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Human coronaviruses (HCoVs) cause respiratory tract infections and are of great importance due to the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Human betacoronavirus OC43 (HCoV-OC43) is an adequate surrogate for SARS-CoV-2 because it infects the human respiratory system, presents a comparable biology, and is transmitted in a similar way. Its use is advantageous since it only requires biosafety level (BSL)-2 infrastructure which minimizes costs and biosafety associated limitations. In this report, we describe a high-throughput screening (HTS) platform to identify compounds that inhibit the propagation of HCoV-OC43. Optimization of assays based on inhibition of the cytopathic effect and virus immunodetection with a specific antibody, has provided a robust methodology for the screening of a selection of microbial natural product extracts from the Fundación MEDINA collection. Using this approach, a subset of 1280 extracts has been explored. Of these, upon hit confirmation and early LC-MS dereplication, 10 extracts were identified that contain potential new compounds. In addition, we report on the novel antiviral activity of some previously described natural products whose presence in bioactive extracts was confirmed by LC/MS analysis., This work was funded by the the European Commission—Next Generation EU (regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global), the Instituto de Salud Carlos III Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014), the MCIN/AEI/10.13039/501100011033 (PID2019-109623RB-I00), the MCIN/AEI/10.13039/501100011033 and FEDER Una manera de hacer Europa (2016-79957-R) and by the Junta de Andalucía (BIO-199)

Inosine triphosphate pyrophosphatases (ITPases) are ubiquitous house-cleaning enzymes that specifically recognize deaminated purine nucleotides and catalyze their hydrolytic cleavage. In this work, we have characterized the Trypanosoma brucei ITPase ortholog (TbITPA). Recombinant TbITPA efficiently hydrolyzes (deoxy)ITP and XTP nucleotides into their respective monophosphate form. Immunolocalization analysis performed in bloodstream forms suggests that the primary role of TbITPA is the exclusion of deaminated purines from the cytosolic nucleoside triphosphate pools. Even though ITPA-knockout bloodstream parasites are viable, they are more sensitive to inhibition of IMP dehydrogenase with mycophenolic acid, likely due to an expansion of IMP, the ITP precursor. On the other hand, TbITPA can also hydrolyze the activated form of the antiviral ribavirin although in this case, the absence of ITPase activity in the cell confers protection against this nucleoside analog. This unexpected phenotype is dependant on purine availability and can be explained by the fact that ribavirin monophosphate, the reaction product generated by TbITPA, is a potent inhibitor of trypanosomal IMP dehydrogenase and GMP reductase. In summary, the present study constitutes the first report on a protozoan inosine triphosphate pyrophosphatase involved in the removal of harmful deaminated nucleotides from the cytosolic pool.

Kinetoplastid and apicomplexan parasites comprise a group of protozoans responsible for human diseases, with a serious impact on human health and the socioeconomic growth of developing countries. Chemotherapy is the main option to control these pathogenic organisms and nucleotide metabolism is considered a promising area for the provision of antimicrobial therapeutic targets. Impairment of thymidylate (dTMP) biosynthesis severely diminishes the viability of parasitic protozoa and the absence of enzymatic activities specifically involved in the formation of dTMP (e.g. dUTPase, thymidylate synthase, dihydrofolate reductase or thymidine kinase) results in decreased deoxythymidine triphosphate (dTTP) levels and the so-called thymineless death. In this process, the ratio of deoxyuridine triphosphate (dUTP) versus dTTP in the cellular nucleotide pool has a crucial role. A high dUTP/dTTP ratio leads to uracil misincorporation into DNA, the activation of DNA repair pathways, DNA fragmentation and eventually cell death. The essential character of dTMP synthesis has stimulated interest in the identification and development of drugs that specifically block the biochemical steps involved in thymine nucleotide formation. Here, we review the available literature in relation to drug discovery studies targeting thymidylate biosynthesis in kinetoplastid (genera Trypanosoma and Leishmania) and apicomplexan (Plasmodium spp and Toxoplasma gondii) protozoans. The most relevant findings concerning novel inhibitory molecules with antiparasitic activity against these human pathogens are presented herein.

To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity., This work was funded by the Junta de Andalucía (BIO 2059; BIO-199), the Plan Nacional de Investigación Científica (SAF2016-79957-R) and FEDER. We thank Dr Beáta Vértessy for assistance and advice in the implementation of the Pfu-based measurements of uracil-DNA, and Aurora Constán for technical assistance.

The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA replication fidelity.

The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA replication fidelity. Trypanosoma brucei makes use of both the salvage and de novo pathways for the provision of pyrimidine dNTPs. In this respect, the sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) appears to be the most relevant dNTPase controlling dNTP/deoxynucleoside homeostasis in mammalian cells. Here, we have characterized the role of a unique trypanosomal SAMHD1 orthologue denominated TbHD52. Our results show that TbHD52 is a mitochondrial enzyme essential in bloodstream forms of T. brucei. Knockout cells are pyrimidine auxotrophs that exhibit strong defects in genomic integrity, cell cycle progression, and nuclear DNA and kinetoplast segregation in the absence of extracellular thymidine. The lack of TbHD52 can be counteracted by the overexpression of human dCMP deaminase, an enzyme that is directly involved in dUMP formation yet absent in trypanosomes. Furthermore, the cellular dNTP quantification and metabolomic analysis of TbHD52 null mutants revealed perturbations in the nucleotide metabolism with a substantial accumulation of dCTP and cytosine-derived metabolites while dTTP formation was significantly reduced. We propose that this HD-domain-containing protein unique to kinetoplastids plays an essential role in pyrimidine dNTP homeostasis and contributes to the provision of deoxycytidine required for cellular dTTP biosynthesis.

In the fight against Malaria, new strategies need to be developed to avoid resistance of the parasite to pharmaceutics and other prevention barriers. Recently, a Host Directed Therapy approach based on the suppression of the starting materials uptake from the host by the parasite has provided excellent results. In this article, we propose the synthesis of bioisosteric compounds that are capable of inhibiting Plasmodium falciparum Choline Kinase and therefore to reduce choline uptake, which is essential for the development of the parasite. Of the 41 bioisosteric compounds reported herein, none showed any influence of the linker on the antimalarial and enzyme inhibitory activity, whereas an effect of the type of cationic heads used could be observed. SARs determined that the thienopyrimidine substituted in 4 by a pyrrolidine is the best scaffold, independently of the chosen linker. The decrease in lipophilicity seems to improve the antimalarial activity but to cause an opposite effect on the inhibition of the enzyme. While potent compounds with similar good inhibitory values have been related to the proposed mechanism of action, some of them still show discrepancies and further studies are needed to determine their specific molecular target.

A novel family of four potent antimalarial macrolides, strasseriolides A-D (

A novel family of four potent antimalarial macrolides, strasseriolides A−D (1−4), has been isolated from cultures of Strasseria geniculata CF-247251, a fungal strain obtained from plant tissues. The structures of these compounds, including their absolute configurations, were elucidated by HRMS, NMR spectroscopy, and X-ray single-crystal diffraction. The four compounds gave respective IC values of 9.810, 0.013, 0.123, and 0.128 μM against Plasmodium falciparum 3D7 parasites with no significant cytotoxicity against the HepG2 cell line., This work was funded by the European Commission FP7 Marie Curie Initial Training Network “ParaMet” [grant agreement 290080], the Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014, RD16/0027/0015, and RD12/0018/0005), and the Plan Nacional (SAF2016-79957-R). The polarimeter, HPLC, IR, NMR equipment, and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovación [Grants Nos. PCT-010000-2010-4 (NMR), INP-2011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR), and PCT-01000-ACT7, 2011-13 (plate reader)]. D.Ch.-L. acknowledges funding by project No. PGC2018-102047-B-I00 (MCIU/AEI/FEDER, UE).

Human African trypanosomiasis is a neglected tropical disease (NTD) that is fatal if left untreated. Although approximately 13 million people live in moderate- to high-risk areas for infection, current treatments are plagued by problems with safety, efficacy, and emerging resistance. In an effort to fill the drug development pipeline for HAT, we have expanded previous work exploring the chemotype represented by the compound NEU-1090, with a particular focus on improvement of absorption, distribution, metabolism and elimination (ADME) properties. These efforts resulted in several compounds with substantially improved aqueous solubility, although these modifications typically resulted in a loss of trypanosomal activity. We herein report the results of our investigation into the antiparasitic activity, toxicity, and ADME properties of this class of compounds in the interest of informing the NTD drug discovery community and avoiding duplication of effort.

The levels of the four deoxynucleoside triphosphates (dNTPs) are under strict control in the cell, as improper or imbalanced dNTP pools may lead to growth defects and oncogenesis. Upon treatment of cancer cells with therapeutic agents, changes in the canonical dNTPs levels may provide critical information for evaluating drug response and mode of action. The radioisotope-labeling enzymatic assay has been commonly used for quantitation of cellular dNTP levels. However, the disadvantage of this method is the handling of biohazard materials. Here, we described the use of click chemistry to replace radioisotope-labeling in template-dependent DNA polymerization for quantitation of the four canonical dNTPs. Specific oligomers were designed for dCTP, dTTP, dATP and dGTP measurement, and the incorporation of 5-ethynyl-dUTP or C8-alkyne-dCTP during the polymerization reaction allowed for fluorophore conjugation on immobilized oligonucleotides. The four reactions gave a linear correlation coefficient >0.99 in the range of the concentration of dNTPs present in 106 cells, with little interference of cellular rNTPs. We present evidence indicating that data generated by this methodology is comparable to radioisotope-labeling data. Furthermore, the design and utilization of a robust microplate assay based on this technology evidenced the modulation of dNTPs in response to different chemotherapeutic agents in cancer cells., We thank Hsin-Yen Wang for maintaining K562 cells. This study was financially supported by the “Center of Precision Medicine” from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by the grant, MOST 107-3017-F-002-002, MOST 107-2321-B-002-041, and MOST 106-2113-M-002-021-MY3 from the Ministry of Science and Technology, Taiwan, (R.O.C.).

Uracil-DNA glycosylase (UNG) initiates the base excision repair pathway by excising uracil from DNA. We have previously shown that Trypanosoma brucei cells defective in UNG exhibit reduced infectivity thus demonstrating the relevance of this glycosylase for survival within the mammalian host. In the early steps of the immune response, nitric oxide (NO) is released by phagocytes, which in combination with oxygen radicals produce reactive nitrogen species (RNS). These species can react with DNA generating strand breaks and base modifications including deaminations. Since deaminated cytosines are the main substrate for UNG, we hypothesized that the glycosylase might confer protection towards nitrosative stress. Our work establishes the occurrence of genotoxic damage in Trypanosoma brucei upon exposure to NO in vitro and shows that deficient base excision repair results in increased levels of damage in DNA and a hypermutator phenotype. We also evaluate the incidence of DNA damage during infection in vivo and show that parasites recovered from mice exhibit higher levels of DNA strand breaks, base deamination and repair foci compared to cells cultured in vitro. Notably, the absence of UNG leads to reduced infectivity and enhanced DNA damage also in animal infections. By analysing mRNA and protein levels, we found that surviving UNG-KO trypanosomes highly express tryparedoxin peroxidase involved in trypanothione/tryparedoxin metabolism. These observations suggest that the immune response developed by the host enhances the activation of genes required to counteract oxidative stress and emphasize the importance of DNA repair pathways in the protection to genotoxic and oxidative stress in trypanosomes., This work was funded by the Junta de Andalucía (BIO-199); the Plan Nacionalde Investigación Científica, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014) and FEDER; and the Plan Nacional (SAF2016-79957-R). We thank Dr Carlos Robello for kindly providing the anti-TbTRYP1 antibody and Dr David Horn for the anti-TbγH2A antibody.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2′-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 A. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

Kinetoplastid parasites are responsible for serious diseases in humans and livestock such as Chagas disease and sleeping sickness (caused by Trypanosoma cruzi and Trypanosoma brucei, respectively), and the different forms of cutaneous, mucocutaneous and visceral leishmaniasis (produced by Leishmania spp). The limited number of antiparasitic drugs available together with the emergence of resistance underscores the need for new therapeutic agents with novel mechanisms of action. The use of agents binding to surface glycans has been recently suggested as a new approach to antitrypanosomal design and a series of peptidic and non-peptidic carbohydrate-binding agents have been identified as antiparasitics showing efficacy in animal models of sleeping sickness. Here we provide an overview of the nature of surface glycans in three kinetoplastid parasites, T. cruzi, T. brucei and Leishmania. Their role in virulence and host cell invasion is highlighted with the aim of identifying specific glycan–lectin interactions and carbohydrate functions that may be the target of novel carbohydrate-binding agents with therapeutic applications., Financial support was received from the Junta de Andalucía (BIO-199 P12-BIO-2059); the Plan Nacional, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014) and the Plan Nacional de Investigación Científica (SAF2016-79957-R) and the FEDER funds from the EU.

[Background] Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5′-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported., [Methods] To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein., [Results] Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3′ replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5′-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol., [Conclusion] These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages., This work was funded by the Junta de Andalucía (BIO-199); the Plan Nacional de Investigación Científica, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014); The European Union (contract 037587); and the Plan Nacional (SAF2016-79957-R).

In this article we present a series of non-cytotoxic potent human choline kinase (CK) inhibitors that exhibit nanomolar antiplasmodial activity in vitro. The most active antiplasmodial compounds, 10a-b, bearing a pyridinium cationic head were inactive against CK, while compounds 10g and 10j with a quinolinium moiety exhibit moderate inhibition of both the parasite and the enzyme. The results point towards an additional mechanism of action unrelated to CK inhibition that remains to be established., This work was funded by the University of Granada, Cei-Biotic Project CEI2013-MP-1; the Junta de Andalucia (BIO-199); the Plan Nacional de Investigacion Cientifica, Instituto de Salud Carlos III-Subdireccion General de Redes y Centros de Investigacion Cooperativa-Red de Investigacion Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014); and the Plan Nacional (SAF2016-79957-R).

A potent antiplasmodial polycyclic xanthone, MDN-0185 (1), was isolated from an unidentified species of the genus Micromonospora. The planar structure of 1 was established as a seven-ring polycyclic xanthone with partial structures very similar to two known natural products, namely, xantholipin and Sch 54445. Using ROESY correlations, the relative stereochemistry of the two independent stereoclusters of compound 1 could be determined. Mosher analysis and comparison of the specific rotation of compound 1 with that of xantholipin allowed the determination of its absolute configuration. Compound 1 exhibited an IC50 of 9 nM against Plasmodium falciparum 3D7 parasites., This work was funded by the European Commission FP7 Marie Curie Initial Training Network >ParaMet> (FP7-PEOPLE-2011-ITN, GA290080), the Junta de Andalucia (BIO-199, P12-BIO-2059), the Plan Nacional de Investigacion Cientifica, Instituto de Salud Carlos III-Subdireccion General de Redes y Centros de Investigacion Cooperativa-Red de Investigacion Cooperativa en Enfermedades Tropicales (RICET: RD16/0027/0014 and RD16/0027/0015), and the Plan Nacional (SAF2016-79957-R). The polarimeter, HPLC, IR, NMR equipment, and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovation [Grant Nos. PCT-010000-2010-4 (NMR), INP-2011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR), and PCT-01000-ACTT, 2011-13 (plate reader)].

Bioassay-guided compound isolation led to the discovery of two new scalarane sesterterpenes (1 and 2) and two new triterpenes (3 and 4) from two mushroom species, Pleurotus ostreatus (edible) and Scleroderma areolatum, collected from Ghana. Their structures, including absolute stereochemistry, were established by spectroscopic methods, particularly (+)-ESI-TOF mass spectrometry and 1D and 2D NMR. The four compounds exhibited IC50 values of 1.65-7.63 μM against Plasmodium falciparum 3D7 and 5.04-13.65 μM against Trypanosoma cruzi Tulahuen C4 parasites and were also non-cytotoxic against HepG2 tumoral human liver cells. This is the first report describing the isolation of sesterterpenes belonging to the scalarane structural class from a terrestrial source., The present work was funded by the European Commission FP7 Marie Curie Initial Training Network >ParaMet> (grant agreement 290080), ISCIII Project >RD16/0027/0015> Tropical Diseases Network, Subprogram RETICS from Plan Estatal de I + D + I 2013-2016 and co-financed by FEDER: A way to make Europe. The polarimeter, HPLC, IR, NMR equipment, and plate reader used in this work were purchased via grants for scientific and technological infrastructures from the Ministerio de Ciencia e Innovacion [Grants No. PCT-010000-2010-4 (NMR), INP-2011-0016-PCT-010000 ACT6 (polarimeter, HPLC, and IR), and PCT-01000-ACTT, 2011-13 (plate reader)].

In 2014, a published report of the high-throughput screen of>42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery., Boettcher Foundation's Webb-Waring Biomedical Research Program; Boettcher Collaboration Grant; Research Corporation Cottrell College Science Award; National Institute of Allergy and Infectious Diseases (US):R01AI114685; Ministerio de Economia, Industria y Competitividad (España) SAF2015-71444-P; SAF2015-68042-R; SAF2016-79957-R; Junta de Andalucia CTS-7282; Subdireccion General de Redes y Centros de Investigacion Cooperativa (RICET) RD16/0027/0019, DG-P RD16/0027/0014; NSF-MRI CHE-1429567

Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS fromLeishmania major(the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca2+ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg2+ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures ofL. majorFPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

The Tritryps Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani are responsible for great morbidity and mortality in developing countries. Their dimeric dUTPases are members of the all-α NTP pyrophosphohydrolase family and represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared with the trimeric human enzyme. In the present paper we describe the structure of the T. brucei enzyme in open and closed conformations. Furthermore, we probe the reaction mechanism through the binding of transition state mimics both in solution and in the crystal. 31P-NMR and tryptophan fluorescence quenching in the presence of AlF3 and MgF3− identified which phosphate is subject to nucleophilic attack by a water molecule. The structures in complex with two transition state analogues confirm that the nucleophilic attack occurs on the β-phosphate in contrast with the α-phosphate in the trimeric enzymes. These results establish the structural basis of catalysis of these important housekeeping enzymes and has ramifications for the wider all-α NTP pyrophosphohydrolase family.

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T.brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

Trypanosomal all-alpha dUTPases are homodimeric enzymes that catalyze the hydrolysis of dUTP and dUDP to dUMP and PPi. Trypanosomes lack dCTP/dCMP deaminase and therefore strongly depend on dUDP/dUTP hydrolysis for dUMP production. Here we have addressed by gene replacement the consequences of elimination of dUTPase activity in bloodstream forms of Trypanosoma brucei. We first generated conditional DUT-knockout strains that allowed an effective decrease of dUTPase resulting in proliferation arrest, although gene repression could not be sustained long enough to cause lethality. Alternatively, DUT null mutants could be isolated in the presence of high levels of thymidine while exogenous supplementation with uracil, uridine or deoxyuridine could not complement metabolically the dUTPase deficiency. Upon thymidine removal, trypanosomes exhibited impaired proliferation and eventually died. These data establish a strict requirement for dUTPase in T. brucei viability and support a major role of the enzyme in the provision of pyrimidine nucleotides in kinetoplastids.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT., Funding: This work was supported by grants from the VI Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III - Subdirección General de Redes y Centros de Investigación Cooperativa, Red de Investigación Cooperativa en Enfermedades Tropicales (RICET) (RD06/0021), the Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016 (SAF2013-48999-R) (http://www.idi.mineco.gob.es), and the Junta de Andalucía (BIO-199, P09-CVI-5367) to DGP. Research of JB and SL was supported by the Katholieke Universiteit Leuven (PF 10/18). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Decitabine (5-aza-2′-deoxycytidine, aza-dCyd) is an anti-cancer drug used clinically for the treatment of myelodysplastic syndromes and acute myeloid leukaemia that can act as a DNA-demethylating or genotoxic agent in a dose-dependent manner. On the other hand, DCTPP1 (dCTP pyrophosphatase 1) and dUTPase are two ‘house-cleaning’ nucleotidohydrolases involved in the elimination of non-canonical nucleotides. In the present study, we show that exposure of HeLa cells to decitabine up-regulates the expression of several pyrimidine metabolic enzymes including DCTPP1, dUTPase, dCMP deaminase and thymidylate synthase, thus suggesting their contribution to the cellular response to this anti-cancer nucleoside. We present several lines of evidence supporting that, in addition to the formation of aza-dCTP (5-aza-2′-deoxycytidine-5′-triphosphate), an alternative cytotoxic mechanism for decitabine may involve the formation of aza-dUMP, a potential thymidylate synthase inhibitor. Indeed, dUTPase or DCTPP1 down-regulation enhanced the cytotoxic effect of decitabine producing an accumulation of nucleoside triphosphates containing uracil as well as uracil misincorporation and double-strand breaks in genomic DNA. Moreover, DCTPP1 hydrolyses the triphosphate form of decitabine with similar kinetic efficiency to its natural substrate dCTP and prevents decitabine-induced global DNA demethylation. The data suggest that the nucleotidohydrolases DCTPP1 and dUTPase are factors involved in the mode of action of decitabine with potential value as enzymatic targets to improve decitabine-based chemotherapy.

Plasmodium falciparum thymidylate kinase (PfTMPK) is a key enzyme in pyrimidine nucleotide biosynthesis. 3-Trifluoromethyl-4-chloro-phenyl-urea-α-thymidine has been reported as an inhibitor of Mycobacterium tuberculosis TMPK (MtTMPK). Starting from this point, we designed, synthesized and evaluated a number of thymidine analogues as antimalarials. Both 5'-urea-α- and β-thymidine derivatives were moderate inhibitors of PfTMPK and furthermore showed moderate inhibition of parasite growth. The structure of several enzyme-inhibitor complexes provides a basis for improved inhibitor design. However, we found that certain 5'-urea-α-thymidine analogues had antimalarial activity where inhibition of PfTMPK is not the major mode of action. Optimization of this series resulted in a compound with potent antimalarial activity (EC(50) = 28 nM; CC(50) = 29 μM).