B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Open Access
Abstract: Monkeypox virus (MPXV) is a double-stranded DNA virus from the family Poxviridae, which is endemic in West and Central Africa. Various human outbreaks occurred in the 1980s, resulting from a cessation of smallpox vaccination. Recently, MPXV cases have reemerged in non-endemic nations, and the 2022 outbreak has been declared a public health emergency. Treatment optionsare limited, and many countries lack the infrastructure to provide symptomatic treatments. The development of cost-effective antivirals could ease severe health outcomes. G-quadruplexes have been a target of interest in treating viral infections with different chemicals. In the present work, a genomic-scale mapping of different MPXV isolates highlighted two conserved putative quadruplex-forming sequences MPXV-exclusive in 590 isolates. Subsequently, we assessed the G-quadruplex formation using circular dichroism spectroscopy and solution small-angle X-ray scattering. Furthermore, biochemical assays indicated the ability of MPXV quadruplexes to be recognized by two specific G4-binding partners—Thioflavin T and DHX36. Additionally, our work also suggests that a quadruplex binding small-molecule with previously reported antiviral activity, TMPyP4, interacts with MPXV G-quadruplexes with nanomolar affinity in the presence and absence of DHX36. Finally, cell biology experiments suggests that TMPyP4 treatment substantially reduced gene expression of MPXV proteins. In summary, our work provides insights into the G-quadruplexes from the MPXV genome that can be further exploited to develop therapeutics.
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May 2023
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I03-Macromolecular Crystallography
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Takashi
Miura
,
Tika R.
Malla
,
C. David
Owen
,
Anthony
Tumber
,
Lennart
Brewitz
,
Michael A.
Mcdonough
,
Eidarus
Salah
,
Naohiro
Terasaka
,
Takayuki
Katoh
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
Halina
Mikolajek
,
Martin A.
Walsh
,
Akane
Kawamura
,
Christopher J.
Schofield
,
Hiroaki
Suga
Diamond Proposal Number(s):
[27088]
Open Access
Abstract: γ-Amino acids can play important roles in the biological activities of natural products; however, the ribosomal incorporation of γ-amino acids into peptides is challenging. Here we report how a selection campaign employing a non-canonical peptide library containing cyclic γ2,4-amino acids resulted in the discovery of very potent inhibitors of the SARS-CoV-2 main protease (Mpro). Two kinds of cyclic γ2,4-amino acids, cis-3-aminocyclobutane carboxylic acid (γ1) and (1R,3S)-3-aminocyclopentane carboxylic acid (γ2), were ribosomally introduced into a library of thioether-macrocyclic peptides. One resultant potent Mpro inhibitor (half-maximal inhibitory concentration = 50 nM), GM4, comprising 13 residues with γ1 at the fourth position, manifests a 5.2 nM dissociation constant. An Mpro:GM4 complex crystal structure reveals the intact inhibitor spans the substrate binding cleft. The γ1 interacts with the S1′ catalytic subsite and contributes to a 12-fold increase in proteolytic stability compared to its alanine-substituted variant. Knowledge of interactions between GM4 and Mpro enabled production of a variant with a 5-fold increase in potency.
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May 2023
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B21-High Throughput SAXS
B23-Circular Dichroism
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Philip
Bardelang
,
Ewan J.
Murray
,
Isobel
Blower
,
Sara
Zandomeneghi
,
Alice
Goode
,
Rohanah
Hussain
,
Divya
Kumari
,
Giuliano
Siligardi
,
Katsuaki
Inoue
,
Jeni
Luckett
,
James
Doutch
,
Jonas
Emsley
,
Weng C.
Chan
,
Philip
Hill
,
Paul
Williams
,
Boyan B.
Bonev
Diamond Proposal Number(s):
[5098, 12923, 13185, 13634, 15146]
Open Access
Abstract: Virulence gene expression in the human pathogen, S. aureus is regulated by the agr (accessory gene regulator) quorum sensing (QS) system which is conserved in diverse Gram-positive bacteria. The agr QS signal molecule is an autoinducing peptide (AIP) generated via the initial processing of the AgrD pro-peptide by the transmembrane peptidase AgrB. Since structural information for AgrB and AgrBD interactions are lacking, we used homology modelling and molecular dynamics (MD) annealing to characterise the conformations of AgrB and AgrD in model membranes and in solution. These revealed a six helical transmembrane domain (6TMD) topology for AgrB. In solution, AgrD behaves as a disordered peptide, which binds N-terminally to membranes in the absence and in the presence of AgrB. In silico, membrane complexes of AgrD and dimeric AgrB show non-equivalent AgrB monomers responsible for initial binding and for processing, respectively. By exploiting split luciferase assays in Staphylococcus aureus, we provide experimental evidence that AgrB interacts directly with itself and with AgrD. We confirmed the in vitro formation of an AgrBD complex and AIP production after Western blotting using either membranes from Escherichia coli expressing AgrB or with purified AgrB and T7-tagged AgrD. AgrB and AgrD formed stable complexes in detergent micelles revealed using synchrotron radiation CD (SRCD) and Landau analysis consistent with the enhanced thermal stability of AgrB in the presence of AgrD. Conformational alteration of AgrB following provision of AgrD was observed by small angle X-ray scattering from proteodetergent micelles. An atomistic description of AgrB and AgrD has been obtained together with confirmation of the AgrB 6TMD membrane topology and existence of AgrBD molecular complexes in vitro and in vivo.
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May 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Josephine H. R.
Maidment
,
Motoki
Shimizu
,
Adam R.
Bentham
,
Sham
Vera
,
Marina
Franceschetti
,
Apinya
Longya
,
Clare E. M.
Stevenson
,
Juan Carlos
De La Concepcion
,
Aleksandra
Bialas
,
Sophien
Kamoun
,
Ryohei
Terauchi
,
Mark J.
Banfield
Diamond Proposal Number(s):
[13467, 18565]
Open Access
Abstract: A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.
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May 2023
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Luiz Carlos
Saramago
,
Marcos V.
Santana
,
Bárbara Figueira
Gomes
,
Rafael Ferreira
Dantas
,
Mario R.
Senger
,
Pedro Henrique
Oliveira Borges
,
Vivian Neuza
Dos Santos Ferreira
,
Alice
Dos Santos Rosa
,
Amanda Resende
Tucci
,
Milene
Dias Miranda
,
Petra
Lukacik
,
Claire
Strain-Damerell
,
C. David
Owen
,
Martin A.
Walsh
,
Sabrina
Baptista Ferreira
,
Floriano Paes
Silva-Junior
Abstract: SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global pandemic. The viral genome contains 5 major open reading frames of which the largest ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into 16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp’s, making it essential for viral replication and has been successfully targeted for the development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid. Increasing the arsenal of antivirals and development of protease inhibitors and other antivirals with a varied mode of action remains a priority to reduce the likelihood for resistance emerging. Here, we report results from an artificial intelligence-driven approach followed by in vitro validation, allowing the identification of five fragment-like Mpro inhibitors with IC50 values ranging from 1.5 to 241 μM. The three most potent molecules (compounds 818, 737, and 183) were tested against SARS-CoV-2 by in vitro replication in Vero E6 and Calu-3 cells. Compound 818 was active in both cell models with an EC50 value comparable to its measured IC50 value. On the other hand, compounds 737 and 183 were only active in Calu-3, a preclinical model of respiratory cells, showing selective indexes twice as high as those for compound 818. We also show that our in silico methodology was successful in identifying both reversible and covalent inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of 8-methyl-γ-carboline, while compound 737 is an N-pyridyl-isatin that covalently inhibits Mpro. Given the small molecular weights of these fragments, their high binding efficiency in vitro and efficacy in blocking viral replication, these compounds represent good starting points for the development of potent lead molecules targeting the Mpro of SARS-CoV-2.
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Apr 2023
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I04-Macromolecular Crystallography
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Filip
Mihalič
,
Leandro
Simonetti
,
Girolamo
Giudice
,
Marie Rubin
Sander
,
Richard
Lindqvist
,
Marie Berit Akpiroro
Peters
,
Caroline
Benz
,
Eszter
Kassa
,
Dilip
Badgujar
,
Raviteja
Inturi
,
Muhammad
Ali
,
Izabella
Krystkowiak
,
Ahmed
Sayadi
,
Eva
Andersson
,
Hanna
Aronsson
,
Ola
Söderberg
,
Doreen
Dobritzsch
,
Evangelia
Petsalaki
,
Anna K.
Överby
,
Per
Jemth
,
Norman E.
Davey
,
Ylva
Ivarsson
Diamond Proposal Number(s):
[23773]
Open Access
Abstract: Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.
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Apr 2023
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I24-Microfocus Macromolecular Crystallography
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Peter T.
Buckley
,
Rita
Chan
,
Jeffrey
Fernandez
,
Jinquan
Luo
,
Keenan A.
Lacey
,
Ashley L.
Dumont
,
Aidan
O’malley
,
Randall J.
Brezski
,
Songmao
Zheng
,
Thomas
Malia
,
Brian
Whitaker
,
Adam
Zwolak
,
Angela
Payne
,
Desmond
Clark
,
Martin
Sigg
,
Eilyn R.
Lacy
,
Anna
Kornilova
,
Debra
Kwok
,
Steve
Mccarthy
,
Bingyuan
Wu
,
Brian
Morrow
,
Jennifer
Nemeth-Seay
,
Ted
Petley
,
Sam
Wu
,
William R.
Strohl
,
Anthony S.
Lynch
,
Victor J.
Torres
Open Access
Abstract: Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein (“mAbtyrin”) that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.
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Apr 2023
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I04-Macromolecular Crystallography
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Clive S.
Mason
,
Tim
Avis
,
Chenlin
Hu
,
Nabeetha
Nagalingam
,
Manikhandan
Mudaliar
,
Chris
Coward
,
Khurshida
Begum
,
Kathleen
Gajewski
,
M. Jahangir
Alam
,
Eugenie
Bassères
,
Stephen
Moss
,
Stefanie
Reich
,
Esther
Duperchy
,
Keith R.
Fox
,
Kevin W.
Garey
,
David J.
Powell
Open Access
Abstract: Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.
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Apr 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
Krios II-Titan Krios II at Diamond
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Andre
Schutzer Godoy
,
Aline Minalli
Nakamura
,
Alice
Douangamath
,
Yun
Song
,
Gabriela
Dias Noske
,
Victor
Oliveira Gawriljuk
,
Rafaela
Sachetto Fernandes
,
Humberto
D'Muniz Pereira
,
Ketllyn irene
Zagato Oliveira
,
Daren
Fearon
,
Alexandre
Dias
,
Tobias
Krojer
,
Michael
Fairhead
,
Alisa
Powell
,
Louise
Dunnett
,
Jose
Brandao-Neto
,
Rachael
Skyner
,
Rod
Chalk
,
Dávid
Bajusz
,
Miklós
Bege
,
Anikó
Borbás
,
György Miklós
Keserű
,
Frank
Von Delft
,
Glaucius
Oliva
Diamond Proposal Number(s):
[27083, 27023]
Open Access
Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. However, the complexity of the enzyme's structure and kinetics, along with the broad range of recognition sequences and lack of structural complexes, hampers the development of inhibitors. Here, we performed enzymatic characterization of NendoU in its monomeric and hexameric form, showing that hexamers are allosteric enzymes with a positive cooperative index, and with no influence of manganese on enzymatic activity. Through combining cryo-electron microscopy at different pHs, X-ray crystallography and biochemical and structural analysis, we showed that NendoU can shift between open and closed forms, which probably correspond to active and inactive states, respectively. We also explored the possibility of NendoU assembling into larger supramolecular structures and proposed a mechanism for allosteric regulation. In addition, we conducted a large fragment screening campaign against NendoU and identified several new allosteric sites that could be targeted for the development of new inhibitors. Overall, our findings provide insights into the complex structure and function of NendoU and offer new opportunities for the development of inhibitors.
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Apr 2023
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B21-High Throughput SAXS
I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Open Access
Abstract: Bornaviruses are RNA viruses with a mammalian, reptilian, and avian host range. The viruses infect neuronal cells and in rare cases cause a lethal encephalitis. The family Bornaviridae are part of the Mononegavirales order of viruses, which contain a nonsegmented viral genome. Mononegavirales encode a viral phosphoprotein (P) that binds both the viral polymerase (L) and the viral nucleoprotein (N). The P protein acts as a molecular chaperone and is required for the formation of a functional replication/transcription complex. In this study, the structure of the oligomerization domain of the phosphoprotein determined by X-ray crystallography is reported. The structural results are complemented with biophysical characterization using circular dichroism, differential scanning calorimetry and small-angle X-ray scattering. The data reveal the phosphoprotein to assemble into a stable tetramer, with the regions outside the oligomerization domain remaining highly flexible. A helix-breaking motif is observed between the α-helices at the midpoint of the oligomerization domain that appears to be conserved across the Bornaviridae. These data provide information on an important component of the bornavirus replication complex.
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Mar 2023
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