resistant and sensitive P. falciparum strains and field isolates. Unfortunately, 30 lacked oral bioavailability, which could not be mitigated with a prodrug approach. Nevertheless, learnings from this series will assist future KRS programs in delivering a clinical candidate with this novel mode of action.
|
Jun 2026
|
|
Krios IV-Titan Krios IV at Diamond
|
Diamond Proposal Number(s):
[25452, 32707]
Open Access
Abstract: Antimicrobial resistance is driving the search for new antibiotics and a greater understanding of their mechanism of action. Doxycycline is amongst the most-prescribed antimicrobials. It demonstrates a particularly low minimum inhibitory concentration against the zoonotic pathogen Coxiella burnetii. Doxycycline canonically targets the bacterial ribosome by blocking tRNA binding at the decoding centre (A site) of the small subunit. Using cryo-electron microscopy, we analysed doxycycline binding to C. burnetii and Escherichia coli ribosomes. Both structures reveal doxycycline binding at the exit tunnel in the large subunit. In C. burnetii three doxycycline molecules stack to block the tunnel. In E. coli one doxycycline molecule triggers a major change in the conformation of the ribosome. This rearrangement of the peptidyl transferase centre blocks tRNA binding and nascent chain accommodation, abolishing interactions that are fundamental to ribosome function. We identify a distinct ribosomal protein in the C. burnetii large subunit and characterise an additional member of the prokaryotic ribosome hibernation-promoting factor family. These insights into ribosome function and antibiotic action may aid the development of new ribosome inhibitor antibiotics.
|
Jun 2026
|
|
|
|
Sakshi
Sharma
,
Peter A. C.
Wing
,
Wojtek
Trede
,
Shyam
Basak
,
Simeon D.
Draganov
,
Taylah
Andrews-Clark
,
Eidarus
Salah
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Adán
Pinto‐fernández
,
Martin A.
Walsh
,
Fernanda
Duarte
,
Christopher J.
Schofield
,
Lennart
Brewitz
Open Access
Abstract: The SARS-CoV-2 papain-like protease (PLpro) and the main protease (Mpro) catalyze hydrolysis of the viral polyproteins pp1a/1ab into functional nonstructural proteins. PLpro and Mpro are medicinal chemistry targets, with Mpro inhibitors being used for COVID-19 treatment. PLpro also catalyzes hydrolysis of ubiquitin and interferon-stimulated gene 15 (ISG15) from post-translationally modified human proteins. Here we report how screening of reported deubiquitinase inhibitors using solid-phase extraction coupled to mass spectrometry assays with oligopeptide substrates based on pp1a/1ab and on an ISG15-modified human protein enabled the identification of substrate-selective PLpro inhibitors. The results reveal that the deubiquitinase inhibitor ML364 selectively inhibits the deISGylase activity of isolated PLpro over its pp1a/1ab-processing activity. Structure-activity relationship and computational studies support the assignment of ML364 and derivatives as substrate-selective PLpro inhibitors. The combined results provide proof-of-concept for developing substrate-selective inhibitors of PLpro and, by implication, related proteolytic enzymes, including deubiquitinases.
|
Jun 2026
|
|
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Daming
Zhou
,
Abhay
Kotecha
,
James T.
Kelly
,
Peng-Nien
Huang
,
Yi-Yin
Chen
,
Thomas S.
Walter
,
Helen M. E.
Duyvesteyn
,
Raymond J.
Owens
,
Shu-Yuan
Ho
,
Tzou-Yien
Lin
,
Elizabeth E.
Fry
,
Jingshan
Ren
,
Kuan-Ying A.
Huang
,
David I.
Stuart
Diamond Proposal Number(s):
[14744]
Open Access
Abstract: EV-A71 has been responsible for recent severe HFMD outbreaks. We report structures for 12 potently neutralizing human anti–EV-A71 monoclonal antibody Fabs, alone and complexed with virus. Most recognize the native antigenic state with epitopes that span interfaces, together covering 85% of the capsid surface. The majority (8 of 12) bind the canyon, while the others cluster around the icosahedral two- and threefold axes. Blocking SCARB2 receptor binding likely contributes to neutralization for all, and a subset induces empty particles. A predominant gene family (IGHV4-39) does not dictate a common binding pose. Long CDR-H3 loops are frequently key to binding, especially at the canyon, suggesting that antigenicity data based on antibodies with shorter CDR3s (e.g., murine) may be misleading. This dataset reveals neutralization mechanisms for recently circulating EV-A71 genotypes, which will inform immunotherapies. We demonstrate synergy in vitro between canyon binding and both two- and threefold binding antibodies to increase neutralization potency.
|
Jun 2026
|
|
I03-Macromolecular Crystallography
|
Abstract: The widespread emergence of bacterial resistance to β-lactam antibiotics, driven primarily by serine β-lactamases (SBLs), continues to threaten the clinical utility of this cornerstone class of antibacterial agents. Although β-lactamase inhibitors such as sulbactam and tazobactam have historically restored the activity of partner antibiotics, their effectiveness has been eroded by the proliferation of inhibitor-resistant enzymes, particularly class C (AmpC) and class D (OXA) β-lactamases. Consequently, there remains a pressing need for new inhibitor scaffolds that combine potent enzyme inhibition with improved chemical stability and tunable physicochemical properties.
This thesis explores sulfoximine substitution on the penam scaffold as a strategy to expand the chemical and biological properties of β-lactamase inhibitors. Sulfoximines offer distinct advantages over classical sulfones, including stereochemical control, dual hydrogen-bond donor/acceptor capability, and enhanced opportunities for structural diversification. Leveraging recent advances in nitrene-transfer chemistry, robust and stereocontrolled synthetic routes to penam-sulfoximines were developed from sulbactam-derived sulfoxides. Both (S)- and (R)-configured penam sulfoximines were prepared, including synthetically challenging NH-sulfoximines that closely mimic the parent sulbactam framework. Single-crystal X-ray diffraction confirmed stereochemical integrity and structural assignments.
The scope and limitations of sulfoximine N-functionalization were systematically investigated, enabling access to amide, urea, and N-aryl derivatives while revealing intrinsic stability constraints of the penam core under basic or reductive conditions. These studies establish a versatile synthetic platform for the generation of structurally diverse penam-sulfoximines suitable for biological evaluation.
Biochemical and microbiological studies demonstrated that penam-sulfoximines are potent inhibitors of clinically relevant SBLs, including AmpC and OXA-type enzymes. A free NH penam-sulfoximine displayed particularly strong enzyme inhibition and measurable antibacterial activity against Acinetobacter baumannii, supported by enzyme inhibition assays, protein-binding studies, antimicrobial susceptibility testing, and structural characterization of enzyme–inhibitor complexes. However, many substituted sulfoximines exhibited reduced stability under physiological conditions, highlighting a trade-off between potency and chemical robustness.
Overall, this work establishes penam-sulfoximines as a chemically accessible and biologically active class of β-lactamase inhibitors, delineates key structure–stability–activity relationships, and provides a foundation for the future design of sulfoximine-based agents targeting resistant Gram-negative pathogens.
|
Jun 2026
|
|
I03-Macromolecular Crystallography
|
Huazhang
Shu
,
Julian M.
Ludäscher
,
Sushma
Sharma
,
Seher
Alam
,
Lilian
Frank
,
Emma
Scaletti Hutchinson
,
Marianna
Tampere
,
Chloé
Lévêque
,
André B. P.
Van Kuilenburg
,
Nicholas C. K.
Valerie
,
Mikael
Altun
,
Andrei
Chabes
,
Pal
Stenmark
,
Sean G.
Rudd
,
Si Min
Zhang
Diamond Proposal Number(s):
[36026]
Open Access
Abstract: Molnupiravir is a nucleoside analogue antiviral drug against RNA viruses, including its clinical indication SARS-CoV-2. Whilst its mechanism-of-action is well defined, host factors that regulate its therapeutic responses have not been thoroughly deciphered and characterized. Here we show that uridine cytidine kinases (UCKs), key enzymes in pyrimidine salvage, effectively phosphorylate and thereby bioactivate N4-hydroxycytidine (NHC) – the active compound of molnupiravir, thus dictating its anti-SARS-CoV-2 efficacy and furthermore selectivity. In vitro, both isoforms of UCKs (UCK1 and UCK2) effectively phosphorylated NHC, where the structural basis of the catalysis was further deciphered via the first complete substrate bound co-crystal structure of UCK, i.e., UCK1-NHC-AMPPNP. In SARS-CoV-2-infected cells, UCK2 knockdown via siRNA hampered the intracellular accumulation of the tri-phosphorylated antiviral metabolite of NHC, resulting in a 10-fold reduction of the antiviral efficacy, and surprisingly, 2-fold reduction of its selectivity, which were critically recapitulated in a dose-dependent manner using a pan-UCK inhibitor. Altogether, this work underscores UCKs as pivotal players in upholding molnupiravir efficacy and therapeutic window of molnupiravir, and furthermore as pharmacologically tractable targets for tailoring the drug response.
|
May 2026
|
|
I03-Macromolecular Crystallography
|
Sarah
Hijazi
,
Francesco
Marchesani
,
Marialaura
Marchetti
,
Valeria
Buoli Comani
,
Paul
Brear
,
Barbara
Campanini
,
Luca
Ronda
,
Serena
Faggiano
,
Eleonora
Gianquinto
,
Somayeh
Asgharpour Hassankiade
,
Barbara
Rolando
,
Francesca
Spyrakis
,
Carlotta
Compari
,
Loretta
Lazzarato
,
Omar
De Bei
,
Emanuela
Frangipani
,
Stefano
Bettati
Diamond Proposal Number(s):
[25402]
Open Access
Abstract: Infections caused by Staphylococcus aureus depend on its ability to access essential nutrients, including acquiring iron from human hemoglobin (Hb) through the iron-regulated surface determinant (Isd) system. The compound 4-[(2-{[5-(1H-indol-3-yl)-1,3,4-oxadiazol-2-yl]sulfanyl}acetyl)amino]benzoic acid (C35) was recently identified as a promising antimicrobial agent for its ability to bind Hb and hamper its interaction with the staphylococcal hemophore IsdB in vitro. Here, we show that C35 inhibits S. aureus growth by targeting the hemophore-driven iron-acquisition system, highlighting its potential as an inhibitor and validating hemophores as antibacterial targets. Furthermore, for drug design purposes, we solved the X-ray structure of Hb:C35 complex. In contrast to the predicted binding pose, C35 binds tetrameric Hb in a cleft between the α subunits, stabilizing a relaxed conformation (R2) and increasing Hb oxygen affinity. This serendipitous result hints to C35 as a promising scaffold for developing compounds with diverse, or even dual, therapeutic aims, with antimicrobial and Hb-modulating activity.
|
May 2026
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[39641]
Open Access
Abstract: Nicotinamidases (PncA) catalyze the hydrolysis of nicotinamide to nicotinic acid, a key step in NAD+ salvage pathways. In the Lyme disease spirochete Borrelia burgdorferi, the plasmid-encoded gene bbe22 encodes a PncA enzyme that is essential for survival in both mammalian and tick hosts. Previous genetic and biochemical studies demonstrated that translation of B. burgdorferi PncA initiates from a rare non-canonical AUU start codon, resulting in a protein that is 24 residues longer than the sequence currently annotated in major databases. Despite these findings, public resources such as UniProt and KEGG still list a truncated protein beginning at residue 36, which lacks part of the N-terminal region required for enzymatic activity. Here we report the crystal structure of full-length B. burgdorferi PncA determined at 3.2 Å resolution. The structure reveals the canonical fold of bacterial nicotinamidases and clear electron density for a ligand in the active site consistent with nicotinic acid, the product of the enzymatic reaction. Structural comparison with homologous PncA enzymes demonstrates conservation of the catalytic architecture, including residues involved in substrate binding and catalysis. Importantly, the experimentally determined structure confirms that the longer N-terminal sequence described previously is required for formation of the correct fold and active-site geometry, whereas the truncated annotation is structurally inconsistent with the observed fold and with AlphaFold predictions. Our results provide the first structural characterization of B. burgdorferi PncA and resolve the long-standing annotation discrepancy for bbe22, validating the correct protein sequence and providing the structural basis for nicotinamidase activity in this essential metabolic enzyme.
|
May 2026
|
|
B21-High Throughput SAXS
Krios III-Titan Krios III at Diamond
|
Abstract: In conclusion, this work provides a comprehensive and detailed view of the
functioning of Tse5, establishing it as a unique paradigm within the Rhs toxins of the T6SS
system. Tse5 integrates a highly specialized delivery machinery, capable of recognizing
and anchoring to the target cell membrane, with a potent effector domain that
executes cell death through the formation of depolarizing pores. These findings not only
significantly expand our understanding of the mechanistic diversity of bacterial
weapons used in intercellular competition, but they also offer a solid molecular basis
for the future development of new biotechnological and antimicrobial strategies inspired
by sophisticated bacterial secretion systems.
|
May 2026
|
|
|
|
Open Access
Abstract: Angiotensin-converting enzyme 2 (ACE2) is a key node in the protective axis of the renin-angiotensin-aldosterone system (RAAS) for blood pressure and hydroelectrolyte regulation and the receptor recognized by the spike glycoproteins of the severe acute respiratory syndrome (SARS) coronaviruses (CoV) SARS-CoV and SARS-CoV-2. We identified the macrocyclic peptide WJL-63 by mRNA display and characterized it biochemically and with respect to ACE2-binding. The crystal structure of the extracellular region of ACE2 in complex with the peptide at 2.2 Å resolution was elucidated. The structure revealed a binding mode in which WJL-63 is accommodated towards one side of the catalytic cleft of the peptidase domain, without contacting the conserved zinc ion site. WJL-63 residues Q4, R7, R11, and R14 anchor the peptide in the binding pocket. The peptide contacts both peptidase subdomains. The upright binding mode requires an open ACE2 conformation, in contrast to small-molecule carboxypeptidase inhibitors, which typically bind to the closed conformation. One side of WJL-63 is accessible for modification such as the herein reported conjugation of a chelator for radiometal labeling. The radiolabeled DOTA-WJL-63 was evaluated on ACE2-transfected HEK cells, where it exhibited binding with a KD value of 90 ± 28 nM. The ACE2 − WJL-63 complex structure provides a basis for the development of compounds that modulate ACE2 conformation and for the development of imaging agents for visualization of ACE2, including fluorescence or electron microscopy and positron emission tomography (PET).
|
Apr 2026
|
|
