Targeting Toxoplasma gondii CPSF 3 as a new approach to control toxoplasmosis
2017; Springer Nature; Volume: 9; Issue: 3 Linguagem: Inglês
10.15252/emmm.201607370
ISSN1757-4684
AutoresAndrés Palencia, Alexandre Bougdour, Marie‐Pierre Brenier‐Pinchart, Bastien Touquet, Rose‐Laurence Bertini, Cristina Sensi, Gabrielle Gay, Julien Vollaire, Véronique Josserand, Eric E. Easom, Yvonne R. Freund, Hervé Pelloux, Philip J. Rosenthal, S. Cusack, Mohamed‐Ali Hakimi,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoResearch Article1 February 2017Open Access Transparent process Targeting Toxoplasma gondii CPSF3 as a new approach to control toxoplasmosis Andrés Palencia Corresponding Author Andrés Palencia [email protected] orcid.org/0000-0002-1805-319X Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Alexandre Bougdour Corresponding Author Alexandre Bougdour [email protected] orcid.org/0000-0002-5895-0020 Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Marie-Pierre Brenier-Pinchart Marie-Pierre Brenier-Pinchart Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Bastien Touquet Bastien Touquet Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Rose-Laurence Bertini Rose-Laurence Bertini Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Cristina Sensi Cristina Sensi European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Gabrielle Gay Gabrielle Gay Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Julien Vollaire Julien Vollaire Institute for Advanced Biosciences (IAB), OPTIMAL Small Animal Imaging Facility, Grenoble, France Search for more papers by this author Véronique Josserand Véronique Josserand Institute for Advanced Biosciences (IAB), OPTIMAL Small Animal Imaging Facility, Grenoble, France Search for more papers by this author Eric Easom Eric Easom Anacor Pharmaceuticals Inc., Palo Alto, CA, USA Search for more papers by this author Yvonne R Freund Yvonne R Freund Anacor Pharmaceuticals Inc., Palo Alto, CA, USA Search for more papers by this author Hervé Pelloux Hervé Pelloux Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Philip J Rosenthal Philip J Rosenthal Department of Medicine, University of California, San Francisco, CA, USA Search for more papers by this author Stephen Cusack Stephen Cusack European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Mohamed-Ali Hakimi Corresponding Author Mohamed-Ali Hakimi [email protected] orcid.org/0000-0002-2547-8233 Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Andrés Palencia Corresponding Author Andrés Palencia [email protected] orcid.org/0000-0002-1805-319X Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Alexandre Bougdour Corresponding Author Alexandre Bougdour [email protected] orcid.org/0000-0002-5895-0020 Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Marie-Pierre Brenier-Pinchart Marie-Pierre Brenier-Pinchart Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Bastien Touquet Bastien Touquet Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Rose-Laurence Bertini Rose-Laurence Bertini Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Cristina Sensi Cristina Sensi European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Gabrielle Gay Gabrielle Gay Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Julien Vollaire Julien Vollaire Institute for Advanced Biosciences (IAB), OPTIMAL Small Animal Imaging Facility, Grenoble, France Search for more papers by this author Véronique Josserand Véronique Josserand Institute for Advanced Biosciences (IAB), OPTIMAL Small Animal Imaging Facility, Grenoble, France Search for more papers by this author Eric Easom Eric Easom Anacor Pharmaceuticals Inc., Palo Alto, CA, USA Search for more papers by this author Yvonne R Freund Yvonne R Freund Anacor Pharmaceuticals Inc., Palo Alto, CA, USA Search for more papers by this author Hervé Pelloux Hervé Pelloux Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Philip J Rosenthal Philip J Rosenthal Department of Medicine, University of California, San Francisco, CA, USA Search for more papers by this author Stephen Cusack Stephen Cusack European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France Search for more papers by this author Mohamed-Ali Hakimi Corresponding Author Mohamed-Ali Hakimi [email protected] orcid.org/0000-0002-2547-8233 Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France Search for more papers by this author Author Information Andrés Palencia *,1,2,‡, Alexandre Bougdour *,1,‡, Marie-Pierre Brenier-Pinchart1, Bastien Touquet1, Rose-Laurence Bertini1, Cristina Sensi2, Gabrielle Gay1, Julien Vollaire3, Véronique Josserand3, Eric Easom4, Yvonne R Freund4, Hervé Pelloux1, Philip J Rosenthal5, Stephen Cusack2 and Mohamed-Ali Hakimi *,1 1Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U1209, CNRS UMR 5309, Université Grenoble Alpes, Grenoble, France 2European Molecular Biology Laboratory (EMBL), Grenoble Outstation and Unit of Virus Host-Cell Interactions, University of Grenoble-EMBL-Centre National de la Recherche Scientifique, Grenoble Cedex 9, France 3Institute for Advanced Biosciences (IAB), OPTIMAL Small Animal Imaging Facility, Grenoble, France 4Anacor Pharmaceuticals Inc., Palo Alto, CA, USA 5Department of Medicine, University of California, San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +33 476 63 71 09; [email protected] *Corresponding author. Tel: +33 476 63 71 14; [email protected] *Corresponding author. Tel: +33 476 63 74 69; [email protected] EMBO Mol Med (2017)9:385-394https://doi.org/10.15252/emmm.201607370 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii. Parasites selected to be resistant to AN3661 had mutations in TgCPSF3, which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild-type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, TgCPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti-parasitic drugs. Synopsis Benzoxaboroles are boron-containing compounds effective against a wide range of infectious pathogens. The benzoxaborole AN3661 is a novel drug candidate against acute toxoplasmosis. The compound blocks an unprecedented target of Toxoplasma, the endonuclease CPSF3 that cleaves 3′-mRNAs in eukaryotes. AN3661 inhibits Toxoplasma growth in vitro at low micromolar concentrations, with potency comparable to that of the clinically relevant drug pyrimethamine. AN3661 orally administered to mice controls otherwise lethal infections with comparable efficacy to sulfadiazine, the standard of care to treat toxoplasmosis. Mice treated with AN3661 developed protective immunity to subsequent Toxoplasma infections. Forward genetics and CRISPR/Cas9 editing validated CPSF3 as the main target of AN3661 in Toxoplasma. Structural modelling allowed to place the resistant mutations at the Toxoplasma CPSF3 catalytic site and to identify the putative binding site of AN3661. Introduction Toxoplasma gondii chronically infects about 30–50% of the human population (Pappas et al, 2009; Flegr et al, 2014; Parlog et al, 2015). Toxoplasmosis is usually an unapparent or mild disease in immunocompetent individuals, but it is a serious threat in immunocompromised patients, who can experience lethal or chronic cardiac, pulmonary or cerebral pathologies. Moreover, congenital toxoplasmosis can cause a range of problems including foetal malformations and retinochoroiditis. Current therapies for toxoplasmosis are reasonably effective, but they require long durations of treatment, often with toxic side effects (Farthing et al, 1992; Fung & Kirschenbaum, 1996), underlining the need for new classes of drugs to treat this infection (Neville et al, 2015). Benzoxaboroles are boron-containing compounds that have demonstrated efficacy in a number of clinical indications in recent years (Baker et al, 2009; Liu et al, 2014). Notably, Kerydin is an FDA-approved benzoxaborole that inhibits fungal leucyl-tRNA synthetase (LeuRS) and is used for the treatment of onychomycosis. Related compounds are being developed as LeuRS inhibitors of other human pathogens (Hernandez et al, 2013; Palencia et al, 2016a,b). Other benzoxaboroles inhibit phosphodiesterase-4 (Freund et al, 2012), Rho kinase (Akama et al, 2013) and bacterial β-lactamases (Xia et al, 2011). Overall, these compounds are synthetically tractable and show excellent drug-like properties without significant safety liabilities. In this study, we report that the benzoxaborole AN3661 inhibits T. gondii growth in human cells at low micromolar concentrations. Resistant parasites had mutations in a previously unexploited protein target, TgCPSF3. Importantly, all mice treated orally with AN3661 survived an otherwise lethal T. gondii infection and developed protective immunity to subsequent infections. Our results suggest TgCPSF3 is a promising novel target for the generation of new drugs to treat toxoplasmosis. Results A benzoxaborole with potent in vitro activity against Toxoplasma gondii Human foreskin fibroblasts (HFFs) were infected with tachyzoites of the virulent RH strain and treated with benzoxaboroles, pyrimethamine, the standard of care to treat toxoplasmosis, or vehicle (DMSO). We screened a group of 20 representative benzoxaboroles that were previously shown to have activity against bacteria, fungi or other eukaryotic parasites (Rock et al, 2007; Xia et al, 2011; Hernandez et al, 2013; Zhang et al, 2013; Palencia et al, 2016b). Some of these compounds were known to target leucyl-tRNA synthetase (LeuRS). From this group of benzoxaboroles, only two compounds, AN6426 and AN3661, showed activity against Toxoplasma. AN6426 is a LeuRS inhibitor with moderate activity against Toxoplasma and is described in a separated article (Palencia et al, 2016a). However, AN3661 demonstrated very good activity (IC50 = 0.9 μM), with potency comparable to that of pyrimethamine, and without apparent detrimental effects to host cells (Fig 1). Figure 1. AN3661 demonstrates potent activity against Toxoplasma gondii in vitro Activity of AN3661 against Toxoplasma gondii parasites growing intracellularly on human foreskin fibroblasts (HFFs). HFF cells were infected with tachyzoites and incubated with 5 μM AN3661, 2 μM pyrimethamine or 0.1% DMSO (mock control). Cells were fixed at 24 h and 4 days post-infection and then stained with antibodies against the T. gondii inner membrane complex protein 1 (IMC1, red) and rhoptry protein toxofilin (green) to define the parasite periphery and apical complex, respectively. Nuclei were labelled with Hoechst dye (blue). Scale bars represent 10 μm. Determination of IC50s against wild-type and engineered T. gondii mutant strains. Dose–response curves are shown for the indicated T. gondii clones treated with AN3661 (top) or pyrimethamine (bottom). Parasitic vacuoles were counted by using anti-GRA1 Toxoplasma antibodies and parasite nuclei by Hoechst. Data information: In (B), IC50s were determined with GraphPad Prism as the average of three independent experiments, each performed in triplicate. Error bars represent the standard errors. Download figure Download PowerPoint Selection of Toxoplasma gondii parasite lines resistant to AN3661 and target identification To explore the mechanism of action of AN3661, resistant parasites were generated with 7 mM ethyl methanesulphonate (EMS) in four independent chemical mutagenesis experiments, followed by selection in the presence of 5 μM AN3661 (> sixfold the IC50 value) over approximately 4 weeks. This is a useful approach to increase the frequency of mutations in Toxoplasma, which is otherwise very low (Farrell et al, 2014). The resistant parasite lines were then cloned by serial dilution. In a concomitant study, Plasmodium falciparum parasites that were resistant to AN3661 harboured mutations in two genes, pfcpsf3 and pfmdr1 (Sonoiki et al, 2017). CPSF3 encodes a homologue of the metal-dependent endonuclease, subunit 3, of the mammalian cleavage and polyadenylation specificity factor complex (CPSF-73) (Ryan et al, 2004; Xiang et al, 2014), and pfmdr1 encodes for an ABC transporter. Based on previous benzoxaboroles binding to proteins containing bimetal centres, we first decided to sequence Toxoplasma CPSF3 (TGGT1_285200; TgCPSF3), because it has a putative MBL domain with bimetal centre (two zinc ions). In all the AN3661-resistant T. gondii lines that we isolated, we invariably found three single nucleotide polymorphisms (SNPs) leading to one of the following amino acid substitutions: E545K, Y328C and Y483N (Fig 2A). Figure 2. Resistance to AN3661 is mediated by gene variations in TgCPSF3 Strategy used to obtain Toxoplasma gondii resistant lines. The mutations in TgCPSF3 found in four independent resistant mutants are shown. CPSF3 gene editing strategy to introduce mutations into a wild-type parasite. With this methodology, the guide RNA targets the CAS9 editing enzyme to a 20-base pair site on TgCPSF3 in wild-type parasites (green line); after cleavage by CAS9 (vertical dashed line in blue) three nucleotides downstream of the PAM NGG motif (in violet), homology-dependent repair from a 120-base donor oligonucleotide resulted in incorporation of the specific SNP (E545K, Y483N or Y328C). Only E545K (red asterisk) is shown for clarity. The corresponding chromatograms are shown on the right. Nucleotide positions relative to the ATG start codon on genomic DNA are indicated. Download figure Download PowerPoint In humans, CPSF-73 co-assembles in the nucleus into a large complex, including other cleavage/polyadenylation or stimulatory factors and polyadenylate polymerase (PAP). The complex cleaves the 3′-end of pre-mRNAs, which is subsequently polyadenylated (Xiang et al, 2014; Schönemann et al, 2014) before the mRNA is exported into the cytoplasm for translation (Fig EV1A). CPSF-73 provides the endonuclease activity for this complex (Ryan et al, 2004; Dominski et al, 2005; Mandel et al, 2006). When we monitored TgCPSF3 in a T. gondii line expressing the endogenous protein tagged with an HA-FLAG, we found that TgCPSF3 accumulates in the parasite nucleus (Fig EV1B), consistent with a similar function to that of its human counterpart. Click here to expand this figure. Figure EV1. CPSF3 localizes in the nucleus of Toxoplasma gondii Schematic view of the cleavage and polyadenylation specificity complex that, in some eukaryotes, is responsible for processing the newly synthesized pre-mRNAs. CPSF-73, in humans, has the endonuclease activity and acts concertedly with other CPSF subunits, polyadenosine polymerase (PAP) and other factors. Please note that the number of CPSF subunits is dependent on each species and this model is just shown for reference. A homologous recombination-proficient T. gondii strain (RH ku80) was used for endogenous epitope tagging of T. gondii CPSF3-HAFLAG (red) hosted in HFF. Staining of CPSF3-HAFLAG was done with anti-HA antibodies and DNA nuclei with Hoechst (blue). Download figure Download PowerPoint CRISPR/Cas9-mediated point mutations in TgCPSF3 confer resistance to AN3661 To confirm that TgCPSF3 mutations account for resistance to AN3661, we introduced each of the mutations identified in AN3661-resistant parasites into the T. gondii parental strain using CRISPR/Cas9 gene editing (Fig 2B). After co-transfection with oligonucleotides containing the desired mutations, resistant parasites were selected in the presence of 5 μM AN3661 (> sixfold the IC50 value). Emergent resistant parasites were cloned, and DNA sequencing confirmed that the mutations were correctly introduced into TgCPSF3 (Figs 2B and EV2). No resistant parasite lines emerged following transfection with the CRISPR/Cas9 control vectors alone. Compared to wild-type parasites, mutant lines (each containing only one of the above mutations) had markedly decreased susceptibility to AN3661 (Fig 1B). To corroborate that TgCPSF3 is the bona fide target of AN3661, we expressed a mutated copy of TgCPSF3 (TgCPSF3E545K) in wild-type parasites and evaluated whether the transgene would restore parasite growth in the presence of AN3661. The TgCPSF3E545K cassette was inserted by homologous recombination into the locus coding for the surface antigen protein 1 (SAG1), a non-essential gene, using CRISPR/Cas9 gene editing (Fig EV3A). All the resultant transgenic lines contained the TgCPSF3E545K cassette correctly inserted into the SAG1 locus, as confirmed by both immunofluorescence and genomic analysis (Fig EV3A and B). This extra copy efficiently restored parasite growth in the presence of 5 μM AN3661, indicating that the ectopic expression of mutant TgCPSF3E545K conferred resistance to AN3661. Click here to expand this figure. Figure EV2. AN3661-resistant parasites had mutations in Toxoplasma gondii CPSF3 A, B. Chromatograms of the sequences of wild-type TgCPSF3 and mutants Y328C (A) or Y483N (B). Parasites were transfected with a CRISPR/Cas9 vector producing a single guide RNA (sgY328C, sgY483N or sgE545K) to target the Cas9 editing enzyme to 20-bp sites on wild-type CPSF3 (see Fig 2). After cleavage by Cas9, homology-dependent repair from a 120-base donor oligonucleotide (Y328C, Y483N or E545K) resulted in incorporation of the specific SNP (shown in red). For clarity, only chromatograms of Y328 and Y483N are shown. E545K data is shown in Fig 2B. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Insertion of CPSF3E545K (AN3661-resistant) in the SAG1 locus Schematic overview of the SAG1 gene editing strategy with CRISPR/Cas9 and the CPSF3E545K resistance cassette. Wild-type parasites (RH ku80) were transfected with the CRISPR/Cas9 vector, producing sgSAG1 RNA that targets Cas9 to the SAG1 coding sequence. After cleavage by Cas9, homology-dependent repair was directed by a donor PCR amplicon encompassing CPSF3E545K coding sequence with 5′ and 3′ regulatory sequences flanked by 60-bp sequences homologous to SAG1. Transfected parasites were selected in the presence of 5 μM AN3661. Insertion of the CPSF3E545K cassette within SAG1 was verified by PCR analysis in all clones using the indicated primers (in blue); bands at 6,500 bp indicate correct insertion of CPSF3E545K, and the band at 370 bp is specific for wild-type SAG1. Detection of SAG1 by immunofluorescence. The parental wild-type strain (RH ku80) and a SAG1 mutant strain [RH ku80 SAG1::CPSF3E545K, clone #1 in (A)] were stained using anti-SAG1 antibodies. Scale bars represent 10 μm. Download figure Download PowerPoint Mutations conferring resistance are clustered at the endonucleolytic site of TgCPSF3 We built a structural homology model of TgCPSF3 with a pre-mRNA substrate bound into the catalytic site using structures of mammalian CPSF-73 and bacterial J/Z RNases that contained a metallo-β-lactamase (MBL) domain (Fig 3A). Similar to eukaryotic homologues, TgCPSF3 contained an MBL domain, a β-CASP domain and a C-terminal domain with a putative endonuclease site at the interface between the MBL and β-CASP domains (Fig 3A). TgCPSF3 showed strict conservation of the catalytic motifs, including highly conserved histidine and aspartic/glutamic acid residues, which coordinate the two zinc atoms involved in the cleavage of the 3′-end of pre-mRNAs (Fig EV4). The three mutated residues associated with AN3661 resistance (Y328C, Y483N and E545K) clustered to one side of the catalytic site, which, by homology to other CPSF-73 or bacterial RNases, binds the 3′-end of pre-mRNAs (Fig 3A–C). In fact, one of the mutated residues, Y483, was described as important for positioning of the 3′-end of the pre-mRNA by forming the closing gate at the catalytic site of human CPSF-73 (Mandel et al, 2006). To investigate the binding of the inhibitor, we performed in silico molecular docking of AN3661 into the homology model of TgCPSF3, and found that AN3661 favourably fits (docking Glide score ~6 kcal/mol) into the catalytic site of TgCPSF3, and the placement mimics the position of the 3′-end of the mRNA substrate (Fig 3C and D). More specifically, the tetrahedral boron atom of AN3661 occupies the position of the cleavage site phosphate (second to last, Pi−1) of the mRNA substrate near the catalytic site, with one hydroxyl group interacting with a zinc atom (Fig 3C and D). This is consistent with structures for other benzoxaboroles that were shown to bind to the bimetal centres of beta-lactamases and phosphodiesterase-4 (Xia et al, 2011; Freund et al, 2012). In this conformation, the aromatic ring AN3661 favourably binds into the inverted V-shape pocket formed by the aromatic residues Y328 and Y483. In addition, the carboxylic group of AN3661 establishes hydrogen bonds to the side chains of Y366 and S519, and to the main chain backbone atoms of Y483 and A520. Considering the clustered position of the mutations conferring resistance to AN3661 in the catalytic site of TgCPSF3, it is likely that the compound binds into this site and perturbs the pre-mRNA processing activity that is essential for parasite growth. Figure 3. Structural homology model of TgCPSF3 and analysis of resistance mutations A. Domain architecture and structural model of TgCPSF3 with a pre-mRNA substrate bound into the catalytic site. TgCPSF3 residues are shown in cartoon and surface representation, with the following colour code: metallo-β-lactamase in turquoise, β-CASP domain in yellow and C-terminal domain in pink. A pre-mRNA substrate (5-mer) is shown for reference as green sticks, and the two catalytic zinc atoms are shown as spheres. Mutations identified in AN3661-resistant strains of Toxoplasma gondii are in the TgCPSF3 catalytic site and are shown as red sticks. The protein model was built using the structures of eukaryotic/archaeal CPSF homologues [Protein Data Bank (PDB) accession codes: 3AF5, 2I7V] and bacterial RNases Z/J (PDB codes: 3A4Y, 3IEM and 3AF5). B. Comparison of sequences in homologous proteins near the residues mutated in TgCPSF3. The mutated positions found in T. gondii and Plasmodium falciparum parasites that were resistant to AN3661 are pointed by arrows. Sequences shown are from T. gondii (Tg), Hammondia hamondi (Hh), Neospora caninum Liverpool (Nc), Cryptosporidium parvum Iowa II (Cp), Babesia bigemina (Bb), Theileria equi strain WA (Te) and P. falciparum 3D7 (Pf). C. Zoomed-in view of the TgCPSF3 mRNA cleavage site showing the three residues which are changed in the resistant mutants: Y328, Y483 and E545. The 3′-mRNA substrate (shown as green sticks) was docked in the catalytic site by using as templates bacterial RNase complexes with RNA (PDB codes: 3IEM and 5A0T). mRNA phosphates are labelled (Pi) and Pi-1 corresponds to the position of cleavage. The zinc atoms (shown as spheres) were modelled by using the metallo-β-lactamase and β-CASP domains of RNase J (PDB code: 5A0T). D. In silico docking of AN3661 into the catalytic site of the TgCPSF3. The docking position was calculated with Glide in Maestro. Protein surface, resistant mutants and Zn ions are colour-coded as in (A), and AN3661 is shown in sticks-surface-overlapped representation, with carbon in purple sticks, oxygen in red sticks and boron in pink sticks. Protein residues interacting with AN3661 are shown as sticks, and hydrogen bonds are depicted are green-dashed lines. E–G. Modelling of resistance mutations Y328C (F), Y483N (E) and E545K (G). The position of the mutations was modelled in Coot by using the most favourable rotamer conformation. In the case of the mutant Y483N, two rotamers (rot1 and rot2) were similarly favourable. The expected rearrangements as a consequence of the mutations are represented by curved arrows. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Conservation of catalytic residues in Toxoplasma gondii CPSF3 A, B. Coordination of Zn atoms at the catalytic sites of the T. gondii CPSF3 model (A), same model as in Fig 3A, and Streptomyces coelicolor RNase J (B), PDB: 5A0T. The position of the phosphate of the 3′-mRNA at the cleavage position is shown for reference. Key interactions are shown as red dashed lines. C. Sequence alignment of regions constituting the catalytic sites of CPSF3 homologous proteins of Toxoplasma gondii (Tg), Plasmodium falciparum (Pf), Homo sapiens (Hs) and of RNase J from Streptomyces coelicolor (Sc). Download figure Download PowerPoint We then extended this structural analysis to understand how the mutations confer resistance to AN3661. This analysis shows that rather than clashing with AN3661, the mutations Y483N and Y328C distort the geometry of the drug binding pocket and would lead to loss of contacts between the protein and AN3661 that decrease the affinity (Fig 3D–F). The mutation E545K has an indirect effect on the drug binding pocket that is mediated by Y483, again likely via the perturbation of the drug binding pocket (Fig 3D–G). The side chain of E545 is in a favourable conformation at 4.6 Å of Y483. The change to lysine introduces a positive charge and a bulky side chain that would clash to Y483 as it gets as close as 1.8 Å. Therefore, it is likely that the rearrangement of Y483 to prevent the clash with K545 impacts negatively on the affinity of AN3661. As the mutated residues in Plasmodium (Y406, D470) are equivalent to the ones we found in T. gondii (Fig 3B), it is very much possible that the resistance mechanism is shared. It is also interesting to note that all the mutated residues are conserved among other apicomplexan parasites (Fig 3B). In vivo efficacy of AN3661 in a murine model of acute toxoplasmosis and development of protective i
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