Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

A multilayer screening approach toward the discovery of novel Pf-DHFR inhibitors.

A small yet diverse xanthone library was build and computationally docked against wild type Pf-DHFR by Molegro Virtual Docker (MolDock). For analysis of results an integrated approach based on re-ranking, scaling (based on heavy atom counts), pose clustering and visual inspection was implemented. Standard methods such as self-docking (for docking), EF analysis, average rank determinations (for size normalization), and cluster quality indices (for pose clustering) were used for validation of results. Three compounds X5, X113A and X164B displayed contact footprints similar to the known inhibitors with good scores. Finally, 16 compounds were extracted from ZINC data base by similarity based screening, docking score and drug/lead likeness. Out of these 16 compounds, 11 displayed very close contact footprints to experimentally known inhibitors, indicating there potential utility in further drug discovery efforts.

The Lactone form of stachybotrydial: a new inhibitor of dihydrofolate reductase from stachybotrys sp. FN298.

Dihydrofolate reductase (DHFR) has been confirmed to be a novel target for antibacterial drug development. In this study, we determined that a fungal metabolite from Stachybotrys sp. FN298 can inhibit the DHFR of Staphylococcus aureus. Its structure was identified as a lactone form of stachybotrydial using mass spectrometry and nuclear magnetic resonance analysis. This compound inhibited S. aureus DHFR with a half-maximal inhibitory concentration of 41 µM. It also prevented the growth of S. aureus and methicillin-resistant S. aureus (MRSA) with a minimum inhibitory concentration of 32 µg·mL(-1). To our knowledge, this is the first description of a DHFR inhibitor of microbial origin. The inhibitory function of the lactone form of stachybotrydial highlights its potential for development into a new broad-spectrum antibacterial agent and as an agent against MRSA.

Exploration of 2, 4-diaminopyrimidine and 2, 4-diamino-s-triazine derivatives as potential antifilarial agents.

In view of the mandate from the World Health Organization (WHO) for developing novel drug candidates against human lymphatic filariasis, dihydrofolate reductase (DHFR) inhibitors are explored as potential antifilarial agents. The in vitro biological evaluation of an in-house library of 12 diverse antifolate compounds with 2,4-diaminopyrimidine and 2,4-diamino-s-triazine structural features against Brugia malayi is reported. To confirm the DHFR inhibitory potential of these compounds, reversal studies using folic acid and folinic acid were undertaken. Inhibition of DHFR can induce apoptosis; in this light, preliminary evidence of apoptosis by test compounds was detected using ethidium bromide-acridine orange staining and the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibition assay. Among the evaluated compounds, 3 showed significant activity against both microfilariae and adult worms. The effects of 2 of these compounds were mostly reversed by folic acid, validating DHFR inhibitory activity. Partial reversal of the effect of 2 compounds by folinic acid and non-reversal of the effect of the third compound both by folic and folinic acids are discussed. This study opens new avenues for the discovery of lead molecules by exploiting the folate pathway against one of the major neglected tropical diseases, filariasis.

The folate metabolic network of Falciparum malaria.

The targeting of key enzymes in the folate pathway continues to be an effective chemotherapeutic approach that has earned antifolate drugs a valuable position in the medical pharmacopoeia. The successful therapeutic use of antifolates as antimalarials has been a catalyst for ongoing research into the biochemistry of folate and pterin biosynthesis in malaria parasites. However, our understanding of the parasites folate metabolism remains partial and patchy, especially in relation to the shikimate pathway, the folate cycle, and folate salvage. A sizeable number of potential folate targets remain to be characterised. Recent reports on the parasite specific transport of folate precursors that would normally be present in the human host awaken previous hypotheses on the salvage of folate precursors or by-products. As the parasite progresses through its life-cycle it encounters very contrasting host cell environments that present radically different metabolic milieus and biochemical challenges. It would seem probable that as the parasite encounters differing environments it would need to modify its biochemistry. This would be reflected in the folate homeostasis in Plasmodium. Recent drug screening efforts and insights into folate membrane transport substantiate the argument that folate metabolism may still offer unexplored opportunities for therapeutic attack.

Folate metabolism in human malaria parasites--75 years on.

Malaria still poses one of the most serious threats to human health worldwide and the prevailing lack of effective, clinically licensed, vaccines means that prophylaxis and treatment depend heavily on a small number of compounds whose efficacies are progressively compromised at varying rates by the inevitable emergence of drug-resistant parasite populations. Of these antimalarials, those inhibiting steps in folate metabolism, along with chloroquine, are the oldest synthetic compounds, with origins dating back three-quarters of a century. Despite widespread parasite resistance, the antifolates still play an important role in malaria control, and our understanding of the underlying mechanisms of folate metabolism and genesis of drug resistance has increased considerably over the last twenty years. Folate de novo synthesis in the parasite, interconversion of active folate derivatives and their utilisation as multifunctional cofactors involve numerous enzymes, although only two of these have ever served as targets of clinical antimalarial inhibitors. The current application of antifolates, resistance to this class of drugs, new insights into folate metabolism in the parasite, its potential for providing novel targets of inhibition and some of the questions that are still outstanding are reviewed here.

Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening.

BACKGROUND: Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites. METHODS: Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays. RESULTS: Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites. CONCLUSION: With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

Quenched phosphorescence as alternative detection mode in the chiral separation of methotrexate by electrokinetic chromatography.

Quenched phosphorescence was used, for the first time, as detection mode in the chiral separation of methotrexate (MTX) enantiomers by electrokinetic chromatography. The detection is based on dynamic quenching of the strong emission of the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) by MTX under deoxygenated conditions. The use of a background electrolyte with 3 mg/mL 2-hydroxypropyl-ß-cyclodextrin and 20% MeOH in 25 mM phosphate buffer (pH 7.0) and an applied voltage of 30 kV allowed the separation of L-MTX and its enantiomeric impurity D-MTX with sufficient resolution. In the presence of 1 mM BrNS, a detection limit of 3.2 × 10(-7) M was achieved, about an order of magnitude better than published techniques based on UV absorption. The potential of the method was demonstrated with a degradation study and an enantiomeric purity assessment of L-MTX. Furthermore, L-MTX was determined in a cell culture extract as a proof-of-principle experiment to show the applicability of the method to biological samples.

Greeen tea extracts lower serum folates in rats at very high dietary concentrations only and do not affect plasma folates in a human pilot study.

Green tea catechins (GTC) have been shown to inhibit the activities of enzymes involved in folate uptake. Hence, regular green tea drinkers may be at risk of impaired folate status. The present experiments aimed at studying the impact of dietary GTC on folate concentrations and metabolism. In a human pilot study (parallel design) healthy men consumed for 3 weeks 6 capsules (approximately 670 mg GTC) per day (2 capsules with each principal meal) containing aqueous extracts of the leaves of Camellia sinensis (n=17) or placebo (n=16). No differences in plasma folate concentrations were observed between treatments. We further fed groups of 10 male rats diets fortified with 0, 0.05, 0.5, 1, or 5 g GTC/kg for 6 weeks. Only at the highest intake, GTC significantly decreased serum 5-methyl-tetrahydrofolate concentrations in rats, while mRNA concentrations of reduced folate carrier, proton-coupled folate transporter/heme carrier protein 1, and dihydrofolate reductase (DHFR) remained unchanged in intestinal mucosa. Using an in vitro enzyme activity assay, we observed a time- and dose-dependent inhibition of DHFR activity by epigallocatechin gallate and a green tea extract. Our data suggest that regular green tea consumption is unlikely to impair folate status in healthy males, despite the DHFR inhibitory activity of GTC.

Immobilization of malarial (Plasmodium falciparum) dihydrofolate reductase for the selection of tight-binding inhibitors from combinatorial library.

A simple procedure for selection of tight-binding inhibitors of mutant dihydrofolate reductases from Plasmodium falciparum (PfDHFRs) based on preferential binding to the enzyme immobilized on a Sepharose column has been described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a thiopropyl-Sepharose gel via S-S linkage. The amount of immobilized DHFRs was estimated to be 4-5 mg/g of dried gel, and the activities of bound DHFRs were comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on the affinities of each ligand with the enzyme. Free ligands were then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry, and the components with high binding affinity of the library could thus be realized. Results could be confirmed by quantitative analysis of the bound ligands released from the enzyme by guanidine hydrochloride treatment.

Separation methods for methotrexate, its structural analogues and metabolites.

Methotrexate (MTX) is the prototype folate antagonist cytotoxic drug, employed in the therapy of solid tumors and leukaemias, and recently also as an immunosuppressive agent in organ transplantation, in the treatment of some autoimmune diseases and in the therapy of severe asthma. MTX is one of the very few antineoplastic drugs the therapeutic concentration monitoring of which is currently employed in clinical practice and can be routinely measured in biological samples by a number of different analytical techniques, among which are immunoenzymatic and chromatographic methods. Each technique has of course its own advantages in terms of sensitivity, specificity, speed, cost and level of expertise required. Along with therapeutic drug concentration monitoring and clinical pharmacology, fundamental research into the mechanism of action of antifolate drugs is still a field which requires the measurement of MTX, of its new analogues and of their metabolites in biological samples. This review summarizes the instrumental conditions and the performance of several published chromatographic methods employed to measure MTX, its metabolites and some analogues in clinical and biological research. More than 70 papers describing chromatographic assays for MTX and its metabolites have been published in the literature between 1975 and 2000. A wide array of experimental conditions for sample preparation, analyte separation and detection have been employed. According to their chemical properties, MTX, its metabolites and analogue drugs present in several biological samples (plasma, serum, saliva, urine, cerebrospinal fluid, tissue specimens) can be extracted, separated and detected under a variety of chromatographic conditions, i.e. on different stationary phases, under a wide choice of mobile phase conditions (acidic or neutral, employing ion-pair or micellar chromatography), followed by several detection techniques (UV-Vis spectrophotometry, pre- or post-column oxidation and fluorimetry, electrochemistry, mass spectrometry). Optimized methods allow simultaneous measurement within a few minutes of the plasma levels of MTX and its main metabolites at concentrations in the low-nM range. One special field which needs sensitive, fast and inexpensive methods for the detection and measurement of MTX is the monitoring of contamination in workplace environments, such as pharmaceutical industries and oncological hospital pharmacies, and in sewage waters. The measurement of the intracellular gamma-oligo-glutamate metabolites of biological folates, of MTX and of some analogue drugs is of great importance in basic pharmacological research. The existence of empirical quantitative relationships between the retention of individual oligomers under different chromatographic conditions and the number of added glutamic acid units allows identification of the metabolites even when authentic standards are not available.

Dihydrofolate reductase and cell growth activity inhibition by the beta-carboline-benzoquinolizidine plant alkaloid deoxytubulosine from Alangium lamarckii: its potential as an antimicrobial and anticancer agent.

Beta-carboline-benzoquinolizidine plant alkaloid deoxytubulosine (DTB) was evaluated and assessed for the first time for its biochemical and biological activity employing the biomarker dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) as the probe enzyme, a key target in cancer chemotherapy. DHFR, employed in the present investigations was purified from Lactobacillus leichmannii. DTB, isolated from the Indian medicinal plant Alangium lamarckii was demonstrated to exhibit potent cytotoxicity. The alkaloid potently inhibited the cell growth of L. leichmannii and the cellular enzyme activity of DHFR (IC50=40 and 30 microM for the cell growth and enzyme inhibitions, respectively). DTB concentrations >75 microM resulted in a total loss of the DHFR activity, thus suggesting that the beta-carboline-benzoquinolizidine plant alkaloid is a promising potential antitumor agent. Our results are also suggestive of its potential antimicrobial activity. DTB binding to DHFR appears to be slow and reversible. Inhibition kinetics revealed that DHFR has a Ki value of 5x10(-6) M for DTB and that the enzyme inhibition is a simple linear 'non-competitive' type.

Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry.

A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase.

Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid. The products of the enzymatic reactions were purified by semipreparative C18 HPLC. The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y. Under standard reaction conditions, the E. coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position. Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates. The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors.

Renal and hepatic toxicity after high-dose 7-hydroxymethotrexate in the rat.

To examine directly the hepatic and renal toxicity of 7-hydroxymethotrexate (7-OH-MTX) without interference of the parent compound methotrexate (MTX), we purified and gave 100 mg/kg 7-OH-MTX to rats, a dose resulting in serum levels of 7-OH-MTX comparable with those achieved in the clinic after the administration of high-dose MTX (HD-MTX). After only 5 h, the 7-OH-MTX-treated rats demonstrated 2.6-fold increases in serum creatinine values and 2-fold elevations in serum aspartate aminotransferase (ASAT) levels as compared with the controls. Morphologic evidence of toxicity, however, was apparent only in the kidneys. Intraluminal cellular debris containing membranous material and deteriorated organelles was seen, but no precipitate of the delivered drug. The peak serum concentration of 7-OH was up to 939 microM, and concentrations of 7-OH-MTX declined triphasically, showing a t1/2 alpha value of 2.45 min, a t1/2 beta value of 30.5 min, and a terminal half-life (t1/2 gamma) of 240 min. The total clearance value was 14.5 ml min-1 kg, and the postdistributional volume of distribution (V beta) was 5070 ml/kg. Our results may indicate a direct toxic effect of 7-OH-MTX on kidney and liver cells.

Synthesis and biological properties of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid.

The synthesis of the antifolate 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) has been modified. It is prepared from 2-acetamido-6-formyl-4(3H)-pyrido[2,3-b]pyrimidone and [P-(N-[1,3-bis(ethoxycarbonyl)propan-1-yl]aminocarbonyl)] phenylmethyl]-triphenylphosphonium bromide. The synthesis proceeds via a sodium hydride promoted Wittig condensation in 1-methyl-2-pyrrolidone followed by catalytic reduction, mild base hydrolysis, and acid precipitation of the product. Synthesis of DDATHF is achieved in a total of seven steps from commercially available reagents. DDATHF is transported effectively into CCRF-CEM cells and inhibits growth of both human (CEM) and murine (L1210) cells in culture. Studies reported here support the view that methotrexate and DDATHF are transported via a shared transport mechanism.

Screening for new antifolates of microbial origin and a new antifolate AM-8402.

A screening method was established for new specific inhibitors of folate metabolism. Culture broths of soil isolates were selected based on relative microbial activity. A culture, to be retained, must be active against Enterococcus faecium grown in a medium which contains a limited amount of pteroic acid but lacks activity against the microorganism grown in a medium supplemented with thymidine. By this screening method, three new antibiotics, diazaquinomycins A and B and AM-8402 were selected from eight thousand soil isolates. The isolation and structures of diazaquinomycins have been reported. AM-8402 is a new antifolate active against Gram-positive bacteria and mycoplasmas. It consists of a nanaomycin D moiety as chromophore and a deoxysugar and is structurally related to medermycin.

Structural features of 4-amino antifolates required for substrate activity with mammalian folylpolyglutamate synthetase.

The activity of a series of folic acid analogues as substrates for partially purified mouse liver folylpolyglutamate synthetase was determined and the effects of substituents on the binding to, and catalytic processes of, this enzyme were inferred. A 4-amino group improved substrate activity primarily by decreasing the apparent Km while N10-methyl substitution substantially diminished utilization as a substrate, again, by effects on Km. Isosteric replacement of N-10 altered substrate activity. A free alpha-carboxyl group in the amino acid side chain was required for catalysis as was the presence of the side chain amide carbonyl group. Modification of the amino acid side chain length profoundly affected activity. Several observations were made that may be relevant to chemotherapy with folate antimetabolites: 1) 7-hydroxymethotrexate was a substrate for this enzyme; 2) substrate activity and substrate inhibition were observed with CB 3717, a potent inhibitor of thymidylate synthase; 3) potent classical dihydrofolate reductase inhibitors were identified that were either not substrates for mouse liver folylpolyglutamate synthetase (e.g., 4-amino-4-deoxy-N10-methylpteroyl-L-alpha-aminoadipate) or were much better substrates than methotrexate for this enzyme (e.g., aminopterin); and 4) leucovorin and methotrexate appeared to be substrates for the same synthetase, but leucovorin saturated the reaction at much lower concentrations. These results have implications for the design of folylpolyglutamate synthetase inhibitors and for the selection of dihydrofolate reductase inhibitors that are either not polyglutamated or are efficiently polyglutamated in vivo.