Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 µM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Identification of the binding modes of N-phenylphthalimides inhibiting bacterial thymidylate synthase through X-ray crystallography screening.

To identify specific bacterial thymidylate synthase (TS) inhibitors, we exploited phenolphthalein (PTH), which inhibits both bacterial and human enzymes. The X-ray crystal structure of Lactobacillus casei TS (LcTS) that binds PTH showed multiple binding modes of the inhibitor, which prevented a classical structure-based drug design approach. To overcome this issue, we synthesized two phthalimidic libraries that were tested against TS enzymes and then we performed X-ray crystallographic screening of the active compounds. Compounds 6A, 8A, and 12A showed 40-fold higher affinity for bacterial TS than human TS. The X-ray crystallographic screening characterized the binding mode of six inhibitors in complexes with LcTS. Of these, 20A, 23A, and 24A showed a common unique binding mode, whereas 8A showed a different, unique binding mode. A comparative analysis of the LcTS X-ray complexes that were obtained with the pathogenic TS enabled the selection of compounds 8A and 23A as specific compounds and starting points to be exploited for the specific inhibition of pathogen enzymes.

Virtual screening identification of nonfolate compounds, including a CNS drug, as antiparasitic agents inhibiting pteridine reductase.

Folate analogue inhibitors of Leishmania major pteridine reductase (PTR1) are potential antiparasitic drug candidates for combined therapy with dihydrofolate reductase (DHFR) inhibitors. To identify new molecules with specificity for PTR1, we carried out a virtual screening of the Available Chemicals Directory (ACD) database to select compounds that could interact with L. major PTR1 but not with human DHFR. Through two rounds of drug discovery, we successfully identified eighteen drug-like molecules with low micromolar affinities and high in vitro specificity profiles. Their efficacy against Leishmania species was studied in cultured cells of the promastigote stage, using the compounds both alone and in combination with 1 (pyrimethamine; 5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine). Six compounds showed efficacy only in combination. In toxicity tests against human fibroblasts, several compounds showed low toxicity. One compound, 5c (riluzole; 6-(trifluoromethoxy)-1,3-benzothiazol-2-ylamine), a known drug approved for CNS pathologies, was active in combination and is suitable for early preclinical evaluation of its potential for label extension as a PTR1 inhibitor and antiparasitic drug candidate.

Curcumin derivatives: molecular basis of their anti-cancer activity.

Curcumin, a phenolic compound from the plant Curcuma longa L., has shown a wide-spectrum of chemopreventive, antioxidant and antitumor properties. Although its promising chemotherapeutic activity, preclinical and clinical studies highlight Curcumin limited therapeutic application due to its instability in physiological conditions. To improve its stability and activity, many derivatives have been synthesized and studied, among which bis-DemethoxyCurcumin (bDMC) and diAcetylCurcumin (DAC). In this report, we show that both bDMC and DAC are more stable than Curcumin in physiological medium. To explore the mechanism of their chemotherapeutic effect, we studied their role in proliferation in the HCT116 human colon cancer cells. We correlated kinetic stability and cellular uptake data to their biological effects. Both bDMC and DAC impair correct spindles formation and induce a p53- and p21(CIP1/WAF1)-independent mitotic arrest, which is more stable and long-lasting for bDMC. A subsequent p53/p21(CIP1/WAF1)-dependent inhibition of G1 to S transition is triggered by Curcumin and DAC as a consequence of the mitotic slippage, preventing post-mitotic cells from re-entering the cell cycle. Conversely, the G1/S arrest induced by bDMC is a direct effect of the drug and concomitant to the mitotic block. Finally, we demonstrate that bDMC induces rapid DNA double-strand breaks, moving for its possible development in anti-cancer clinical applications.

New synthetic glucosyl-curcuminoids, and their (1)H and (13)C NMR characterization, from Curcuma longa L.

Turmeric extracts, among which curcumin and bis-demethoxycurcumin, are by far known for their therapeutic activities. In this study we propose easy and low cost synthetic pathways in order to obtain glucosyl-curcuminoids, safe and water soluble potential drugs and dyes, which may be implied in different fields ranging from pharmacology to food chemistry. The complete (1)H and (13)C NMR characterization of naturally occurring curcumin, bis-demethoxycurcumin and new synthetic glucosyl-curcuminoids is reported.

Synthesis, cytotoxic and combined cDDP activity of new stable curcumin derivatives.

New curcumin derivatives are synthesized in order to improve chemical properties of curcumin. The aromatic ring glycosylation of curcumin provides more water-soluble compounds with a greater kinetic stability which is a fundamental feature for drug bioavailability. The glycosylation reaction is quite simple, low cost, with high yield and minimum waste. NMR data show that the ability of curcumin to coordinate metal ion, in particular Ga(III), is maintained in the synthesized products. Although the binding of glucose to curcumin reduces the cytotoxicity of the derivatives towards cisplatin (cDDP)-sensitive and -resistant human ovarian carcinoma cell lines, the compounds display a good selectivity since they are much less toxic against non-tumourigenic Vero cells. The combination of cDDP with the most active glycosyl-curcuminoid drug against both cDDP-sensitive and -resistant as well as against Vero cell lines is tested. The results show an improvement of cDDP efficacy with higher selectivity towards cancer cells than non-cancer cells. These studies indicate the need for developing new valid components of drug treatment protocols to cDDP-resistant cells as well.

Synthesis and characterization of glucosyl-curcuminoids as Fe3+ suppliers in the treatment of iron deficiency.

The Fe(3+) chelating ability of some curcumin glucosyl derivatives (Glc-H; Glc-OH; Glc-OCH(3)) is tested by means of UV and NMR study. The pK(a) values of the ligands and the overall stability constants of Fe(3+) and Ga(3+) complexes are evaluated from UV spectra. The only metal binding site of the ligand is the beta-diketo moiety in the keto-enolic form; the glucosyl moiety does not interact with metal ion but it contributes to the stability of metal/ligand 1:2 complexes by means of hydrophilic interactions. These glucosyl derivatives are able to bind Fe(3+) in a wide pH rage, forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. In addition they demonstrate to have a poor affinity for competitive biological metal ions such as Ca(2+). All ligands and their iron complexes have a good lypophilicity (log P > -0.7) suggesting an efficient gastrointestinal absorption in view of their possible use as iron supplements in oral therapy. The ligand molecules are also tested for their antioxidant properties in "ex vivo" biological system.

Synthesis, chemical and biological studies on new Fe(3+)-glycosilated beta-diketo complexes for the treatment of iron deficiency.

A simple synthetic pathway to obtain glycosilated beta-diketo derivatives is proposed. These compounds show a good iron(III) affinity therefore we may suggest the use of their Fe(3+)-complexes as oral iron supplements in the treatment of anaemia. The glycosilated compounds (6-GlcH, 6-GlcOH and 6-GlcOCH(3)) are characterized by means of spectroscopic (UV, (1)H and (13)C NMR) and potentiometric techniques; they have a good water solubility, are kinetically stable in physiological condition (t(1/2)>100h) and show a low cytotoxicity also in high concentrations (IC(50)>400 microM). They are able to bind Fe(3+) ion in acid condition (pH approximately 2) forming complex species thermodynamically more stable than those of other ligands commonly used in the treatment of iron deficiency. The iron complexes show also a good kinetic stability both in acidic and physiological pH and have a good lypophilicity (logP>-0.7) that suggests an efficient gastrointestinal absorption in view of their possible use in oral therapy. In addition they demonstrate a poor affinity for competitive biological metal ion such as Ca(2+), and in particular 6-GlcOCH(3) is able to inhibit lipid peroxidation.

Synthesis and characterization of new beta-diketo derivatives with iron chelating ability.

Here we report the synthesis, the characterization and a theoretical study on new glycosylated phenyl substituted beta-diketones; two classes of compounds are obtained according to the condensation reaction: central and side derivatives. Their iron(III) chelating ability is tested by means of UV-visible (UV-vis), potentiometric and NMR techniques. The conformation of central derivatives does not allow any metal chelation, while side derivatives bind iron(III) through the beta-dioxo moiety. The glycosyl moiety does not interact with metal ion but it helps to stabilize metal/ligand (1/3) complexes by means of hydrophylic interactions. The pK(a) of the ligands and the stability constants of their Fe(III) complexes are evaluated by means of UV-vis spectroscopy and potentiometry. A comparison with other iron-chelating agents, on the basis of lipophilicity and the pFe(III), is finally reported.