I04-1-Macromolecular Crystallography (fixed wavelength)
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Xiaomin
Ni
,
R. Blake
Richardson
,
Andre
Schutzer Godoy
,
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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Open Access
Abstract: The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1–3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2′ position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2′ contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.
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Oct 2023
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I03-Macromolecular Crystallography
I23-Long wavelength MX
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Kamel
El Omari
,
Ramona
Duman
,
Vitaliy
Mykhaylyk
,
Christian M.
Orr
,
Merlyn
Latimer-Smith
,
Graeme
Winter
,
Vinay
Grama
,
Feng
Qu
,
Kiran
Bountra
,
Hok Sau
Kwong
,
Maria
Romano
,
Rosana
Reis
,
Lutz
Vogeley
,
Luca
Vecchia
,
C. David
Owen
,
Sina
Wittmann
,
Max
Renner
,
Miki
Senda
,
Naohiro
Matsugaki
,
Yoshiaki
Kawano
,
Thomas A.
Bowden
,
Isabel
Moraes
,
Jonathan M.
Grimes
,
Erika J.
Mancini
,
Martin A.
Walsh
,
Cristiane R.
Guzzo
,
Raymond J.
Owens
,
E. Yvonne
Jones
,
David G.
Brown
,
Dave I.
Stuart
,
Konstantinos
Beis
,
Armin
Wagner
Open Access
Abstract: Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.
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Oct 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Vijil
Chenthamarakshan
,
Samuel C.
Hoffman
,
C. David
Owen
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Daren
Fearon
,
Tika R.
Malla
,
Anthony
Tumber
,
Christopher J.
Schofield
,
Helen M. E.
Duyvesteyn
,
Wanwisa
Dejnirattisai
,
Loic
Carrique
,
Thomas S.
Walter
,
Gavin R.
Screaton
,
Tetiana
Matviiuk
,
Aleksandra
Mojsilovic
,
Jason
Crain
,
Martin A.
Walsh
,
David I.
Stuart
,
Payel
Das
Diamond Proposal Number(s):
[27995]
Open Access
Abstract: Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions—unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.
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Jun 2023
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