Identification of Novel Potential Inhibitors of Pteridine Reductase 1 in Trypanosoma brucei via Computational Structure-Based Approaches and in Vitro Inhibition Assays

Pteridine reductase 1 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 TbDHFR and 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 potential TbPTR1 inhibitors that showed low micromolar Trypanosome growth inhibition in in vitro experiments with no significant human cell cytotoxicity. Compounds RUBi004, RUBi007, RUBi014, and RUBi018 displayed moderate to strong antagonism 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. Our results provide a basis for scaffold optimization for further studies in the development of HAT antifolates.

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Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00332-10 ◽

2010 ◽

Vol 54 (7) ◽

pp. 2893-2900 ◽

Cited By ~ 69

Author(s):

Antoaneta Y. Sokolova ◽

Susan Wyllie ◽

Stephen Patterson ◽

Sandra L. Oza ◽

Kevin D. Read ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Parent Drug ◽

Cross Resistance ◽

Pharmacokinetic Studies ◽

African Trypanosomiasis ◽

Trypanosoma Brucei Brucei ◽

Resistant Lines

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

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In SilicoScreening, Synthesis andIn VitroEvaluation of Some Quinazolinone and Pyridine Derivatives as Dihydrofolate Reductase Inhibitors for Anticancer Activity

E-Journal of Chemistry ◽

10.1155/2009/506576 ◽

2009 ◽

Vol 6 (s1) ◽

pp. S97-S102 ◽

Cited By ~ 6

Author(s):

A. G. Nerkar ◽

A. K. Saxena ◽

S. A. Ghone ◽

A. K. Thaker

Keyword(s):

Anticancer Activity ◽

Dihydrofolate Reductase ◽

De Novo ◽

Human Cancer ◽

Microwave Assisted Synthesis ◽

Human Cancer Cell Lines ◽

Reductase Inhibitors ◽

Base Derivative ◽

Dhfr Inhibitors

Dihydrofolate reductase (DHFR) is the important target for anticancer drugs belonging to the class of antimetabolites as the enzyme plays important role in the de novo purine synthesis. We here report thein silicoscreening to obtain best fit molecules as DHFR inhibitors, synthesis of some ʻbest fitʼ quinazolinone from 2-phenyl-3-(substituted-benzilidine-amino) quinazolinones (Quinazolinone Shiff's bases) QSB1-5and pyridine-4-carbohydrazide Shiff's bases (ISB1-5) derivatives and theirin vitroanticancer assay. Synthesis of the molecules was performed using microwave assisted synthesis. The structures of these molecules were elucidated by IR and1H-NMR. These compounds were then subjected forin vitroanticancer evaluation against five human cancer cell-lines for anticancer cyto-toxicity assay. Methotrexate (MTX) was used as standard for this evaluation to give a comparable inhibition of the cell proliferation by DHFR inhibition. Placlitaxel, adriamycin and 5-fluoro-uracil were also used as standard to give a comparable activity of these compounds with other mechanism of anticancer activity. ISB3(4-(N, N-dimethyl-amino)-phenyl) Schiff''s base derivative of pyridine carbohydrazide showed equipotent activity with the standards used inin vitroanticancer assay as per the NCI (National Cancer Institute) guidelines.

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Flavin-Dependent Thymidylate Synthase ThyX Activity: Implications for the Folate Cycle in Bacteria

Journal of Bacteriology ◽

10.1128/jb.01380-07 ◽

2007 ◽

Vol 189 (23) ◽

pp. 8537-8545 ◽

Cited By ~ 23

Author(s):

Damien Leduc ◽

Frédéric Escartin ◽

H. Frederik Nijhout ◽

Michael C. Reed ◽

Ursula Liebl ◽

...

Keyword(s):

Dihydrofolate Reductase ◽

Thymidylate Synthase ◽

Insertional Mutagenesis ◽

Rhodobacter Capsulatus ◽

De Novo ◽

Reductase Activity ◽

Direct Proof ◽

Cellular Functions ◽

Thymidylate Synthesis

ABSTRACT Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised.

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RNA turnover in Trypanosoma brucei

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1242-1249.1987 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1242-1249

Author(s):

B Ehlers ◽

J Czichos ◽

P Overath

Keyword(s):

Protein Synthesis ◽

Trypanosoma Brucei ◽

Half Life ◽

De Novo ◽

State Level ◽

Surface Glycoprotein ◽

Beta Tubulin ◽

Fold Reduction ◽

Tubulin Mrna

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

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Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

Scientific Reports ◽

10.1038/s41598-021-91747-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Eva Doleželová ◽

Tomáš Klejch ◽

Petr Špaček ◽

Martina Slapničková ◽

Luke Guddat ◽

...

Keyword(s):

Trypanosoma Brucei ◽

De Novo ◽

Major Effect ◽

Nucleotide Analogs ◽

Rnai Silencing ◽

Acyclic Nucleoside Phosphonates ◽

A Cell ◽

Actual Target ◽

Promising Source

AbstractAll medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

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In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

Molecular and Biochemical Parasitology ◽

10.1016/0166-6851(86)90045-9 ◽

1986 ◽

Vol 18 (1) ◽

pp. 3-15 ◽

Cited By ~ 29

Author(s):

H SCOTT ◽

A GERO ◽

W OSULLIVAN

Keyword(s):

Plasmodium Falciparum ◽

De Novo ◽

Pyrimidine Biosynthesis ◽

In Vitro Inhibition

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Autoimmunity to phosphatidylserine and anemia in African Trypanosome infections

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009814 ◽

2021 ◽

Vol 15 (9) ◽

pp. e0009814

Author(s):

Juan Rivera-Correa ◽

Joseph Verdi ◽

Julian Sherman ◽

Jeremy M. Sternberg ◽

Jayne Raper ◽

...

Keyword(s):

Plasma Membrane ◽

B Cells ◽

Trypanosoma Cruzi ◽

Trypanosoma Brucei ◽

Mouse Models ◽

Human African Trypanosomiasis ◽

Annexin V ◽

Trypanosome Infection ◽

Erythrocyte Plasma Membrane

Anemia caused by trypanosome infection is poorly understood. Autoimmunity during Trypanosoma brucei infection was proposed to have a role during anemia, but the mechanisms involved during this pathology have not been elucidated. In mouse models and human patients infected with malaria parasites, atypical B-cells promote anemia through the secretion of autoimmune anti-phosphatidylserine (anti-PS) antibodies that bind to uninfected erythrocytes and facilitate their clearance. Using mouse models of two trypanosome infections, Trypanosoma brucei and Trypanosoma cruzi, we assessed levels of autoantibodies and anemia. Our results indicate that acute T. brucei infection, but not T. cruzi, leads to early increased levels of plasma autoantibodies against different auto antigens tested (PS, DNA and erythrocyte lysate) and expansion of atypical B cells (ABCs) that secrete these autoantibodies. In vitro studies confirmed that a lysate of T. brucei, but not T. cruzi, could directly promote the expansion of these ABCs. PS exposure on erythrocyte plasma membrane seems to be an important contributor to anemia by delaying erythrocyte recovery since treatment with an agent that prevents binding to it (Annexin V) ameliorated anemia in T. brucei-infected mice. Analysis of the plasma of patients with human African trypanosomiasis (HAT) revealed high levels of anti-PS antibodies that correlated with anemia. Altogether these results suggest a relation between autoimmunity against PS and anemia in both mice and patients infected with T. brucei.

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RNA turnover in Trypanosoma brucei.

Molecular and Cellular Biology ◽

10.1128/mcb.7.3.1242 ◽

1987 ◽

Vol 7 (3) ◽

pp. 1242-1249 ◽

Cited By ~ 57

Author(s):

B Ehlers ◽

J Czichos ◽

P Overath

Keyword(s):

Protein Synthesis ◽

Trypanosoma Brucei ◽

Half Life ◽

De Novo ◽

State Level ◽

Surface Glycoprotein ◽

Beta Tubulin ◽

Fold Reduction ◽

Tubulin Mrna

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procyclic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27 degrees C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, beta-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of beta-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (t1/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t1/2, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37 degrees Celsius caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while beta-tubulin mRNA was stabilized 2- to 3-fold and mRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.

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Sesquiterpene Lactones with Dual Inhibitory Activity against the Trypanosoma brucei Pteridine Reductase 1 and Dihydrofolate Reductase

Molecules ◽

10.3390/molecules27010149 ◽

2021 ◽

Vol 27 (1) ◽

pp. 149

Author(s):

Katharina Possart ◽

Fabian Herrmann ◽

Joachim Jose ◽

Maria Costi ◽

Thomas Schmidt

Keyword(s):

Trypanosoma Brucei ◽

Dihydrofolate Reductase ◽

Inhibitory Activity ◽

In Silico ◽

Gene Knockout ◽

Sesquiterpene Lactones ◽

Treatment Options ◽

Cattle Disease ◽

Ic50 Values

The parasite Trypanosoma brucei (T. brucei) is responsible for human African trypanosomiasis (HAT) and the cattle disease “Nagana” which to this day cause severe medical and socio-economic issues for the affected areas in Africa. So far, most of the available treatment options are accompanied by harmful side effects and are constantly challenged by newly emerging drug resistances. Since trypanosomatids are auxotrophic for folate, their pteridine metabolism provides a promising target for an innovative chemotherapeutic treatment. They are equipped with a unique corresponding enzyme system consisting of the bifunctional dihydrofolate reductase-thymidylate synthase (TbDHFR-TS) and the pteridine reductase 1 (TbPTR1). Previously, gene knockout experiments with PTR1 null mutants have underlined the importance of these enzymes for parasite survival. In a search for new chemical entities with a dual inhibitory activity against the TbPTR1 and TbDHFR, a multi-step in silico procedure was employed to pre-select promising candidates against the targeted enzymes from a natural product database. Among others, the sesquiterpene lactones (STLs) cynaropicrin and cnicin were identified as in silico hits. Consequently, an in-house database of 118 STLs was submitted to an in silico screening yielding 29 further virtual hits. Ten STLs were subsequently tested against the target enzymes in vitro in a spectrophotometric inhibition assay. Five compounds displayed an inhibition over 50% against TbPTR1 as well as three compounds against TbDHFR. Cynaropicrin turned out to be the most interesting hit since it inhibited both TbPTR1 and TbDHFR, reaching IC50 values of 12.4 µM and 7.1 µM, respectively.

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