Identification of Innovative Folate Inhibitors Leveraging the Amino Dihydrotriazine Motif from Cycloguanil for Their Potential as Anti-Trypanosoma brucei Agents.

Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.

Investigation of the Anticancer Effect of α-Aminophosphonates and Arylidine Derivatives of 3-Acetyl-1-aminoquinolin-2(1H)-one on the DMBA Model of Breast Cancer in Albino Rats with In Silico Prediction of Their Thymidylate Synthase Inhibitory Effect.

Breast cancer is a major cause of death in women worldwide. In this study, 60 female rats were classified into 6 groups; negative control, α-aminophosphonates, arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one, DMBA, DMBA & α-aminophosphonates, and DMBA & arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. New α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one were synthesized and elucidated by different spectroscopic and elemental analysis. Histopathological examination showed marked proliferation of cancer cells in the DMBA group. Treatment with α-aminophosphonates mainly decreased tumor mass. Bcl2 expression increased in DMBA-administered rats and then declined in the treated groups, mostly with α-aminophosphonates. The level of CA15-3 markedly declined in DMBA groups treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. Gene expression of GST-P, PCNA, PDK, and PIK3CA decreased in the DMBA group treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one, whereas PIK3R1 and BAX increased in the DMBA group treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. The molecular docking postulated that the investigated compounds can inhibt the Thymidylate synthase TM due to high hydrophobicity charachter.

Synthesis of acyclic nucleoside phosphonates targeting flavin-dependent thymidylate synthase in Mycobacterium tuberculosis.

Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural's cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 µM.

Thymidylate synthase is essential for efficient HIV-1 replication in macrophages.

Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.

Folic acid alleviates age-related cognitive decline and inhibits apoptosis of neurocytes in senescence-accelerated mouse prone 8: deoxythymidine triphosphate biosynthesis as a potential mechanism.

Disturbed deoxythymidine triphosphate biosynthesis due to the inhibition of thymidylate synthase (TS) can lead to uracil accumulation in DNA, eventually, lead to neurocytes apoptosis and cognitive decline. Folic acid supplementation delayed cognitive decline and neurodegeneration in senescence-accelerated mouse prone 8 (SAMP8). Whether folic acid, one of nutrition factor, the effect on the expression of TS is unknown. The study aimed to determine if folic acid supplementation could alleviate age-related cognitive decline and apoptosis of neurocytes by increasing TS expression in SAMP8 mice. According to folic acid concentration in diet, four-month-old male SAMP8 mice were randomly divided into three different diet groups by baseline body weight in equal numbers. Moreover, to evaluate the role of TS, a TS inhibitor was injected intraperitoneal. Cognitive test, apoptosis rates of neurocytes, expression of TS, relative uracil level in telomere, and telomere length in brain tissue were detected. The results showed that folic acid supplementation decreased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, alleviated telomere length shorting, increased expression of TS, then decreased apoptosis rates of neurocytes, and alleviated cognitive performance in SAMP8 mice. Moreover, at the same concentration of folic acid, TS inhibitor raltitrexed increased deoxyuridine monophosphate accumulation, uracil misincorporation in telomere, and exacerbated telomere length shorting, decreased expression of TS, then increased apoptosis rates of neurocytes, and decreased cognitive performance in SAMP8 mice. In conclusion, folic acid supplementation alleviated age-related cognitive decline and inhibited apoptosis of neurocytes by increasing TS expression in SAMP8 mice.

Molecular Mechanism of Thymidylate Synthase Inhibition by N4-Hydroxy-dCMP in View of Spectrophotometric and Crystallographic Studies.

Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.

Design, synthesis, and molecular docking studies of thiazolidinediones as PPAR-γ agonists and thymidylate synthase inhibitors.

New thiazolidine-2,4-dione hybrids were designed and synthesized as potential peroxisome proliferator-activated receptor (PPAR)-γ agonists and thymidylate synthase inhibitors. All the synthesized compounds follow Lipinski's and Veber's rules and possess the desired pharmacokinetics properties. The PPAR-γ transactivation results displayed that compounds 12 (78.9%) and 11 (73.4%) were the most active compounds and they increased PPAR-γ gene expression by 2.2- and 2.4-fold, respectively. Compounds 12, 11, and 8 showed promising cytotoxicity, with IC50 values ranging from 1.4 to 4.5 µM against MCF-7 cells and from 1.8 to 8.4 µM against HCT-116 cells. Compounds 11 and 12 also inhibited thymidylate synthase with IC50 values of 5.1 and 3.2 µM, respectively, confirming their mode of action as thymidylate synthase inhibitors. Finally, molecular docking studies supported the in vitro biological activity results.

Design, synthesis and molecular docking studies of thymol based 1,2,3-triazole hybrids as thymidylate synthase inhibitors and apoptosis inducers against breast cancer cells.

Natural product produced by plants has been the backbone for numerous anticancer agents. In the present work, natural bioactive thymol based 1,2,3-triazole hybrids have been synthesized and evaluated for anticancer activity in MCF-7 and MDA-MB-231 cancer cells. The synthesized molecules displayed desired pharmacokinetic predictions for an orally available drug. Among the synthesized hybrids, compound 4-((2-isopropyl-5-methylphenoxy)methyl)-1-o-tolyl-1H-1,2,3-triazole (10) was the most potent (IC50 6.17 µM) showing comparable cytotoxity to tamoxifen (IC50 5.62 µM) and 3.2 fold inhibition to 5-fluorouracil (IC50 20.09 µM) against MCF-7 cancer cells. Whereas against MDA-MB-231 cancer cells, compound 10 (IC50 10.52 µM) and 3-(4-((2-isopropyl-5-methylphenoxy)methyl)-1H-1,2,3-triazol-1-yl)benzoic acid (12) (IC50 11.41 µM) displayed 1.42 and 1.3 fold inhibition, respectively to tamoxifen (IC50 15.01 µM) whereas 2.4 fold and 2.2 activity to 5-Florouracil (IC50 25.31 µM). Furthermore, 10 and 12 significantly inhibited thymidylate synthase enzyme with 2.4 and 1.26 fold activity to standard drug, Pemetrexed (IC50 5.39 µM) suggesting their mode of action as thymidylate synthase inhibitors. Cell cycle arrest and annexin V induced apoptosis study of compound 10 showed cell cycle arrest at the G2/M phase and induction of apoptosis in MCF-7 cells. The molecular docking was accomplished onto thymidylate synthase (TS) protein. The active compounds exhibited promising binding interactions and binding affinities into active sites. Finally, density functional theory (DFT) calculations including chemical reactivity and molecular electrostatic potential (MEP) have been performed to confirm the data obtained from docking and biological experiments. The results from this study inferred that compound 10 could be served as a lead molecule for the treatment of breast cancer.

Folic Acid-Peptide Conjugates Combine Selective Cancer Cell Internalization with Thymidylate Synthase Dimer Interface Targeting.

Drug-target interaction, cellular internalization, and target engagement should be addressed to design a lead with high chances of success in further optimization stages. Accordingly, we have designed conjugates of folic acid with anticancer peptides able to bind human thymidylate synthase (hTS) and enter cancer cells through folate receptor α (FRα) highly expressed by several cancer cells. Mechanistic analyses and molecular modeling simulations have shown that these conjugates bind the hTS monomer-monomer interface with affinities over 20 times larger than the enzyme active site. When tested on several cancer cell models, these conjugates exhibited FRα selectivity at nanomolar concentrations. A similar selectivity was observed when the conjugates were delivered in synergistic or additive combinations with anticancer agents. At variance with 5-fluorouracil and other anticancer drugs that target the hTS catalytic pocket, these conjugates do not induce overexpression of this protein and can thus help combating drug resistance associated with high hTS levels.

Identification of Potential Targets for Thymidylate Synthase and Amp-C ß-lactamase from Non-alkaloidal Fractions of Moringa oleifera Leaves.

AIM: The aim of present investigation is to identify the potential targets for Thymidylate Synthase and Amp-C ß-lactamase from non-alkaloidal fractions of Moringa oleifera leaves. BACKGROUND: Bioactive constituents from medicinal plants, either as pure compounds or as crude forms, provide vast opportunities for new drug discoveries. Due to an increasing demand for chemical diversity in screening programs, seeking therapeutic drugs from natural products, mainly from edible plants, has grown throughout the world. Moringa oleifera has an impressive range of medicinal uses with high nutritional value. Therefore, this medicinal plant has been used widely in traditional Indian medicine for anti-inflammation, anticancer and antibacterial infections. OBJECTIVES: The primary objective is to identify the phytoconstituents present in the maximum proportion in non-alkaloidal fractions of ethanolic leaf extract of Moringa oleifera. Then, the identified phytoconstituents were used to ensure the potential target molecules for binding affinity towards the target proteins viz. Thymidylate Synthase (1HVY) and Amp-C beta-lactamase (1FSY) by docking analysis. METHODS: In present investigation, ethanolic extract of Moringa leaves was prepared and then fractionated on the basis of presence/absence of alkaloids. The antimicrobial activity of different fractions of ethanolic leaf extract was evaluated against various pathogens. Later, after this, bioactive molecules present in the non-alkaloidal fractions of ethanolic leaf extract were accomplished through GC-MS analysis, and finally, the identified phytocompounds were analyzed through docking studies to evaluate their affinity for target proteins viz. Thymidylate Synthase (1HVY) and Amp-C ß-lactamase (1FSY). RESULTS: The antimicrobial activity of non-alkaloidal fractions of ethanolic leaf extract was evaluated against various pathogens which exhibited significant antimicrobial activity. Twenty phytocompounds were identified as gas chromatogram of non-alkaloidal fractions (chloroform and ethyl acetate) of leaf extract of M. oleifera; Four most prominent compounds having highest peak area percentage were identified as Ethane, 1,1,2,2-tetrachloro, (46.45%) 2-Propanone, 1,1,3-trichloro, (13.77%) Heptasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11,13,13-tetradecamethyl (17.87%) and 2,4-Dichlorodiphenylsulfone (17.64%). Other notable compounds were 9,12-Octadecadienoic acid (Z,Z) (14.06%), Oleic acid, 3- (octadecyloxy)propyl ester (12.41%), Fluoranthene (6.98%), Phenol, 2,4-bis( 1,1-dimethylethyl) (4.16%) and Phthalic acid, butyl nonyl ester (3.47%). Only, five compounds viz. 2,6-Bis(1,1- dimethylethyl)phenol(C1), Dodecamethylcyclohexasiloxane(C2), Chlorodimethylethylsilane(C3), Fluoranthene(C4) and Hexadecanoic acid, methyl ester(C5) showed the maximum interaction with 1HVY with highest docking score of -178.51Kcal/mol, - 231.65Kcal/mol, -129.18Kcal/mol, - 173.10Kcal/mol and -220.78Kcal/mol, respectively. In addition, three compounds viz. Dodecamethylcyclohexasiloxane( C2), Fluoranthene(C4) and Hexadecanoic acid, methyl ester(C5) showed the maximum interaction with 1FSY with highest docking score of -137.23Kcal/mol, -54.34Kcal/mol and -153.84Kcal/mol, respectively. CONCLUSION: Moringa plant may provide incredible capabilities to develop pharmacological products. The present finding demonstrated that Moringa oleifera is an excellent plant candidate to be used for improving the health of communities.

dUTPase inhibition confers susceptibility to a thymidylate synthase inhibitor in DNA-repair-defective human cancer cells.

Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5-Fluorouracil (5-FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5-FU and uracil misincorporation, we examined cancer cells treated with 2'-deoxy-5-fluorouridine (FdUrd) in the presence of TAS-114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS-114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5-FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5-FU incorporation into RNA. TAS-114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short-term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5-FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA-treated HeLa cells. These results suggested that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5-FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR.

Multiaction Platinum(IV) Prodrug Containing Thymidylate Synthase Inhibitor and Metabolic Modifier against Triple-Negative Breast Cancer.

Multifunctional platinumIV anticancer prodrugs have the potential to enrich the anticancer properties and overcome the clinical problems of drug resistance and side effects of platinumII anticancer agents. Herein, we develop dual and triple action platinumIV complexes with targeted and biological active functionalities. One complex (PFL) that consists of cisplatin, tegafur, and lonidamine exhibits strong cytotoxicity against triple negative breast cancer (TNBC) cells. Cellular uptake and distribution studies reveal that PFL mainly accumulates in mitochondria. As a result, PFL disrupts the mitochondrial ultrastructure and induces significant alterations in the mitochondrial membrane potential, which further leads to an increase in production of reactive oxygen species (ROS) and a decrease in ATP synthesis in MDA-MB-231 TNBCs. Western blot analysis reveals the formation of ternary complex of thymidylate synthase, which shows the intracellular conversion of tegafur into 5-FU after its release from PFL. Furthermore, treatment with PFL impairs the mitochondrial function, leading to the inhibition of glycolysis and mitochondrial respiration and induction of apoptosis through the mitochondrial pathway. The RNA-sequencing experiment shows that PFL can perturb the pathways involved in DNA synthesis, DNA damage, metabolism, and transcriptional activity. These findings demonstrate that PFL intervenes in several cellular processes including DNA damage, thymidylate synthase inhibition, and perturbation of the mitochondrial bioenergetics to kill the cancer cells. The results highlight the significance of a triple-action prodrug for efficient anticancer therapy for TNBCs.

Targeting the TS dimer interface in bifunctional Cryptosporidium hominis TS-DHFR from parasitic protozoa: Virtual screening identifies novel TS allosteric inhibitors.

Effective therapies are lacking to treat gastrointestinal infections caused by the genus Cryptosporidium, which can be fatal in the immunocompromised. One target of interest is Cryptosporidium hominis (C. hominis) thymidylate synthase-dihydrofolate reductase (ChTS-DHFR), a bifunctional enzyme necessary for DNA biosynthesis. Targeting the TS-TS dimer interface is a novel strategy previously used to identify inhibitors against the related bifunctional enzyme in Toxoplasma gondii. In the present study, we target the ChTS dimer interface through homology modeling and high-throughput virtual screening to identifying allosteric, ChTS-specific inhibitors. Our work led to the discovery of methylenedioxyphenyl-aminophenoxypropanol analogues which inhibit ChTS activity in a manner that is both dose-dependent and influenced by the conformation of the enzyme. Preliminary results presented here include an analysis of structure activity relationships and a ChTS-apo crystal structure of ChTS-DHFR supporting the continued development of inhibitors that stabilize a novel pocket formed in the open conformation of ChTS-TS.

A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells.

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

Alvaxanthone, a Thymidylate Synthase Inhibitor with Nematocidal and Tumoricidal Activities.

With the aim to identify novel inhibitors of parasitic nematode thymidylate synthase (TS), we screened in silico an in-house library of natural compounds, taking advantage of a model of nematode TS three-dimensional (3D) structure and choosing candidate compounds potentially capable of enzyme binding/inhibition. Selected compounds were tested as (i) inhibitors of the reaction catalyzed by TSs of different species, (ii) agents toxic to a nematode parasite model (C. elegans grown in vitro), (iii) inhibitors of normal human cell growth, and (iv) antitumor agents affecting human tumor cells grown in vitro. The results pointed to alvaxanthone as a relatively strong TS inhibitor that causes C. elegans population growth reduction with nematocidal potency similar to the anthelmintic drug mebendazole. Alvaxanthone also demonstrated an antiproliferative effect in tumor cells, associated with a selective toxicity against mitochondria observed in cancer cells compared to normal cells.

Design, synthesis and in vitro antiproliferative activity of new thiazolidinedione-1,3,4-oxadiazole hybrids as thymidylate synthase inhibitors.

Thymidylate synthase (TS) has been an attention-grabbing area of research for the treatment of cancers due to their role in DNA biosynthesis. In the present study, we have synthesised a library of thiazolidinedione-1,3,4-oxadiazole hybrids as TS inhibitors. All the synthesised hybrids followed Lipinski and Veber rules which indicated good drug likeness properties upon oral administration. Among the synthesised hybrids, compound 9 and 10 displayed 4.5 and 4.4 folds activity of 5-Fluorouracil, respectively against MCF-7 cell line whereas 3.1 and 2.5 folds cytotoxicity against HCT-116 cell line. Furthermore, compound 9 and 10 also inhibited TS enzyme with IC50 = 1.67 and 2.21 µM, respectively. Finally, the docking studies of 9 and 10 were found to be consistent with in vitro TS results. From these studies, compound 9 and 10 has the potential to be developed as TS inhibitors.

Thymidylate synthase inhibitor raltitrexed can induce high levels of DNA damage in MYCN-amplified neuroblastoma cells.

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and nonamplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

Knocking down of the DHFR-TS gene in Toxoplasma gondii using siRNA and assessing the subsequences on toxoplasmosis in mice.

Toxoplasma gondii (T. gondii), an obligatory intracellular parasite, is the etiologic agent of toxoplasmosis. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) is one of the most important enzymes in toxoplasma folic acid cycle. Due to the emergence of resistance in RH strain of T. gondii against pyrimethamine that acts via DHFR-TS inhibition and also the crucial role of small interference RNA (siRNA) technology in gene silencing, we aimed to use siRNA to knock down DHFR-TS gene expression in T. gondii as a therapeutic target against toxoplasmosis in a mouse model. Based on the DHFR-TS gene sequence, siRNA was designed. The siRNAs were transfected into the parasites by electroporation. Total RNA was extracted using RNX-Plus kit. The viability of parasite was assessed by methylthiazole tetrazolium (MTT). The survival time of mice challenged with siRNA-treated T.gondii were compared to the control group infected with the same amount of wild-type tachyzoites. The viability of siRNA-embedded parasites was 70.7% (29.3% decreased) compared to the wild-type parasite as control (P = 0.0001). The transcription level of siRNA-transfected parasites was reduced to 17.4% (82.6% inhibition) (P = 0.016). The in vivo assessment showed that the mean survival time of the mice inoculated with modified parasites was increased about 2 days after the death of all mice in the control group. The designed siRNAs in the current study were able to silence the DHFR-TS gene efficiently. This silencing led to a decrease in viability of the parasites and an increase in the survival time of the parasites-treated mice.

Mycobacterium tuberculosis thymidylate synthase (ThyX) is a target for plumbagin, a natural product with antimycobacterial activity.

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

The Dual-Targeting Activity of the Metabolite Substrate of Para-amino Salicyclic Acid in the Mycobacterial Folate Pathway: Atomistic and Structural Perspectives.

Therapeutic targeting of folate biosynthetic pathway has recently been explored as a viable strategy in the treatment of tuberculosis. The bioactive metabolite substrate of Para-amino salicyclic acid (PAS-M) reportedly dual-targets dihydrofolate reductase (DHFR) and flavin-dependent thymidylate synthase (FDTS), two essential enzymes in folate biosynthetic pathway. However, the molecular mechanisms and structural dynamics of this dual inhibitory activity of the PAS-M remain elusive. Molecular dynamics simulations revealed that binding of PAS-M towards DHFR is characterized by a recurrence of strong conventional hydrogen bond interactions between a peculiar DHFR binding site residue (Asp27) and the 2-amino-decahydropteridin-4-ol group of PAS-M. Similarly, the binding of PAS-M towards FDTS also involved consistent strong conventional hydrogen bond interactions between some specific residues (Tyr101, Arg172, Thr4, Gln103, Arg87 and Gln106) and, the 2-amino-decahydropteridin-4-ol group, thus establishing the cruciality of the group. Structural dynamics of the bound complexes of both enzymes revealed that, upon binding, PAS-M is anchored at the entrance of hydrophobic pockets by strong hydrogen bond interactions while the rest of the structure gains access to deeper hydrophobic residues to engage in favorable interactions. Further analysis of atomistic changes of both enzymes showed increased C-α atom deviations as well as an increase C-α atoms radius of gyration consistent with structural disorientations. These conformational changes possibly interfered with the biological functions of the enzymes and hence their inhibition as experimentally reported. Structural Insights provided could open up a novel paradigm of structure-based design of multi-targeting inhibitors of biological targets in the folate biosynthetic pathway toward tuberculosis therapy.