VMXi-Versatile Macromolecular Crystallography in situ
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Elnaz
Aledavood
,
Sandra
Ramos-Inza
,
Jannis
Born
,
Megan
Lambert
,
Hannes
Berg
,
Jan Michael
Köhler
,
Anna
Niesteruk
,
Sridhar
Sreeramulu
,
Amy J.
Thompson
,
Halina
Mikolajek
,
James
Sandy
,
Juan
Sanchez-Weatherby
,
Siyi
Wang
,
Eleanor P.
Williams
,
Martin A.
Walsh
,
Michael A.
Hough
,
Harald
Schwalbe
,
Ana
Martínez
,
Carmen
Gil
Diamond Proposal Number(s):
[32304]
Open Access
Abstract: Fragment-based drug discovery (FBDD) is an effective approach for exploring chemical space using small, low-affinity fragments as starting points to facilitate development of lead compounds. Strategies to improve fragment potency include fragment merging and linking to generate higher-affinity inhibitors. Recently, artificial intelligence (AI) and machine learning (ML) have accelerated this process through structure-based optimization and generative compound design. Here, we present an AI-assisted FBDD workflow applied to the SARS-CoV-2 macrodomain (Mac1), a conserved viral protein involved in immune evasion and ADP-ribose metabolism. Using available structural data and previously identified fragments, we combined deep learning with molecular docking to design novel Mac1 binders. Selected compounds were synthesized and validated by NMR spectroscopy and X-ray crystallography, demonstrating improved binding relative to the original fragment hits with KD values in the range of 299-990 µM. This study demonstrates the advantages of integrating AI with FBDD to streamline molecular design, providing a data-driven framework for discovering new Mac1 inhibitors and guiding future antiviral drug development.
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Jul 2026
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Open Access
Abstract: Streptococcus pneumoniae relies on sialic acid uptake for nutrition and human respiratory tract colonisation. The ATP binding cassette (ABC) transporter SatABC-MsmK facilitates this, with SatA being the substrate-binding protein (SBP). We show that SatA specifically recognises the α-anomer of N-acetylneuraminic acid (α-Neu5Ac). Crystallographic analysis, mutagenesis and binding affinity measurements identify conserved residues Phe87, Arg113, Gln216, Arg404 as critical for α-Neu5Ac coordination. Nuclear magnetic resonance spectroscopy confirms selective binding of α-Neu5Ac in buffered solution, despite its low equilibrium abundance. Isothermal titration calorimetry shows high affinity of SatA for the anomeric mixture of Neu5Ac (Kd ≈ 270 nM). The α-anomer preference of SatA, not previously observed in other SBPs, may confer selective advantage to S. pneumoniae by enabling uptake of α-Neu5Ac, the immediate product of sialidase-mediated glycan cleavage.
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Jul 2026
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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.
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Jun 2026
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Xiaomin
Ni
,
R. Blake
Richardson
,
Andre
Schutzer Godoy
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Matteo P.
Ferla
,
Caroline
Kikawa
,
Jenke
Scheen
,
William W.
Hannon
,
Eda
Capkin
,
Noa
Lahav
,
Blake H.
Balcomb
,
Peter G.
Marples
,
Michael
Fairhead
,
Siyi
Wang
,
Eleanor P.
Williams
,
Charles W. E.
Tomlinson
,
Jasmin C.
Aschenbrenner
,
Ryan
Lithgo
,
Max
Winokan
,
Charline
Giroud
,
Isabela
Dolci
,
Rafaela Sachetto
Fernandes
,
Glaucius
Oliva
,
Anu V.
Chandran
,
Mary-Ann
Xavier
,
Martin A.
Walsh
,
Warren
Thompson
,
Jesse D.
Bloom
,
Nathaniel T.
Kenton
,
Alpha A.
Lee
,
Annette
Von Delft
,
Haim
Barr
,
Karla
Kirkegaard
,
Lizbe
Koekemoer
,
Daren
Fearon
,
Matthew J.
Evans
,
Frank
Von Delft
Diamond Proposal Number(s):
[32627]
Open Access
Abstract: The Zika viral protease NS2B-NS3 is essential for the cleavage of viral polyprotein precursor into individual structural and non-structural (NS) proteins and is therefore an attractive drug target. Generation of a robust crystal system of co-expressed NS2B-NS3 protease has enabled us to perform a crystallographic fragment screening campaign with 1076 fragments. 46 fragments with diverse scaffolds are identified to bind in the active site of the protease, with another 6 fragments observed in a potential allosteric site. To identify binding sites that are intolerant to mutation and thus suppress the outgrowth of viruses resistant to inhibitors developed from bound fragments, we perform deep mutational scanning of the NS2B-NS3 protease. Merging fragment hits yields an extensive set of ‘mergers’, defined as synthetically accessible compounds that recapitulate constellations of observed fragment-protein interactions. In addition, the highly sociable fragment hits enable rapid exploration of chemical space via algorithmic calculation and thus yield diverse possible starting points. In this work, we maximally explore the binding opportunities to NS2B-NS3 protease, facilitating its resistance-resilient antiviral development.
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Oct 2025
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Jul 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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Open Access
Abstract: The switch between planktonic and biofilm lifestyle correlates with intracellular concentration of the second messenger bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). While bacteria possess cyclase and phosphodiesterase enzymes to catalyse formation or hydrolysis of c-di-GMP, both enzymatic domains often occur in a single protein. It is tacitly assumed that one of the two enzymatic activities is dominant, and that additional domains and protein interactions enable responses to environmental conditions and control activity. Here we report the structure of the phosphodiesterase domain of the membrane protein RbdA (regulator of biofilm dispersal) in a dimeric, activated state and show that phosphodiesterase activity is controlled by the linked cyclase. The phosphodiesterase region around helices α5/α6 forms the dimer interface, providing a rationale for activation, as this region was seen in contact with the cyclase domain in an auto-inhibited structure previously described. Kinetic analysis supports this model, as the activity of the phosphodiesterase alone is lower when linked to the cyclase. Analysis of a computed model of the RbdA periplasmatic domain reveals an all-helical architecture with a large binding pocket that could accommodate putative ligands. Unravelling the regulatory circuits in multi-domain phosphodiesterases like RbdA is important to develop strategies to manipulate or disperse bacterial biofilms.
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Aug 2024
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Ana
Martínez Gascueña
,
Haiyang
Wu
,
Rui
Wang
,
C. David
Owen
,
Pedro J.
Hernando
,
Serena
Monaco
,
Matthew
Penner
,
Ke
Xing
,
Gwenaelle
Le Gall
,
Richard
Gardner
,
Didier
Ndeh
,
Paulina A.
Urbanowicz
,
Daniel I. R.
Spencer
,
Martin
Walsh
,
Jesus
Angulo
,
Nathalie
Juge
Open Access
Abstract: Microbial α-L-fucosidases catalyse the hydrolysis of terminal α-L-fucosidic linkages and can perform transglycosylation reactions. Based on sequence identity, α-L-fucosidases are classified in glycoside hydrolases (GHs) families of the carbohydrate-active enzyme database. Here we explored the sequence-function space of GH29 fucosidases. Based on sequence similarity network (SSN) analyses, 15 GH29 α-L-fucosidases were selected for functional characterisation. HPAEC-PAD and LC-FD-MS/MS analyses revealed substrate and linkage specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated oligosaccharides and glycoconjugates, consistent with their SSN clustering. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1,6 linkages and FA2G2 N-glycan was determined by X-ray crystallography and STD NMR. The capacity of GH29 fucosidases to carry out transfucosylation reactions with GlcNAc and 3FN as acceptors was evaluated by TLC combined with ESI–MS and NMR. These experimental data supported the use of SSN to further explore the GH29 sequence-function space through machine-learning models. Our lightweight protein language models could accurately allocate test sequences in their respective SSN clusters and assign 34,258 non-redundant GH29 sequences into SSN clusters. It is expected that the combination of these computational approaches will be used in the future for the identification of novel GHs with desired specificities.
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Jun 2024
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Gianluigi A.
Botton
,
Peter
Coll
,
Andrew J.
Dent
,
Sarnjeet
Dhesi
,
Sky
French
,
David R.
Hall
,
Joseph
Hriljac
,
Sarah
Macdonell
,
Sofia
Diaz-Moreno
,
Chris
Nicklin
,
Paul
Quinn
,
Robert
Rambo
,
Kawal
Sawhney
,
Richard P.
Walker
,
Martin A.
Walsh
,
Molly
Pekarik-Fry
Open Access
Abstract: A look at the Diamond-II upgrade programme
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Mar 2024
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I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
Lennart
Brewitz
,
Anthony
Tumber
,
Eidarus
Salah
,
Kang Ju
Lee
,
Naohiro
Terasaka
,
C. David
Owen
,
Claire
Strain-Damerell
,
Petra
Lukacik
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Takayuki
Katoh
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: Due to their constrained conformations, cyclic β2,3-amino acids (cβAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cβAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cβAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating three kinds of cβAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (β1), (1S,2S)-2-aminocyclohexane carboxylic acid (β2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one β2 and two β1, exhibited potent inhibitory activities with IC50 values of 40 nM and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 h and >168 h, respectively. Notably, BM3A and BM7A, wherein the cβAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties.
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Mar 2024
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