I04-1-Macromolecular Crystallography (fixed wavelength)
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Muhamamd
Faheem
,
Napoleao
Fonseca Valadares
,
Jose
Brandao-Neto
,
Domenico
Bellini
,
Patrick
Collins
,
Nicholas M.
Pearce
,
Louise
Bird
,
Juliana
Torini De Souza
,
Raymond
Owens
,
Humberto
Pereira
,
Frank
Von Delft
,
João Alexandre Ribeiro Gonçalves
Barbosa
Diamond Proposal Number(s):
[11175]
Open Access
Abstract: Several Schistosoma species cause Schistosomiasis, an endemic disease in 78 countries that is ranked second amongst the parasitic diseases in terms of its socioeconomic impact and human health importance. The drug recommended for treatment by the WHO is praziquantel (PZQ), but there are concerns associated with PZQ, such as the lack of information about its exact mechanism of action, its high price, its effectiveness – which is limited to the parasite’s adult form – and reports of resistance. The parasites lack the de novo purine pathway, rendering them dependent on the purine salvage pathway or host purine bases for nucleotide synthesis. Thus, the Schistosoma purine salvage pathway is an attractive target for the development of necessary and selective new drugs. In this study, the purine nucleotide phosphorylase II (PNP2), a new isoform of PNP1, was submitted to a high-throughput fragment-based hit discovery using a crystallographic screening strategy. PNP2 was crystallized and crystals were soaked with 827 fragments, a subset of the Maybridge 1000 library. X-ray diffraction data was collected and structures were solved. Out of 827-screened fragments we have obtained a total of 19 fragments that show binding to PNP2. 14 of these fragments bind to the active site of PNP2, while five were observed in three other sites. Here we present the first fragment screening against PNP2.
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Sep 2021
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069]
Open Access
Abstract: The kinetochore is the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Evolutionarily divergent kinetoplastids have an unconventional set of kinetochore proteins. It remains unknown how kinetochores assemble at centromeres in these organisms. Here, we characterize KKT2 and KKT3 in the kinetoplastid parasite Trypanosoma brucei. In addition to the N-terminal kinase domain and C-terminal divergent polo boxes, these proteins have a central domain of unknown function. We show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: Plasmodium falciparum invades erythrocytes and extensively modifies them in a manner that increases the virulence of this malaria parasite. A single heat-shock 70 kDa-type chaperone, PfHsp70-x, is among the parasite proteins exported to the host cell. PfHsp70-x assists in the formation of a key protein complex that underpins parasite virulence and supports parasite growth during febrile episodes. Previous work resolved the crystallographic structures of the PfHsp70-x ATPase and substrate-binding domains, and showed them to be highly similar to those of their human counterparts. Here, 233 chemical fragments were screened for binding to the PfHsp70-x ATPase domain, resulting in three crystallographic structures of this domain in complex with ligands. Two binding sites were identified, with most ligands binding proximal to the ATPase nucleotide-binding pocket. Although amino acids participating in direct ligand interactions are conserved between the parasite and human erythrocytic chaperones, one nonconserved residue is also present near the ligand. This work suggests that PfHsp70-x features binding sites that may be exploitable by small-molecule ligands towards the specific inhibition of the parasite chaperone.
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Aug 2021
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I03-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Carmen
Fernandez-Rodriguez
,
Iker
Oyenarte
,
Carolina
Conter
,
Irene
Gonzalez-Recio
,
Reyes
Núñez-Franco
,
Claudia
Gil-Pitarch
,
Iban
Quintana
,
Gonzalo
Jiménez-Osés
,
Paola
Dominici
,
Maria Luz
Martinez-Chantar
,
Alessandra
Astegno
,
Luis Alfonso
Martinez-Cruz
Diamond Proposal Number(s):
[28360]
Open Access
Abstract: Cysteine plays a major role in the redox homeostasis and antioxidative defense mechanisms of many parasites of the phylum Apicomplexa. Of relevance to human health is Toxoplasma gondii, the causative agent of toxoplasmosis. A major route of cysteine biosynthesis in this parasite is the reverse transsulfuration pathway involving two key enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). CBS from T. gondii (TgCBS) catalyzes the pyridoxal-5́-phosphate-dependent condensation of homocysteine with either serine or O-acetylserine to produce cystathionine. The enzyme can perform alternative reactions that use homocysteine and cysteine as substrates leading to the endogenous biosynthesis of hydrogen sulfide, another key element in maintaining the intracellular redox equilibrium. In contrast with human CBS, TgCBS lacks the N-terminal heme binding domain and is not responsive to S-adenosylmethionine. Herein, we describe the structure of a TgCBS construct that lacks amino acid residues 466-491 and shows the same activity of the native protein. TgCBS Δ466-491 was determined alone and in complex with reaction intermediates. A complementary molecular dynamics analysis revealed a unique domain organization, similar to the pathogenic mutant D444N of human CBS. Our data provides one missing piece in the structural diversity of CBSs by revealing the so far unknown three-dimensional arrangement of the CBS-type of Apicomplexa. This domain distribution is also detected in yeast and bacteria like Pseudomonas aeruginosa. These results pave the way for understanding the mechanisms by which TgCBS regulates the intracellular redox of the parasite, and have far-reaching consequences for the functional understanding of CBSs with similar domain distribution.
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Aug 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Jan
Kotál
,
Stephanie G. I.
Polderdijk
,
Helena
Langhansová
,
Monika
Ederová
,
Larissa A.
Martins
,
Zuzana
Beránková
,
Adéla
Chlastáková
,
Ondřej
Hajdušek
,
Michail
Kotsyfakis
,
James A.
Huntington
,
Jindřich
Chmelař
Open Access
Abstract: Tick saliva is a rich source of antihemostatic, anti-inflammatory, and immunomodulatory molecules that actively help the tick to finish its blood meal. Moreover, these molecules facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-8, a salivary serpin from the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Iripin-8 displayed blood-meal-induced mRNA expression that peaked in nymphs and the salivary glands of adult females. Iripin-8 inhibited multiple proteases involved in blood coagulation and blocked the intrinsic and common pathways of the coagulation cascade in vitro. Moreover, Iripin-8 inhibited erythrocyte lysis by complement, and Iripin-8 knockdown by RNA interference in tick nymphs delayed the feeding time. Finally, we resolved the crystal structure of Iripin-8 at 1.89 Å resolution to reveal an unusually long and rigid reactive center loop that is conserved in several tick species. The P1 Arg residue is held in place distant from the serpin body by a conserved poly-Pro element on the P′ side. Several PEG molecules bind to Iripin-8, including one in a deep cavity, perhaps indicating the presence of a small-molecule binding site. This is the first crystal structure of a tick serpin in the native state, and Iripin-8 is a tick serpin with a conserved reactive center loop that possesses antihemostatic activity that may mediate interference with host innate immunity.
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Aug 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11088]
Abstract: Chagas disease, an infectious condition caused by Trypanosoma cruzi, lacks treatment with drugs with desired efficacy and safety profiles. To address this unmet medical need, a set of trypanocidal compounds were identified through a large multicenter phenotypic-screening initiative and assembled in the GSK Chagas Box. In the present work, we report the screening of the Chagas Box against T. cruzi malic enzymes (MEs) and the identification of three potent inhibitors of its cytosolic isoform (TcMEc). One of these compounds, TCMDC-143108 (1), came out as a nanomolar inhibitor of TcMEc, and 14 new derivatives were synthesized and tested for target inhibition and efficacy against the parasite. Moreover, we determined the crystallographic structures of TcMEc in complex with TCMDC-143108 (1) and six derivatives, revealing the allosteric inhibition site and the determinants of specificity. Our findings connect phenotypic hits from the Chagas Box to a relevant metabolic target in the parasite, providing data to foster new structure–activity guided hit optimization initiatives.
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Jul 2021
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I04-Macromolecular Crystallography
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Dayana C.
Farhat
,
Matthew W.
Bowler
,
Guillaume
Communie
,
Dominique
Pontier
,
Lucid
Belmudes
,
Caroline
Mas
,
Charlotte
Corrao
,
Yohann
Couté
,
Alexandre
Bougdour
,
Thierry
Lagrange
,
Mohamed-Ali
Hakimi
,
Christopher
Swale
Diamond Proposal Number(s):
[21969]
Open Access
Abstract: Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.
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Jul 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11690]
Open Access
Abstract: Trypanosoma and Leishmania parasites are the etiological agents of various threatening neglected tropical diseases (NTDs), including human African trypanosomiasis (HAT), Chagas disease, and various types of leishmaniasis. Recently, meaningful progresses in the treatment of HAT, due to Trypanosoma brucei (Tb), have been achieved by the introduction of fexinidazole and the combination therapy eflornithine–nifurtimox. Nevertheless, due to drug resistance issues and the exitance of animal reservoirs, the development of new NTD treatments is still required. For this purpose, we explored the combined targeting of two key folate enzymes, dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1). We formerly showed that the TbDHFR inhibitor cycloguanil (CYC) also targets TbPTR1, although with reduced affinity. Here, we explored a small library of CYC analogues to understand how their substitution pattern affects the inhibition of both TbPTR1 and TbDHFR. Some novel structural features responsible for an improved, but preferential, ability of CYC analogues to target TbPTR1 were disclosed. Furthermore, we showed that the known drug pyrimethamine (PYR) effectively targets both enzymes, also unveiling its binding mode to TbPTR1. The structural comparison between PYR and CYC binding modes to TbPTR1 and TbDHFR provided key insights for the future design of dual inhibitors for HAT therapy.
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Jun 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19946]
Abstract: Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS) represent a promising therapeutic anti‐malarial target. Cladosporin, a tool compound was identified as a selective and potent PfKRS inhibitor but lacks metabolic stability. Here, we report chemical synthesis, biological evaluation and structural characterization of analogues where the tetrahydropyran (THP) frame of cladosporin is replaced with the piperidine ring bearing functional group variations. Thermal binding, enzymatic, kinetic and parasitic assays complemented with x‐ray crystallography reveals compounds to be of moderate potency. Co‐crystals of Cla‐B and Cla‐C with PfKRS reveals key atomic configurations that allow drug binding to, and inhibition of the enzyme. Collectively, these piperidine ring scaffold inhibitors lays a framework for further structural editing and functional modifications of cladosporin scaffold to obtain a potent lead.
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May 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
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José T.
Moreira-Filho
,
Arthur C.
Silva
,
Rafael F.
Dantas
,
Barbara F.
Gomes
,
Lauro R.
De Souza Neto
,
Jose
Brandao-Neto
,
Raymond J.
Owens
,
Nicholas
Furnham
,
Bruno J.
Neves
,
Floriano P.
Silva-Junior
,
Carolina H.
Andrade
Diamond Proposal Number(s):
[16978]
Open Access
Abstract: Schistosomiasis is a parasitic disease caused by trematode worms of the genus Schistosoma and affects over 200 million people worldwide. The control and treatment of this neglected tropical disease is based on a single drug, praziquantel, which raises concerns about the development of drug resistance. This, and the lack of efficacy of praziquantel against juvenile worms, highlights the urgency for new antischistosomal therapies. In this review we focus on innovative approaches to the identification of antischistosomal drug candidates, including the use of automated assays, fragment-based screening, computer-aided and artificial intelligence-based computational methods. We highlight the current developments that may contribute to optimizing research outputs and lead to more effective drugs for this highly prevalent disease, in a more cost-effective drug discovery endeavor.
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May 2021
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