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SUMMARY: Plant intracellular immune receptors, primarily nucleotide‐binding, leucine‐rich repeat proteins (NLRs), detect pathogen effector proteins and activate NLR‐triggered immunity (NTI). Recently, 'sensor' NLRs have been reported to function with 'helper' NLRs to activate immunity. We investigated the role of two helper NLRs, Nrc2 and Nrc3, on immunity in tomato to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) mediated by the sensor NLR Prf and the Pto kinase. An nrc2/nrc3 mutant no longer activated Prf/Pto‐mediated NTI to Pst containing the effectors AvrPto and AvrPtoB. An nrc3 mutant showed intermediate susceptibility between wild‐type plants and a Prf mutant, while an nrc2 mutant developed only mild disease. These observations indicate that Nrc2 and Nrc3 act additively in Prf‐/Pto‐mediated immunity. We examined at what point Nrc2 and Nrc3 act in the Prf/Pto‐mediated immune response. In the nrc2/3 mutant, programmed cell death (PCD) normally induced by constitutively active variants of AvrPtoB, Pto, or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active Nrc3 was also abolished in a Nicotiana benthamiana line with reduced expression of Prf. MAPK activation triggered by expression of AvrPto in the wild‐type tomato plants was completely abolished in the nrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for PCD triggered by Ptr1, another sensor NLR‐mediating Pst resistance, although these helper NLRs do appear to be involved in resistance to certain Pst race 1 strains [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. A centenary approach to antimonial-based drugs was first initiated with the synthesis of urea stibamine by Upendranath Brahmachari in 1922. The need for new drug development led to resistance toward antimoniates. New drug development to treat leishmaniasis is urgently needed. In this way, searching for new substances with antileishmanial activity, we synthesized ten anthranyl phenylhydrazide and three quinazolinone derivatives and evaluated them against promastigotes and the intracellular amastigotes of Leishmania amazonensis. Three compounds showed good activity against promastigotes 1b, 1d, and 1g, with IC50 between 1 and 5 μM. These new phenylhydrazides were tested against Leishmania arginase, but they all failed to inhibit this parasite enzyme, as we have shown in a previous study. To explain the possible mechanism of action, we proposed the enzyme PTR1 as a new target for these compounds based on in silico analysis. In conclusion, the new anthranyl hydrazide derivatives can be a promising scaffold for developing new substances against the protozoa parasite. [ABSTRACT FROM AUTHOR]

• Expression of DHFR and PTR1 genes. • Real-time PCR method for gene expression. • The effect of OLE on the expression of genes in leishmania major. Among neglected tropical diseases (NTD) following malaria, leishmaniosis has claimed the largest mortality rate (Georgiadou, Makaritsis, and Dalekos 2015). None of the current anti-leishmanial drugs can be considered ideal (Minodier and Parola 2007; Mohapatra 2014). The dihydrofolate reductase- thymidylate synthase (DHFR-TS) leads to DNA synthesis in the parasite and its inhibition causes the death of the parasite. Meanwhile, pteridine reductase1 (PTR1) enzyme, because of having a similar role to DHFR in the parasite, causes by-pass of DHFR enzyme inhibition thereby resulting an incidence of drug resistance in the parasite (Nare et al. 1997b). Considering the current problems regarding leishmaniosis treatment, developing new drugs with less toxicity and greater efficacy plus lower costs has changed into a necessity. The present study examined the effect of olive leaf extract (OLE) at concentrations 6.25, 12.5, 25, 50, and 100 µg/ml on the expression of DHFR and PTR1 genes in Leishmania major parasite. The parasite samples were incubated in RPMI 1640 culture medium in the presence of five mentioned concentrations of OLE. A quantitative real-time PCR technique was used to analyze the DHFR and PTR1 gene expressions. The results revealed that the expression of DHFR gene at 6.25 µg/ml extract increased significantly (p <0.05), while it diminished significantly at 25 µg/ml. At other studied concentrations, DHFR gene showed underexpression compared to the control group, which was not statistically significant (p >0.05). The expression of PTR1 gene at concentrations 6.25, 50, and 100 µg/ml of the olive leaf extract increased significantly (p <0.05). The expression of PTR1 gene at 12.5 µg/ml concentration decreased compared to the control group, while it increased at 25 µg/ml, though the changes in the gene expression at these two concentrations were not statistically significant (p >0.05). The findings of the present study suggest that OLE can reduce DHFR expression, but its effect of reducing PTR1 expression was not very notable, with its influence being greater on DHFR underexpression rather than PTR1. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. A centenary approach to antimonial-based drugs was first initiated with the synthesis of urea stibamine by Upendranath Brahmachari in 1922. The need for new drug development led to resistance toward antimoniates. New drug development to treat leishmaniasis is urgently needed. In this way, searching for new substances with antileishmanial activity, we synthesized ten anthranyl phenylhydrazide and three quinazolinone derivatives and evaluated them against promastigotes and the intracellular amastigotes of Leishmania amazonensis. Three compounds showed good activity against promastigotes 1b, 1d, and 1g, with IC50 between 1 and 5 μM. These new phenylhydrazides were tested against Leishmania arginase, but they all failed to inhibit this parasite enzyme, as we have shown in a previous study. To explain the possible mechanism of action, we proposed the enzyme PTR1 as a new target for these compounds based on in silico analysis. In conclusion, the new anthranyl hydrazide derivatives can be a promising scaffold for developing new substances against the protozoa parasite.

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.

Pteridine reductase 1 (PTR1) is a trypanosomatid multifunctional enzyme that provides a mechanism for escape of dihydrofolate reductase (DHFR) inhibition. This is because PTR1 can reduce pterins and folates. Trypanosomes require folates and pterins for survival and are unable to synthesize them de novo. Currently there are no anti-folate based Human African Trypanosomiasis (HAT) chemotherapeutics in use. Thus, successful dual inhibition of Trypanosoma brucei dihydrofolate reductase (TbDHFR) and Trypanosoma brucei pteridine reductase 1 (TbPTR1) has implications in the exploitation of anti-folates. We carried out molecular docking of a ligand library of 5742 compounds against TbPTR1 and identified 18 compounds showing promising binding modes. The protein-ligand complexes were subjected to molecular dynamics to characterize their molecular interactions and energetics, followed by in vitro testing. In this study, we identified five compounds which showed low micromolar Trypanosome growth inhibition in in vitro experiments that might be acting by inhibition of TbPTR1. Compounds RUBi004, RUBi007, RUBi014, and RUBi018 displayed moderate to strong antagonism (mutual reduction in potency) when used in combination with the known TbDHFR inhibitor, WR99210. This gave an indication that the compounds might inhibit both TbPTR1 and TbDHFR. RUBi016 showed an additive effect in the isobologram assay. Overall, our results provide a basis for scaffold optimization for further studies in the development of HAT anti-folates.

There is a need for improved and generally applicable scoring functions for fragment-based approaches to ligand design. Here, we evaluate the performance of a computationally efficient model for inhibitory activity estimation, which is composed only of multipole electrostatic energy and dispersion energy terms that approximate long-range ab initio quantum mechanical interaction energies. We find that computed energies correlate well with inhibitory activity for a compound series with varying substituents targeting two subpockets of the binding site of Trypanosoma brucei pteridine reductase 1. For one subpocket, we find that the model is more predictive for inhibitory activity than the ab initio interaction energy calculated at the MP2 level. Furthermore, the model is found to outperform a commonly used empirical scoring method. Finally, we show that the results for the two subpockets can be combined, which suggests that this simple nonempirical scoring function could be applied in fragment-based drug design. [ABSTRACT FROM AUTHOR]

Background:Protozoa related to Trypanosome family including Leishmania,synthesize enzymes to escape from drug therapy.One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase(DHFR)Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more.Methods: PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E.coli strain M15.SDS-PAGE and western blot analysis were carried out to analyze the expression.Results:Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done.SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells.Conclusion:Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

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]

Background: Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase (DHFR). Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. Methods: PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Results: Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Conclusion: Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid.

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]

Abstract: Reduced pteridines are required for a number of important cellular functions. Trypanosomatid parasites, unlike their mammalian hosts, are pteridine auxotrophs and salvage the precursor pteridines from the host and reduce them to the respective biologically active tetrahydro forms using parasite-encoded enzymes. These enzymes may offer selective drug targets. In Leishmania, pteridine reductase 1 (PTR1), the primary enzyme for reducing pterins, is also responsible for resistance to antifolate drugs. Typically, PTR1 is more active with fully oxidized biopterin and folate than with their reduced counterparts. We have identified an enzyme, TcPTR2 of Trypanosoma cruzi, which though very similar to PTR1 in its primary sequence, can reduce only dihydrobiopterin and dihydrofolate and not oxidized pteridines. The structures of an inhibitor (methotrexate) and a substrate (dihydrofolate) complex of this enzyme demonstrate that the orientation of the substrate and the inhibitor in the active site of TcPTR2 are different from each other. However, the orientation of each ligand is similar to that of the corresponding ligand in Leishmania major PTR1 complexes. [Copyright &y& Elsevier]

Pteridine reductase 1 (PTR1) is a trypanosomatid multifunctional enzyme that provides a mechanism for escape of dihydrofolate reductase (DHFR) inhibition. This is because PTR1 can reduce pterins and folates. Trypanosomes require folates and pterins for survival and are unable to synthesize them de novo. Currently there are no anti-folate based Human African Trypanosomiasis (HAT) chemotherapeutics in use. Thus, successful dual inhibition of Trypanosoma brucei dihydrofolate reductase (TbDHFR) and Trypanosoma brucei pteridine reductase 1 (TbPTR1) has implications in the exploitation of anti-folates. We carried out molecular docking of a ligand library of 5742 compounds against TbPTR1 and identified 18 compounds showing promising binding modes. The protein-ligand complexes were subjected to molecular dynamics to characterize their molecular interactions and energetics, followed by in vitro testing. In this study, we identified five compounds which showed low micromolar Trypanosome growth inhibition in in vitro experiments that might be acting by inhibition of TbPTR1. Compounds RUBi004, RUBi007, RUBi014, and RUBi018 displayed moderate to strong antagonism (mutual reduction in potency) when used in combination with the known TbDHFR inhibitor, WR99210. This gave an indication that the compounds might inhibit both TbPTR1 and TbDHFR. RUBi016 showed an additive effect in the isobologram assay. Overall, our results provide a basis for scaffold optimization for further studies in the development of HAT anti-folates. [ABSTRACT FROM AUTHOR]