Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[34172]
Open Access
Abstract: Plasmodium falciparum is a eukaryotic pathogen responsible for the majority of malaria-related fatalities. Plasmodium belongs to the phylum Apicomplexa and, like most members of this phylum, contains a non-photosynthetic plastid called the apicoplast. The apicoplast has its own genome, replicated by a dedicated replisome. Unlike other cellular replisomes, the apicoplast replisome uses a single DNA polymerase (apPol). This suggests that apPol can multitask and catalyse both replicative and lesion bypass synthesis. Replicative synthesis relies on a restrictive active site for high accuracy while lesion bypass typically requires an open active site. This raises the question: how does apPol combine the structural features of multiple DNA polymerases in a single protein? Using single-particle electron cryomicroscopy (cryoEM), we have solved the structures of apPol bound to its undamaged DNA and nucleotide substrates in five pre-chemistry conformational states. We found that apPol can accommodate a nascent base pair with the fingers in an open configuration, which might facilitate the lesion bypass activity. In the fingers-open state, we identified a nascent base pair checkpoint that preferentially selects Watson–Crick base pairs, an essential requirement for replicative synthesis. Taken together, these structural features might explain how apPol balances replicative and lesion bypass synthesis.
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Oct 2025
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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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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[32633]
Open Access
Abstract: Dengue viruses (DENVs) infect approximately 400 million people each year, and currently, there are no effective therapeutics available. To explore potential starting points for antiviral drug development, we conducted a large-scale crystallographic fragment screen targeting the RNA-dependent RNA polymerase (RdRp) domain of the nonstructural protein 5 (NS5) from DENV serotype 2. Our screening, which involved 1108 fragments, identified 60 hit compounds across various known binding sites, including the active site, N pocket, and RNA tunnel. Additionally, we discovered a novel binding site and a fragment-binding hot spot in thumb site II. These structural findings open amenable avenues for developing non-nucleoside inhibitors and offer valuable insights for future structure-based drug design aimed at DENV and other flaviviral RdRps.
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Sep 2025
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I02-Macromolecular Crystallography
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Open Access
Abstract: The trematode liver fluke Fasciola hepatica causes the neglected tropical disease fascioliasis in humans and is associated with significant losses in agricultural industry due to reduced animal productivity. Triosephosphate isomerase (TPI) is a glycolytic enzyme that has been researched as a drug target for various parasites, including F. hepatica. The high-resolution crystal structure of F. hepatica TPI (FhTPI) has been solved at 1.51 Å resolution in its monoclinic form. The structure has been used to perform molecular-docking studies with the most successful fasciolocide triclabendazole (TCBZ), which has recently been suggested to target FhTPI. Two FhTPI residues, Lys50 and Asp51, are located at the dimer interface and are found in close proximity to the docked TCBZ. These residues are not conserved in mammalian hosts.
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Sep 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[26855]
Open Access
Abstract: The dengue virus (DENV) poses a significant threat to human health, accounting for approximately 400 million infections each year. Its genome features a circular structure that facilitates replication through long-range RNA-RNA interactions, utilizing cyclization sequences located in the untranslated regions (UTRs). To gain new insights into the organization of the DENV genome, we purified the 5′ and 3′ UTRs of DENV in vitro and examined their structural and binding properties using various biophysical techniques combined with computational methods. Through our biophysical characterization, we determined the 5’ and 3’ UTR regions to bind with an affinity of 40 nM in a 1:1 stoichiometry. By using small-angle X-ray scattering (SAXS), we provide the first structural characterization of the 3’ and 5’ UTR regions, revealing several plausible conformations that the viral UTRs may adopt during replication. This comprehensive investigation revealed key features that provide mechanistic insights into the different structural states during DENV replication, as tracked through the accessibility of various RNA conformations. Overall, our research enhances the understanding of DENV cyclization, emphasising the structural adaptability, dynamic folding, and flexibility of these RNA molecules in solution. By uncovering details at the atomic level, we aim to contribute to the development of targeted drugs that can disrupt crucial stages of viral replication.
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Sep 2025
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I03-Macromolecular Crystallography
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Joanna
Panecka-Hofman
,
Pasquale
Linciano
,
Ina
Pöhner
,
Edyta
Dyguda-Kazimierowicz
,
Wiktoria
Jedwabny
,
Giacomo
Landi
,
Nuno
Santarem
,
Gesa
Witt
,
Bernhard
Ellinger
,
Maria
Kuzikov
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Rosaria
Luciani
,
Stefania
Ferrari
,
Daniele
Aiello
,
Stefano
Mangani
,
Cecilia
Pozzi
,
Anabela
Cordeiro-Da-Silva
,
Sheraz
Gul
,
Maria Paola
Costi
,
Rebecca C.
Wade
Diamond Proposal Number(s):
[15832]
Open Access
Abstract: Pteridine reductase 1 (PTR1) is a folate pathway enzyme essential for pathogenic trypanosomatids and a promising drug target for diseases such as sleeping sickness and leishmaniasis. Previous studies have shown that the 2-aminobenzothiazole moiety targets the PTR1 biopterin pocket, while 3,4-dichlorophenyl-containing compounds, such as I bind a different region of the Trypanosoma brucei PTR1 (TbPTR1) pocket. This study combines both moieties via various linkers, creating two compound series screened in silico against TbPTR1 and Leishmania major PTR1 (LmPTR1). In the first series, five compounds were synthesized, and 1a and 1b emerged as potent TbPTR1 inhibitors, with 1b also being active against LmPTR1 and moderately effective against Leishmania infantum. Furthermore, structure–activity relationship analysis, supported by quantum calculations and crystallography, revealed meta-halogenation to be more favorable than para, although single halogenation reduced antiparasite effects. Our fragment hybridization approach led to less toxic, more effective compounds than I.
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Sep 2025
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B21-High Throughput SAXS
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Sagar
Batra
,
Francisco
Olmo
,
Timothy J.
Ragan
,
Merve
Kaplan
,
Valeria
Calvaresi
,
Asger
Meldgaard Frank
,
Claudia
Lancey
,
Mahya
Assadipapari
,
Cuifeng
Ying
,
Weston B.
Struwe
,
Emma L.
Hesketh
,
Lea
Barfod
,
Ivan
Campeotto
,
John M.
Kelly
Open Access
Abstract: Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a significant global public health concern. Despite its profound health impact in both endemic and non-endemic areas, no vaccine is available, and the existing therapies are outdated, producing severe side effects. The 80 kDa prolyl oligopeptidase of Trypanosoma cruzi (TcPOP) has been identified as a leading candidate for Chagas vaccine development. Here we report the three-dimensional structure of TcPOP in open and closed conformation, at a global resolution of 3.8 and 3.6 Å, respectively, determined using single-particle cryo-electron microscopy. Multiple conformations were observed and further characterized using plasmonic optical tweezers and hydrogen-deuterium exchange mass spectrometry. To assess the immunogenic potential of TcPOP, we immunized female mice and evaluated both polyclonal and monoclonal responses against the TcPOP antigen and its homologues. The anti-TcPOP polyclonal response demonstrates invasion blocking properties via parasite lysis. Polyclonal sera were cross-reactive with closely-related POPs but not with human homologues. Collectively, our findings provide structural and functional insights necessary to understand the immunogenicity of TcPOP for future Chagas vaccine development.
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Aug 2025
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I04-Macromolecular Crystallography
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Lindsay B.
Tulloch
,
Hugh
Tawell
,
Annie E.
Taylor
,
Marta Lopes
Lima
,
Alice
Dawson
,
Sandra
Carvalho
,
Richard J.
Wall
,
Victoriano
Corpas-Lopez
,
Gourav
Dey
,
Jack
Duggan
,
Luma Godoy
Magalhaes
,
Leah S.
Torrie
,
Laura
Frame
,
David
Robinson
,
Stephen
Patterson
,
Michele
Tinti
,
George W.
Weaver
,
William J.
Robinson
,
Monica
Cal
,
Marcel
Kaiser
,
Pascal
Mäser
,
Peter
Sjö
,
Benjamin
Perry
,
John M.
Kelly
,
Amanda Fortes
Francisco
,
Avninder S.
Bhambra
,
Susan
Wyllie
Diamond Proposal Number(s):
[26793]
Abstract: The protozoan parasite Trypanosoma cruzi causes Chagas disease, which is among the deadliest parasitic infections in Latin America. Current therapies are toxic and lack efficacy against the chronic stage of infection; thus, new drugs are urgently needed. Here, we describe a previously unidentified series of quinazoline compounds with potential against Trypanosoma cruzi and the related trypanosomatid parasites Trypanosoma brucei and Leishmania donovani. We demonstrated partial efficacy of a lead quinazoline compound in a mouse model of acute Chagas disease. Mechanism of action studies using several orthogonal approaches showed that this quinazoline compound series targeted the ATP-binding pocket of T. cruzi lysyl-tRNA synthetase 1 (KRS1). A high-resolution crystal structure of KRS1 bound to the drug indicated binding interactions that led to KRS1 inhibition. Our study identified KRS1 as a druggable target for treating T. cruzi infection in a mouse model. This quinazoline series shows potential for treating Chagas disease but will require further development to become a future treatment for this neglected disease.
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Jul 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: In Leishmania parasites, as for their hosts, the ubiquitin proteasome system is important for cell viability. As part of a systematic gene deletion study, it was discovered that four cysteine protease type deubiquitinases (DUBs) are essential for parasite survival in the promastigote stage, including DUB16. Here we have purified and characterised recombinant DUB16 from Leishmania donovani, which belongs to the ubiquitin C-terminal hydrolase (UCH) family. DUB16 efficiently hydrolyses C-terminal aminocoumarin and rhodamine conjugates of ubiquitin consistent with proposed cellular roles of UCH-type DUBs in regenerating free monomeric ubiquitin from small molecule ubiquitin adducts arising from adventitious metabolic processes. The crystal structure of DUB16 reveals a typical UCH-type deubiquitinase fold, and a relatively short and disordered crossover loop that appears to restrict access to the catalytic cysteine. At close to stoichiometric enzyme to substrate ratios, DUB16 exhibits deubiquitinase activity towards diubiquitins linked through isopeptide bonds between Lys11, Lys48 or Lys63 residues of the proximal ubiquitin and the C-terminus of the distal ubiquitin. With 100-1000-fold higher turnover rates, DUB16 cleaves the ubiquitin-ribosomal L40 fusion protein to give the mature products. A DUB-targeting cysteine-reactive cyanopyrrolidine compound, IMP-1710, inhibits DUB16 activity. IMP-1710 was shown in promastigote cell viability assays to have parasite killing activity with EC50 values of 1-2 M, comparable to the anti-leishmanial drug, miltefosine. L. mexicana parasites engineered to overproduce DUB16 showed a modest increase in resistance to IMP-1710, providing evidence that IMP-1710 inhibits DUB16 in vivo. While it is highly likely that IMP-1710 has additional targets, these results suggest that DUB16 is a potential target for the development of new anti-leishmanial compounds.
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Jun 2025
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[28534]
Abstract: Objective: Along with rising resistance to antimalarials, the emergence of insecticide resistance in Anopheles mosquito species also remains a serious concern. Here, we reveal two potent compounds that show larvicidal and endectocidal activity against malaria vectors, Anopheles culicifacies and Anopheles stephensi, respectively. Methods: We investigated larvicidal activity of two inhibitors against III-instar larvae of Anopheles culicifacies. The survival and fertility of adult female Anopheles stephensi mosquitoes were assessed. Additionally, we purified recombinant prolyl-tRNA synthetase of Anopheles culicifacies and performed enzyme-based assays and structural analysis with the two inhibitors. Results: Our study reveals that the Anopheles culicifacies prolyl-tRNA synthetase (AcProRS) is potently inhibited by halofuginone (HFG) and an ATP mimetic (L95). The evaluation of larvicidal activity of HFG against Anopheles culicifacies III-instar larvae showed a dose-dependent increase in mortality. In adult female Anopheles stephensi mosquitoes, ingestion of HFG via artificial blood feeding resulted in impaired ovary development, reduced egg laying, and decreased overall survival. The potent enzymatic inhibition of AcProRS thus drives the killing of larvae. The co-crystal structure of AcProRS with inhibitors provides a structural basis for improving their potency as future larvicides. Conclusion: Our data suggest the potential for repositioning halofuginone (HFG) and pyrrolidine-based ATP-mimetics (L95) as larvicides. Targeting the vector-encoded aminoacyl-tRNA synthetases provides a new focus for developing effective agents that can control multiple mosquitoe-borne infectious diseases like malaria and dengue.
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Mar 2025
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