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As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcDGRA7 and NcDROP40. For NcDGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcDROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfrts marker were integrated into the genomic DNA during gene editing by CRISPRCas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains. [ABSTRACT FROM AUTHOR]

As for many other organisms, CRISPR-Cas9 mediated genetic modification has gained increasing importance for the identification of vaccine candidates and drug targets in Neospora caninum, an apicomplexan parasite causing abortion in cattle and neuromuscular disease in dogs. A widely used approach for generating knock-out (KO) strains devoid of virulence factors is the integration of a drug selectable marker such as mutated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) into the target gene, thus preventing the synthesis of respective protein and mediating resistance to pyrimethamine. However, CRISPR-Cas9 mutagenesis is not free of off-target effects, which can lead to integration of multiple mdhfr-ts copies into other sites of the genome. To determine the number of integrated mdhfr-ts in N. caninum, a duplex quantitative TaqMan PCR was developed. For this purpose, primers were designed that amplifies a 106 bp fragment from wild-type (WT) parasites corresponding to the single copy wtdhfrs-ts gene, as well as the mutated mdhfrs-ts present in KO parasites that confers resistance and were used simultaneously with primers amplifying the diagnostic NC5 gene. Thus, the dhfr-ts to NC5 ratio should be approximately 1 in WT parasites, while in KO parasites with a single integrated mdhrf-ts gene this ratio is doubled, and in case of multiple integration events even higher. This approach was applied to the Neospora KO strains NcΔGRA7 and NcΔROP40. For NcΔGRA7, the number of tachyzoites determined by dhfr-ts quantification was twice the number of tachyzoites determined by NC5 quantification, thus indicating that only one mdhfr-ts copy was integrated. The results obtained with the NcΔROP40 strain, however, showed that the number of dhfr-ts copies per genome was substantially higher, indicating that at least three copies of the selectable mdhfr-ts marker were integrated into the genomic DNA during gene editing by CRISPR-Cas9. This duplex TaqMan-qPCR provides a reliable and easy-to-use tool for assessing CRISPR-Cas9 mediated mutagenesis in WT N. caninum strains.

The critical enzyme dihydrofolate reductase-thymidylate synthase in Leishmania major (LmDHFR-TS) serves a dual-purpose role and is essential for DNA synthesis, a cornerstone of the parasite's reproductive processes. Consequently, the development of inhibitors against LmDHFR-TS is crucial for the creation of novel anti-Leishmania chemotherapies. In this study, we employed an in-house database containing 314 secondary metabolites derived from cinnamic acid that occurred in the Asteraceae family. We conducted a combined ligand/structure-based virtual screening to identify potential inhibitors against LmDHFR-TS. Through consensus analysis of both approaches, we identified three compounds, i.e., lithospermic acid (237), diarctigenin (306), and isolappaol A (308), that exhibited a high probability of being inhibitors according to both approaches and were consequently classified as promising hits. Subsequently, we expanded the binding mode examination of these compounds within the active site of the test enzyme through molecular dynamics simulations, revealing a high degree of structural stability and minimal fluctuations in its tertiary structure. The in silico predictions were then validated through in vitro assays to examine the inhibitory capacity of the top-ranked naturally occurring compounds against LmDHFR-TS recombinant protein. The test compounds effectively inhibited the enzyme with IC50 values ranging from 6.1 to 10.1 μM. In contrast, other common cinnamic acid derivatives (i.e., flavonoid glycosides) from the Asteraceae family, such as hesperidin, isovitexin 4′-O-glucoside, and rutin, exhibited low activity against this target. The selective index (SI) for all tested compounds was determined using HsDHFR with moderate inhibitory effect. Among these hits, lignans 306 and 308 demonstrated the highest selectivity, displaying superior SI values compared to methotrexate, the reference inhibitor of DHFR-TS. Therefore, continued research into the anti-leishmanial potential of these C6C3-hybrid butyrolactone lignans may offer a brighter outlook for combating this neglected tropical disease. [ABSTRACT FROM AUTHOR]

The goal of this study was to genetically link natural materials derived from Tinospora crispa L with Berberine to dihydrofolate reductase-thymidylate synthase (DHFR-TS). Method: The ligand berberine (CID: 2353) was obtained from Pubchem, while the protein DHFR-TS (PDB ID 2bl9) was obtained from Protein Data Bank. The ligands and proteins interacted with HEX 8.0.0.0 and were visualized with Discovery Studio. The researchers discovered a positive interaction between berberine and DHFR-TS, observed at four amino acid residues that bind to the protein TYR125, ILE121, LEU45, and MET54. Van der Waals interactions, hydrogen bonds, Pi-Sulfur, Pi-Alkyl, and Pi-Stalked interactions all contribute to strength and stability. In conclusion, berberine has the potential to act as a DHFR-TS inhibitor and thus prevent malaria. ABSTRAK: Tujuan penelitian ini adalah memanfaatkan bahan alam yang berasal dari Tinospora crispa L dengan kandungan utama Berberin dengan dihydrofolate reductase-thymidylate synthase (DHFR-TS) secara genetik. Metode, Ligan berberin (CID: 2353 ) diperoleh dari Pubchem sedangkan protein DHFR-TS (PDB ID 2bl9) diperoleh dari Protein Data Bank, ligan dan protein diinteraksikan menggunakan HEX 8.0.0.0 dan divisualisasikan menggunakan discovery studio. Ditemukan interaksi positif antara berberin dan DHFR-TS yang menunjukkan interaksi pada empat residu asam amino yang berikatan dengan protein. Mereka adalah TYR125, ILE121, LEU45 dan MET54. Beberapa interaksi yang dilakukan Van der Waals, ikatan hidrogen, Pi-Sulfur, Pi-Alkyl dan Pi-Stalked juga memberikan dukungan dalam rangka meningkatkan kekuatan dan stabilisasi. Kesimpulannya, berberin memiliki potensi fungsi sebagai penghambat DHFR-TS dan mengarah pada malaria.

The bifunctional enzyme Dihydrofolate reductase-thymidylate synthase (DHFR-TS) plays a crucial role in the survival of the Leishmania parasite, as folates are essential cofactors for purine and pyrimidine nucleotide biosynthesis. However, DHFR inhibitors are largely ineffective in controlling trypanosomatid infections, largely due to the presence of Pteridine reductase 1 (PTR1). Therefore, the search for structures with dual inhibitory activity against PTR1/DHFR-TS is crucial in the development of new anti-Leishmania chemotherapies. In this research, using the Leishmania major DHFR-TS recombinant protein, enzymatic inhibitory assays were performed on four kauranes and two derivatives that had been previously tested against LmPTR1. The structure 302 (6.3 µM) and its derivative 302a (4.5 µM) showed the lowest IC50 values among the evaluated molecules. To evaluate the mechanism of action of these structures, molecular docking calculations and molecular dynamics simulations were performed using a DHFR-TS hybrid model. Results showed that hydrogen bond interactions are critical for the inhibitory activity against LmDHFR-TS, as well as the presence of the p-hydroxyl group of the phenylpropanoid moiety of 302a. Finally, additional computational studies were performed on DHFR-TS structures from Leishmania species that cause cutaneous and mucocutaneous leishmaniasis in the New World (L. braziliensis, L. panamensis, and L. amazonensis) to explore the targeting potential of these kauranes in these species. It was demonstrated that structures 302 and 302a are multi-Leishmania species compounds with dual DHFR-TS/PTR1 inhibitory activity. [ABSTRACT FROM AUTHOR]

Malaria is one of the major causes of health and disability globally, even after tremendous efforts to eradicate it. Till date no highly effective vaccine is available for its control. The primary reason for the low efficacy of vaccines is extensive polymorphism in potential vaccine candidate antigen genes and HLA polymorphisms in the human population. This problem can be resolved by developing a vaccine using promiscuous peptides to combine the number of HLA alleles. This study predicted T and B cell epitopes (promiscuous peptides) by targeting PPPK-DHPS and DHFR-TS proteins of Plasmodium vivax, using different in silico tools. Selected peptides were characterized as promiscuous peptides on the basis of their immunogenicity, antigenicity and hydrophobicity. Furthermore, to confirm their immunogenicity, these peptides were utilized for molecular modelling and docking analysis. For determining the requisite affinity with distinct HLA Class-I, and HLA Class-II alleles, only five peptides for DHFR-TS and 3 peptides for PPPK-DHPS were chosen as promiscuous peptides. The D1 peptide has the maximum binding energy with HLA alleles, according to HLA-peptide complex modelling and binding interaction analyses. These findings could lead to the development of epitope-based vaccinations with improved safety and efficacy. These epitopes could be major vaccine targets in P. vivax as they possess a higher number of promiscuous peptides. Also, the B cell epitopes possess maximum affinity towards different alleles as analyzed by docking scores. However, further investigation is warranted in vitro and in vivo.

• Ec ΔFΔT was generated by double knockout of the folA and thyA in E. coli BL21(DE3). • Ec ΔFΔT strongly correlated to malaria culture for testing of anti-DHFR inhibitors. • TMP affected antifolate sensitivity of E. coli surrogate, but not Ec ΔFΔT surrogate. • Ec ΔFΔT is an improved surrogate model for antifolate antimalarial screening. A new superior bacteria complementation model was achieved for testing antifolate compounds and investigating antifolate resistance in the dihydrofolate reductase (DHFR) enzyme of the malaria parasite. Earlier models depended on the addition of trimethoprim (TMP) to chemically suppress the host Escherichia coli (Ec) DHFR function. However, incomplete suppression of Ec DHFR and potential interference of antibiotics needed to maintain plasmids for complementary gene expression can complicate the interpretations. To overcome such limitations, the folA (F) and thyA (T) genes were genetically knocked out (Δ) in E. coli BL21(DE3). The resulting Ec ΔFΔT cells were thymidine auxotroph where thymidine supplementation or functional complementation with heterologous DHFR-thymidylate synthase (TS) is needed to restore the loss of gene functions. When tested against pyrimethamine (PYR) and its analogs designed to target Plasmodium falciparum (Pf) DHFR-TS, the 50 % inhibitory concentration values obtained from Ec ΔFΔT surrogates expressing wildtype (Pf TM4) or double mutant (Pf K1) DHFR-TS showed strong correlations to the results obtained from the standard in vitro P. falciparum growth inhibition assay. Interestingly, while TMP had little effect on the susceptibility to PYR and analogs in Ec ΔFΔT expressing Pf DHFR-TS, it hypersensitized the chemically knockdown E. coli BL21(DE3) expressing Pf TM4 DHFR-TS but desensitized the one carrying Pf K1 DHFR-TS. The low intrinsic expression level of Pf TM4 in E. coli BL21(DE3) by western blot analysis may explain the hypersensitive to antifolates of chemical knockdown bacteria surrogate. These results demonstrated the usefulness of Ec ΔFΔT surrogate as a new tool for antifolate antimalarial screening with potential application for investigation of antifolate resistance mechanism. [ABSTRACT FROM AUTHOR]

The bifunctional enzyme Dihydrofolate reductase-thymidylate synthase (DHFR-TS) plays a crucial role in the survival of the Leishmania parasite, as folates are essential cofactors for purine and pyrimidine nucleotide biosynthesis. However, DHFR inhibitors are largely ineffective in controlling trypanosomatid infections, largely due to the presence of Pteridine reductase 1 (PTR1). Therefore, the search for structures with dual inhibitory activity against PTR1/DHFR-TS is crucial in the development of new anti-Leishmania chemotherapies. In this research, using the Leishmania major DHFR-TS recombinant protein, enzymatic inhibitory assays were performed on four kauranes and two derivatives that had been previously tested against LmPTR1. The structure 302 (6.3 µM) and its derivative 302a (4.5 µM) showed the lowest IC50 values among the evaluated molecules. To evaluate the mechanism of action of these structures, molecular docking calculations and molecular dynamics simulations were performed using a DHFR-TS hybrid model. Results showed that hydrogen bond interactions are critical for the inhibitory activity against LmDHFR-TS, as well as the presence of the p-hydroxyl group of the phenylpropanoid moiety of 302a. Finally, additional computational studies were performed on DHFR-TS structures from Leishmania species that cause cutaneous and mucocutaneous leishmaniasis in the New World (L. braziliensis, L. panamensis, and L. amazonensis) to explore the targeting potential of these kauranes in these species. It was demonstrated that structures 302 and 302a are multi-Leishmania species compounds with dual DHFR-TS/PTR1 inhibitory activity.

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC 50 values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs., Competing Interests: The authors declare no conflict of interest.

Background The emergence and spread of Plasmodium falciparum parasites that lack HRP2/3 proteins and the resulting decreased utility of HRP2-based malaria rapid diagnostic tests (RDTs) prompted the World Health Organization and other global health stakeholders to prioritize the discovery of novel diagnostic biomarkers for malaria. Methods To address this pressing need, we adopted a dual, systematic approach by conducting a systematic review of the literature for publications on diagnostic biomarkers for uncomplicated malaria and a systematic in silico analysis of P. falciparum proteomics data for Plasmodium proteins with favorable diagnostic features. Results Our complementary analyses led us to 2 novel malaria diagnostic biomarkers compatible for use in an RDT format: glyceraldehyde 3-phosphate dehydrogenase and dihydrofolate reductase-thymidylate synthase. Conclusions Overall, our results pave the way for the development of next-generation malaria RDTs based on new antigens by identifying 2 lead candidates with favorable diagnostic features and partially de-risked product development prospects. [ABSTRACT FROM AUTHOR]

Abstract Objective Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite’s DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. Results A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug.

Three open-source anti-kinetoplastid chemical boxes derived from a whole-cell phenotypic screening by GlaxoSmithKline (Tres Cantos Anti-Kinetoplastid Screening, TCAKS) were exploited for the discovery of a novel core structure inspiring new treatments of parasitic diseases targeting the trypansosmatidic pteridine reductase 1 (PTR1) and dihydrofolate reductase (DHFR) enzymes. In total, 592 compounds were tested through medium-throughput screening assays. A subset of 14 compounds successfully inhibited the enzyme activity in the low micromolar range of at least one of the enzymes from both Trypanosoma brucei and Lesihmania major parasites (pan-inhibitors), or from both PTR1 and DHFR-TS of the same parasite (dual inhibitors). Molecular docking studies of the protein–ligand interaction focused on new scaffolds not reproducing the well-known antifolate core clearly explaining the experimental data. TCMDC-143249, classified as a benzenesulfonamide derivative by the QikProp descriptor tool, showed selective inhibition of PTR1 and growth inhibition of the kinetoplastid parasites in the 5 μM range. In our work, we enlarged the biological profile of the GSK Kinetobox and identified new core structures inhibiting selectively PTR1, effective against the kinetoplastid infectious protozoans. In perspective, we foresee the development of selective PTR1 and DHFR inhibitors for studies of drug combinations.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) and trypanothione reductase (TR) are important enzymes for the metabolism of protozoan parasites from the family Trypanosomatidae (e.g. Trypanosoma spp., Leishmania spp.) that are targets of current drug-design studies. Very limited information exists on the levels of genetic polymorphism of these enzymes in natural populations of any trypanosomatid parasite. We present results of a survey of nucleotide variation in the genes coding for those enzymes in a large sample of strains from Trypanosoma cruzi, the agent of Chagas' disease. We discuss the results from an evolutionary perspective. A sample of 31 strains show 39 silent and five amino acid polymorphisms in DHFR-TS, and 35 silent and 11 amino acid polymorphisms in TR. No amino acid replacements occur in regions that are important for the enzymatic activity of these proteins, but some polymorphisms occur in sites previously assumed to be invariant. The sequences from both genes cluster in four major groups, a result that is not fully consistent with the current classification of T. cruzi in two major groups of strains. Most polymorphisms correspond to fixed differences among the four sequence groups. Two tests of neutrality show that there is no evidence of adaptive divergence or of selective events having shaped the distribution of polymorphisms and fixed differences in these genes in T. cruzi. However, one nearly significant reduction of variation in the TR sequences from one sequence group suggests a recent selective event at, or close to, that locus.

Abstract Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P. knowlesi dihydrofolate reductase-thymidylate synthase enzyme (Pk DHFR-TS) and its sensitivity to antifolates. The putative Pkdhfr gene was PCR amplified from field isolates collected from the Southern Thailand. Molecular analysis showed 11 polymorphisms in the dhfr domain of the bifunctional dhfr-ts gene. Of these, 1 polymorphism was a non-synonymous substitution (R34L) that had previously been reported but not associated with antifolate resistance. The recombinant Pk DHFR-TS enzyme was found to be sensitive to standard antifolates—pyrimethamine and cycloguanil—as well as P218, a registered candidate drug currently first in human clinical trial. Results suggest that antifolates class of compounds should be effective against P. knowlesi infection. Graphical abstract Unlabelled Image Highlights • Pk DHFR-TS is a catalytically efficient enzyme compared with that of other non-falciparum enzyme. • Its efficiency may contribute to high replication rate and high parasitemia. • Pk DHFR-TS enzyme is sensitive to conventional and clinical trial antifolates. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase ( Pf DHFR-TS) is an important target enzyme in malarial chemotherapy. An understanding of how novel inhibitors interact with wild-type (wt Pf DHFR), quadruple-mutant (qm Pf DHFR), and human ( h DHFR) enzymes is required for the development of these compounds as antimalarials. This study is focused on a series of des -Cl and m -Cl phenyl analogs of pyrimethamine with various flexible 6-substituents. The interactions of these compounds with DHFR enzymes were investigated by 3 D-QSAR, MD simulations, MM-PBSA, and DFT calculations. CoMFA and CoMSIA models were developed with good predictive abilities for wt Pf DHFR and qm Pf DHFR. For h DHFR, CoMSIA models combined with clogP descriptor were successfully derived. Binding free energy using MM-PBSA and comparison of per residue decomposition energy analyses with the DFT method at M06-2X/6-31G ++(d,p) level of theory indicated that Asp54 and Phe58 play important roles in the binding of the most potent compound in the series (compound 27) with both wt Pf DHFR and qm Pf DHFR, whereas Arg59 and Arg122 were additionally found to interact with this inhibitor in qm Pf DHFR. For h DHFR, the residues Glu30 and Phe34 but not Arg70, equivalent to Asp54, Phe58, and Arg122 in Pf DHFR, also play role in compound 27 binding through strong hydrophobic interactions (Phe34) and hydrogen bond network with Glu30, Ile7, and Val115. From the key interactions identified in the DHFR-inhibitor complexes, a general scheme is proposed for designing new inhibitors selective for Pf DHFR that is important for the development of novel antifolate antimalarials.Communicated by Ramaswamy H. Sarma.

We propose a theoretical model for pfDHFR-TS, which includes the 55 aminoacid residues ignored in the crystallographic model. The electrostatic potential calculation on the model surface revealed a continuous positive potential region between the two active sites, suggesting an optimized mechanism for dihydrofolate transport.

In a search for new drug candidates for one of the neglected tropical diseases, leishmaniasis, twenty quinoline-isatin hybrids were synthesized and tested for their in vitro antileishmanial activity against Leishmania major strain. All the synthesized compounds showed promising in vitro activity against the promastigote form in a low micromolar range (IC 50 = 0.5084–5.9486 μM) superior to the reference miltefosine (IC 50 = 7.8976 μM). All the target compounds were then tested against the intracellular amastigote form and showed promising inhibition effects (IC 50 = 0.60442–8.2948 μM versus 8.08 μM for miltefosine). Compounds 4e , 4b and 4f were shown to possess the highest antileishmanial activity against both promastigote and amastigote forms. The most active compounds were proven to exhibit their significant antileishmanial effects through antifolate mechanism, targeting DHFR-TS and PTR1. To evaluate the safety profile of the most active derivatives 4e , 4b and 4f, the in vitro cytotoxicity test was carried out and displayed higher selectivity indices than the reference miltefosine. Molecular docking within putative target protein PTR1 confirmed the high potentiality of the most active compounds 4e , 4b and 4f to block the catalytic activity of Lm-PTR1. [Display omitted] • New quinoline-isatin hybrids were synthesized as antileishmanial agents. • Compounds 4e , 4b and 4f showed the highest in vitro activity. • The most active derivatives acted as DHFR-TS/PTR1 inhibitors. • The promising compounds displayed low toxicity in VERO cells. • In silico studies of the promising compounds showed high docking scores. [ABSTRACT FROM AUTHOR]

Highlights: [•] The DHFR-TS of N. caninum was cloned, sequenced and analyzed. [•] Two mutations on DHFR-TS conferred resistance of N. caninum against pyrimethamine. [•] Lac-Z controlled by the tubulin promoter was ligated to the DHFR-TS vector and transfected. [•] The β-galactosidase tachyzoites were detected by CPRG and X-gal precipitation. [ABSTRACT FROM AUTHOR]

Background: Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (pfDHFR-TS) is a well-defined target of anti-malarial drug, such as pyrimethamine and cycloguanil. Emergence of malaria parasites resistant to these drugs has been shown to be associated with point mutations of the gene coding for the target enzymes. Although the 3D-structure of P. falciparum bifunctional pfDHFR-TS has been reported previously, relatively little is known about the interactions between the pfDHFR and pfTS domains and the roles of the junctional region that links the two domains together. Therefore, a thorough understanding of the interaction of the two domains and the role of the junctional region of this target is important as the knowledge could assist the development of new effective anti-malarial drugs aimed at overcoming drug-resistant malaria. Methods: A system was developed to investigate the interaction between pfDHFR and pfTS domains and the role of the junctional region on the activity of the recombinant pfTS. Based on the ability of co-transformed plasmids coding for pfDHFR and pfTS with truncated junctional region to complement the growth of TS-deficient Escherichia coli strain X2913recA(DE3) on minimum media without thymidine supplementation, active pfTS mutants with minimal length of junctional region were identified. Interactions between active pfDHFR and the pfTS domains were demonstrated by using a bacterial two-hybrid system. Results: Using TS-deficient E. coli strain X2913recA(DE3), the authors have shown for the first time that in P. falciparum a junctional region of at least 44 amino acids or longer was necessary for the pfTS domain to be active for the synthesis of thymidylate for the cells. Truncation of the junctional region of the bifunctional pfDHFR-TS further confirmed the above results, and suggested that a critical length of the junctional peptide of pfDHFR-TS would be essential for the activity of TS to catalyze the synthesis of thymidylate. Conclusion: The present study demonstrated the interactions between the pfDHFR and pfTS domains of the bifunctional pfDHFR-TS, and revealed that the junctional region linking the two protein domains is essential for the expression of catalytically active pfTS domain. The findings could be useful since inhibition of the pfDHFR-TS domain-domain interaction could form a basis for the development of new anti-malarial drugs based on targeting the non-active site region of this important enzyme. [ABSTRACT FROM AUTHOR]

Abstract: The host specificity of blood parasites recovered from a survey of 527 birds in Cameroon and Gabon was examined at several levels within an evolutionary framework. Unique mitochondrial lineages of Haemoproteus were recovered from an average of 1.3 host species (maximum=3) and 1.2 host families (maximum=3) while lineages of Plasmodium were recovered from an average of 2.5 species (maximum=27) and 1.6 families (maximum=9). Averaged within genera, lineages of both Plasmodium and Haemoproteus were constrained in their host distribution relative to random expectations. However, while several individual lineages within both genera exhibited significant host constraint, host breadth varied widely among related lineages, particularly within the genus Plasmodium. Several lineages of Plasmodium exhibited extreme generalist host–parasitism strategies while other lineages appeared to have been constrained to certain host families over recent evolutionary history. Sequence data from two nuclear genes recovered from a limited sample of Plasmodium parasites indicated that, at the resolution of this study, inferences regarding host breadth were unlikely to be grossly affected by the use of parasite mitochondrial lineages as a proxy for biological species. The use of divergent host–parasitism strategies among closely related parasite lineages suggests that host range is a relatively labile character. Since host specificity may also influence parasite virulence, these results argue for considering the impact of haematozoa on avian hosts on a lineage-specific basis. [Copyright &y& Elsevier]

Abstract: Pteridine reductase 1 (PTR1, EC 1.5.1.33) is a NADPH dependent short-chain reductase (SDR) responsible for the salvage of pterins in the protozoan parasite Leishmania. This enzyme acts as a metabolic bypass for drugs targeting dihydrofolate reductase, therefore, for successful antifolate chemotherapy to be developed against Leishmania, it must target both enzyme activities. Based on homology model drawn on recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with two compounds of dihydrofolate reductase specificity showing promising antileishmanial activity in vitro. Both the inhibitors appeared to fit well in the active pocket revealing the tight binding of the carboxylic acid ethyl ester group of pyridine moiety to pteridine reductase and identify the important interactions necessary to assist the structure based development of novel pteridine reductase inhibitors. [Copyright &y& Elsevier]

Analyses of mitochondrial cytochrome b diversity among avian blood parasites of the genera Haemoproteus and Plasmodium suggest that there might be as many lineages of parasites as there are species of birds. This is in sharp contrast to the approximately 175 parasite species described by traditional methods based on morphology using light microscopy. Until now it has not been clear to what extent parasite mitochondrial DNA lineage diversity reflects intra- or interspecific variation. We have sequenced part of a fast-evolving nuclear gene, dihydrofolate reductase--thymidylate synthase (DHFR-TS), and demonstrate that most of the parasite mitochondrial DNA lineages are associated with unique gene copies at this locus. Although these parasite lineages sometimes coexist in the same host individual, they apparently do not recombine and could therefore be considered as functionally distinct evolutionary entities, with independent evolutionary potential. Studies examining parasite virulence and host immune systems must consider this remarkable diversity of avian malaria parasites. [ABSTRACT FROM AUTHOR]

The genomic locus and cDNA encoding Babesia bovis dihydrofolate reductase-thymidylate synthase (DHFR-TS) were cloned and sequenced. A single dhfr-ts gene, composed of four exons, encodes a 511 aa protein that is most closely related to Plasmodium falciparum DHFR-TS. The genomic locus is characterized by the presence of four other genes of which at least three are expressed during the erythrocytic cycle. Three of the genes were highly conserved in closely related Theileria species and for two of the genes and dhfr-ts, gene synteny was observed between B. bovis and Theileria parva, B. bovis in vitro cultures displaying approximately 10–20-fold decreased sensitivity towards the antimalarial drugs WR99210 and pyrimethamine were selected repeatedly after prolonged growth in presence of drugs. Five cultures examined in detail were shown to encode a DHFR-TS carrying amino acid substitution S125F. Three-dimensional-modelling, using the P. falciparum DHFR structure as a template, suggests that substitution S125F protrudes into the binding site of NADPH. The S125F mutant could be isolated by growth under pyrimethamine or WR99210 pressure conferring cross-resistance to both drugs. Although opposing selection for pyrimethamine or WR99210 resistance was reported recently using P. falciparum or P. vivax strains carrying wildtype dhfr, the results obtained here are reminiscent of a quadruple mutant of P. falciparum dhfr displaying strong resistance to pyrimethamine and 10-fold enhanced resistance against WR99210. Wildtype B. bovis DHFR carries three mutations present in this mutant possibly explaining the low sensitivity to pyrimethamine and the ease by which moderately WR99210 resistant mutants could be isolated. [Copyright &y& Elsevier]

Plant Growth Promoting Rhizobacteria (PGPR) were utilized to contemplate their impact on the foliage of Moringa oleifera and examined for changes in tocopherols, chlorophyll, carotenoids, and folate in the sixth week. Among the eight treatments, Bacillus subtilis GB03, B. pumilus SE34, B. pumilus T4, and Pseudomonas fluorescens UOM14 improved α-tocopherol (10–14 fold) and β-carotene (1–1.40 fold) altogether significantly (P ≤ 0.05). The most significant improvement in folate content was apparent for B. subtilis IN937B (5.47 fold) trailed by B. pumilus SE34 (5.05 fold) and B. pumilus T4 (5.12 fold) treatments. P. fluorescens UOM14 indicated remarkable improvement in Chl a (0.39 fold) and Chl b (0.44 fold) content. Organisms showing a significant increase for the analyzed molecules in individual treatment were blended in different combinations and were used for the next set of treatments. Of all the three combinations, Combination 2 (COM2- B. pumilus SE34 + B. pumilus T 4 + B. pumilus INR7) showed the maximum increase in α-tocopherol (8.46 fold) and γ-tocopherol (8.45 fold), followed by Combination 3 (COM3- B. pumilus SE34 + B. pumilus T 4 + P. fluorescens UOM14) (5.93 and 3.65 fold). On the whole COM2 containing different strains of B. pumilus was found to enhance the targeted metabolites in foliage significantly. Real-time PCR studies were conducted for the biochemical pathway genes of the targeted molecules, including, γ-tocopherol methyltransferase (γ-TMT), phytoene synthase (PSY), phytoene desaturase (PDS), lycopene β cyclase (LBC) and dihydrofolate reductase thymidylate synthase (DHFR-TS). All the selected genes exhibited an up-regulation compared to control, similar to the biochemical output. Our investigation provides the strong evidence that PGPR can be viably utilized in combination to enhance the quality of the food crops. Image 1 • B. pumilus T4 & P. fluorescens UOM14 significantly enhanced α-tocopherol. • B. subtilis GB03 & P. fluorescens UOM14 significantly enhanced β-carotene. • B. subtilis IN937 B, B. pumilus SE34 & T4 showed significant increase in folate. • Combination containing all organisms from B. pumilus performed significantly. • PGPR's perform significantly in combination compared to individual treatment. [ABSTRACT FROM AUTHOR]

Objective: Present in silico study was carried out to explore the mode of inhibition of Leishmania donovani dihydrofolate reductase-thymidylate synthase (Ld DHFR-TS) enzyme by Withaferin-A, a withanolide isolated from Withania somnifera. Withaferin-A (WA) is known for its profound multifaceted properties, but its antileishmanial activity is not well understood. The parasite's DHFR-TS enzyme is diverse from its mammalian host and could be a potential drug target in parasites. Results: A 3D model of Ld DHFR-TS enzyme was built and verified using Ramachandran plot and SAVES tools. The protein was docked with WA-the ligand, methotrexate (MTX)-competitive inhibitor of DHFR, and dihydrofolic acid (DHFA)-substrate for DHFR-TS. Molecular docking studies reveal that WA competes for active sites of both Hu DHFR and TS enzymes whereas it binds to a site other than active site in Ld DHFR-TS. Moreover, Lys 173 residue of DHFR-TS forms a H-bond with WA and has higher binding affinity to Ld DHFR-TS than Hu DHFR and Hu TS. The MD simulations confirmed the H-bonding interactions were stable. The binding energies of WA with Ld DHFR-TS were calculated using MM-PBSA. Homology modelling, molecular docking and MD simulations of Ld DHFR-TS revealed that WA could be a potential anti-leishmanial drug. [ABSTRACT FROM AUTHOR]

Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV., (Copyright © 2017 Mueller et al.)