I03-Macromolecular Crystallography
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Pasquale
Linciano
,
Cecilia
Pozzi
,
Giusy
Tassone
,
Giacomo
Landi
,
Stefano
Mangani
,
Matteo
Santucci
,
Rosaria
Luciani
,
Stefania
Ferrari
,
Nuno
Santarem
,
Lorenzo
Tagliazucchi
,
Anabela
Cordeiro-Da-Silva
,
Michele
Tonelli
,
Donatella
Tondi
,
Laura
Bertarini
,
Sheraz
Gul
,
Gesa
Witt
,
Carolina B.
Moraes
,
Luca
Costantino
,
Maria Paola
Costi
Diamond Proposal Number(s):
[21741]
Abstract: Pteridine reductase 1 (PTR1) is a catalytic protein belonging to the folate metabolic pathway in Trypanosmatidic parasites. PTR1 is a known target for the medicinal chemistry development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. In previous studies, new nitro derivatives were elaborated as PTR1 inhibitors. The compounds showing a diammino-pyrimidine core structure were previously developed but they showed limited efficacy. Therefore, a new class phenyl-, heteroaryl- and benzyloxy-nitro derivatives of the 2-nitroethyl-2,4,6-triaminopyrimidine scaffold were designed and tested. The compounds were assayed for their ability to inhibit T. brucei and L. major PTR1 enzymes and for their antiparasitic activity towards T. brucei and L. infantum parasites. To understand the structure-activity relationships of the compounds against TbPTR1, the x-ray crystallographic structure of the 2,4,6-triaminopyrimidine (TAP) was obtained and molecular modelling studies were performed. As a next step, only the most effective T. brucei inhibitors were then tested against the amastigote cellular stage of T. cruzi, searching for a broad-spectrum antiprotozoal agent. An early ADME-Tox profile evaluation was performed. The early toxicity profile of this class of compounds was investigated by measuring their inhibition of hERG and five cytochrome P450 isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4), cytotoxicity towards A549 cells and mitochondrial toxicity. Pharmacokinetic studies (SNAP-PK) were performed on selected compounds using hydroxypropyl-β-cyclodextrins (50 % w/v) to preliminarily study their plasma concentration when administered per os at a dose of 20 mg/kg. Finally, compound 1p, selected for the best pharmacodynamic and pharmacokinetic properties, showed promising activity in a mouse model of T. brucei infection. Compound 1p can be considered a good candidate for further bioavailability and efficacy studies.
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Nov 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18598]
Open Access
Abstract: Leishmaniases are a collection of neglected tropical diseases caused by kinetoplastid parasites in the genus Leishmania. Current chemotherapies are severely limited, and the need for new antileishmanials is of pressing international importance. Bromodomains are epigenetic reader domains that have shown promising therapeutic potential for cancer therapy and may also present an attractive target to treat parasitic diseases. Here, we investigate Leishmania donovani bromodomain factor 5 (LdBDF5) as a target for antileishmanial drug discovery. LdBDF5 contains a pair of bromodomains (BD5.1 and BD5.2) in an N-terminal tandem repeat. We purified recombinant bromodomains of L. donovani BDF5 and determined the structure of BD5.2 by X-ray crystallography. Using a histone peptide microarray and fluorescence polarization assay, we identified binding interactions of LdBDF5 bromodomains with acetylated peptides derived from histones H2B and H4. In orthogonal biophysical assays including thermal shift assays, fluorescence polarization, and NMR, we showed that BDF5 bromodomains bind to human bromodomain inhibitors SGC–CBP30, bromosporine, and I-BRD9; moreover, SGC–CBP30 exhibited activity against Leishmania promastigotes in cell viability assays. These findings exemplify the potential BDF5 holds as a possible drug target in Leishmania and provide a foundation for the future development of optimized antileishmanial compounds targeting this epigenetic reader protein.
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Oct 2023
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[25452]
Open Access
Abstract: Translational control is an essential process for the cell to adapt to varying physiological or environmental conditions. To survive adverse conditions such as low nutrient levels, translation can be shut down almost entirely by inhibiting ribosomal function. Here we investigated eukaryotic hibernating ribosomes from the microsporidian parasite Spraguea lophii in situ by a combination of electron cryo-tomography and single-particle electron cryo-microscopy. We show that microsporidian spores contain hibernating ribosomes that are locked in a dimeric (100S) state, which is formed by a unique dimerization mechanism involving the beak region. The ribosomes within the dimer are fully assembled, suggesting that they are ready to be activated once the host cell is invaded. This study provides structural evidence for dimerization acting as a mechanism for ribosomal hibernation in microsporidia, and therefore demonstrates that eukaryotes utilize this mechanism in translational control.
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Sep 2023
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I04-Macromolecular Crystallography
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Prasun
Kundu
,
Deboki
Naskar
,
Shannon J.
Mckie
,
Sheena
Dass
,
Usheer
Kanjee
,
Viola
Introini
,
Marcelo U.
Ferreira
,
Pietro
Cicuta
,
Manoj
Duraisingh
,
Janet E.
Deane
,
Julian C.
Rayner
Diamond Proposal Number(s):
[21426]
Open Access
Abstract: Tryptophan Rich Antigens (TRAgs) are encoded by a multi-gene family found in all Plasmodium species, but are significantly expanded in P. vivax and closely related parasites. We show that multiple P. vivax TRAgs are expressed on the merozoite surface and that one, PVP01_0000100 binds red blood cells with a strong preference for reticulocytes. Using X-ray crystallography, we solved the structure of the PVP01_0000100 C-terminal tryptophan rich domain, which defines the TRAg family, revealing a three-helical bundle that is conserved across Plasmodium and has structural homology with lipid-binding BAR domains involved in membrane remodelling. Biochemical assays confirm that the PVP01_0000100 C-terminal domain has lipid binding activity with preference for sulfatide, a glycosphingolipid present in the outer leaflet of plasma membranes. Deletion of the putative orthologue in P. knowlesi, PKNH_1300500, impacts invasion in reticulocytes, suggesting a role during this essential process. Together, this work defines an emerging molecular function for the Plasmodium TRAg family.
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Sep 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Abstract: Chagas disease and leishmaniasis are two examples of Neglected Tropical Diseases (NTDs), a diverse group of 20 conditions that cause devastating health, social and economic consequences for over one billion people worldwide. Public health control of NTDs is challenging, as they are often related to environmental conditions and have complex epidemiology. Many have complex life cycles and are vector-borne, meaning that there are animal reservoirs. The World Health Organization's One Health initiative recognises that human and animal health are interlinked, and that an integrated, unifying approach is needed to balance and optimise the health of people, animals and the environment. In work recently published in Acta Crystallographica Section D: Structural Biology, researchers from Durham University and the University of Sao Paulo obtained high-resolution structures of cysteine synthase enzymes from the pathogens that cause Chagas disease and leishmaniasis and a related livestock parasite. Their results allow a greater understanding of cysteine synthase in these organisms, and give a starting point for the structure-based design of new drugs to treat these diseases.
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Aug 2023
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Pathogenic parasites of the Trichomonas genus are causative agents of sexually-transmitted diseases affecting millions of individuals worldwide and whose outcome may include stillbirths and enhanced cancer risks and susceptibility to HIV infection. Trichomonas vaginalis relies on imported purine and pyrimidine nucleosides and nucleobases for survival, since it lacks the enzymatic activities necessary for de novo biosynthesis. Here we show that T. vaginalis additionally lacks homologues of the bacterial or mammalian enzymes required for the synthesis of the nicotinamide ring, crucial component in the redox cofactors NAD+ and NADP. Moreover, we show that a yet fully uncharacterized T. vaginalis protein homologous to bacterial and protozoan nucleoside hydrolases (NHs) is active as a pyrimidine nucleosidase, but shows the highest specificity towards the NAD+ metabolite nicotinamide riboside. Crystal structures of the trichomonal riboside hydrolase in different states reveals novel intermediates along the NH-catalyzed hydrolytic reaction, including an unexpected asymmetry in the homotetrameric assembly. The active site structure explains the broad specificity towards different ribosides, and offers precise insights for the engineering of specific inhibitors that may simultaneously target different essential pathways in the parasite.
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Jul 2023
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B21-High Throughput SAXS
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[24557, 27169]
Open Access
Abstract: Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically and structurally characterised nucleosome core particles (NCPs) from the kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened overall DNA binding. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.
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Jul 2023
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B21-High Throughput SAXS
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23459, 31353]
Open Access
Abstract: The symptoms of malaria occur during the blood stage of infection, when the parasite replicates within human red blood cells. The human malaria parasite, Plasmodium vivax, selectively invades reticulocytes in a process which requires an interaction between the ectodomain of the human DARC receptor and the Plasmodium vivax Duffy-binding protein, PvDBP. Previous studies have revealed that a small helical peptide from DARC binds to region II of PvDBP (PvDBP-RII). However, it is also known that sulphation of tyrosine residues on DARC affects its binding to PvDBP and these residues were not observed in previous structures. We therefore present the structure of PvDBP-RII bound to sulphated DARC peptide, showing that a sulphate on tyrosine 41 binds to a charged pocket on PvDBP-RII. We use molecular dynamics simulations, affinity measurements and growth-inhibition experiments in parasites to confirm the importance of this interaction. We also reveal the epitope for vaccine-elicited growth-inhibitory antibody DB1. This provides a complete understanding of the binding of PvDBP-RII to DARC and will guide the design of vaccines and therapeutics to target this essential interaction.
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Jun 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[24948]
Open Access
Abstract: Chagas disease is a neglected tropical disease (NTD) caused by Trypanosoma cruzi, whilst leishmaniasis, which is caused by over 20 species of Leishmania, represents a group of NTDs endemic to most countries in the tropical and subtropical belt of the planet. These diseases remain a significant health problem both in endemic countries and globally. These parasites and other trypanosomatids, including T. theileri, a bovine pathogen, rely on cysteine biosynthesis for the production of trypanothione, which is essential for parasite survival in hosts. The de novo pathway of cysteine biosynthesis requires the conversion of O-acetyl-L-serine into L-cysteine, which is catalysed by cysteine synthase (CS). These enzymes present potential for drug development against T. cruzi, Leishmania spp. and T. theileri. To enable these possibilities, biochemical and crystallographic studies of CS from T. cruzi (TcCS), L. infantum (LiCS) and T. theileri (TthCS) were conducted. Crystal structures of the three enzymes were determined at resolutions of 1.80 Å for TcCS, 1.75 Å for LiCS and 2.75 Å for TthCS. These three homodimeric structures show the same overall fold and demonstrate that the active-site geometry is conserved, supporting a common reaction mechanism. Detailed structural analysis revealed reaction intermediates of the de novo pathway ranging from an apo structure of LiCS and holo structures of both TcCS and TthCS to the substrate-bound structure of TcCS. These structures will allow exploration of the active site for the design of novel inhibitors. Additionally, unexpected binding sites discovered at the dimer interface represent new potential for the development of protein–protein inhibitors.
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Jun 2023
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I03-Macromolecular Crystallography
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Mauricio P.
Contreras
,
Hsuan
Pai
,
Muniyandi
Selvaraj
,
Amirali
Toghani
,
David M.
Lawson
,
Yasin
Tumtas
,
Cian
Duggan
,
Enoch Lok Him
Yuen
,
Clare E. M.
Stevenson
,
Adeline
Harant
,
Abbas
Maqbool
,
Chih-Hang
Wu
,
Tolga O.
Bozkurt
,
Sophien
Kamoun
,
Lida
Derevnina
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.
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May 2023
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