Indole and aminoimidazole moieties appear as key structural units in antiplasmodial molecules

From a library of compounds of natural sources, a big series of molecules was chosen by random sampling to evaluate their in vitro antimalarial activity against Plasmodium falciparum and their antifungal activity against Candida sp. From 184 molecules tested, no molecules were active against Candida sp. (MIC > 10 μg/ml) whereas 13 clearly showed high antiplasmodial activity in vitro, with an IC₅₀ less than 1 μg/ml against the chloroquine-resistant strain of P. falciparum FcM29-Cameroon. The molecules with the best antiplasmodial efficacy were 10-hydroxy-ellipticin (IC₅₀: 0.08 μg/ml), tchibangensin (IC₅₀: 0.13 μg/ml), ellipticin hydrochloride (IC₅₀: 0.17 μg/ml), usambarensin (IC₅₀: 0.23 μg/ml), 7S,3S-ochropposinine oxindole (IC₅₀: 0.25 μg/ml), 3,14-dihydro-ellipticin (IC₅₀: 0.25 μg/ml), tetrahydro-4′,5′,6′17-usambarensin 17S (IC₅₀: 0.26 μg/ml), ellipticine (IC₅₀: 0.28 μg/ml), aricin (IC₅₀: 0.3 μg/ml), 10-methoxy-ellipticin (IC₅₀: 0.32 μg/ml), aplysinopsin (IC₅₀: 0.43 μg/ml), descarbomethoxydihydrogambirtannin (IC₅₀: 0.46 μg/ml) and ochrolifuanin A (IC₅₀: 0.47 μg/ml). Among these 13 promising molecules, all except descarbomethoxydihydrogambirtannin, ochrolifuanine A and usambarensine presented here novel biological activities since they had never been described in the literature for their antiplasmodial activity. In spite of the large diversity of the molecules which have been tested, it is interesting to note that the ones active against Plasmodium are all indole derivatives (and one is both indolic and aminoimidazolic). To find new antiplasmodial compounds, ethnopharmacological approaches studying traditional medicine treatments for malaria is largely used but random research produced here an interesting yield (7%) of new antiplasmodial hits and appears therefore complementary to the traditional medicine way.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

Ganoderol B: A potent α-glucosidase inhibitor isolated from the fruiting body of Ganoderma lucidum

α-Glucosidase inhibitor has considerable potential as a diabetes mellitus type 2 drug because it prevents the digestion of carbohydrates. The search for the constituents reducing α-glucosidase activity led to the finding of active compounds in the fruiting body of Ganoderma lucidum. The CHCl₃ extract of the fruiting body of G. lucidum was found to show inhibitory activity on α-glucosidase in vitro. The neutral fraction, with an IC₅₀ of 88.7 μg/ml, had stronger inhibition than a positive control, acarbose, with an IC₅₀ of 336.7 μg/ml (521.5 μM). The neutral fraction was subjected to silica gel column chromatography and repeated p-HPLC to provide an active compound, (3β,24E)-lanosta-7,9(11),24-trien-3,26-diol (ganoderol B). It was found to have high α-glucosidase inhibition, with an IC₅₀ of 48.5 μg/ml (119.8 μM).     

Characterization of Saccharomyces cerevisiae Atm1p. Functional studies of an ABC7 type transporter

Saccharomyces cerevisiae Atm1p has been cloned, over-expressed and purified from a yeast expression system. The sequence includes both the soluble ATPase and transmembrane-spanning domains. With the introduction of an N-terminal Kozak sequence and a C-terminal (His)₆-tag, a yield of 1 mg of Atm1p was obtained from 3 g wet yeast cells, which is comparable to other membrane-associated proteins isolated from eukaryotic expression systems. The ATPase activity of Atm1p is sensitive to sodium vanadate, a P-type ATPase inhibitor, with an IC₅₀ of 4 μM. MgADP is a product inhibitor for Atm1p with an IC₅₀ of 0.9 mM. The Michaelis–Menten constants V_max, K_M and k_cat of Atm1p were measured as 8.7 ± 0.3 μM/min, 107 ± 16 μM and 1.24 ± 0.06 min^− 1, respectively. A plot of ATPase activity versus concentration of Atm1p exhibits a nonlinear relationship, suggesting an allosteric response and an important role for the transmembrane domain in mediating both ATP hydrolysis and MgADP release. The metal dependence of Atm1p ATPase activity demonstrated a reactivity order of Mg²⁺ > Mn²⁺ > Co²⁺, while each divalent ion was found to be inhibitory at higher concentrations. The activation and inhibitory effect of phospholipids suggest that formation of a lipid–micelle complex is important for enzymatic activity and stability. Structural analysis of Atm1p by CD spectroscopy suggested a similarity of secondary structure to that found for other members of this ABC protein family.     

Oenothein B's contribution to the anti-inflammatory and antioxidant activity of Epilobium sp

Willow herb tea or preparation are available and relatively popular in the European market, and claimed to be effective inter alia because of their anti-inflammatory activity. The present study is therefore aimed at comparing the anti-inflammatory and antioxidant activity of extracts of the three most popular Epilobium species (E. angustifolium, E. hirsutum and E. parviflorum) and at juxtaposing this activity against the dominating compounds from the following extracts: oenothein B (OeB), quercetin-3-O-glucuronide and myricetin-3-O-rhamnoside. The phytochemical analysis of the extracts has shown that OeB quantities vary between 20% and 35%, while flavonoids content does not exceed 2%. All extracts have inhibited the activity of hyaluronidase and lipoxygenase with IC₅₀ around 5 μg/ml and 25 μg/ml. The inhibition of hyaluronidase is related with the presence of OeB, a strong inhibitor of this enzyme (IC₅₀ 1.1 μM). Additionally, the extracts inhibited myeloperoxidase (MPO) release from stimulated neutrophils. OeB inhibited MPO release similarly to the anti-inflammatory drug indomethacin with IC₅₀ 7.7 μM and 15.4 μM, respectively. Tested extracts significantly reduced the production of reactive oxygen species (ROS) from f-MLP and PMA induced neutrophils with IC₅₀ 5 μg/ml and 25 μg/ml, respectively. The flavonoids content seems to exert little influence on extracts’ activity, contrary to OeB, whose high concentration explains the activity of extract obtained from Epilobium. Tested currently marketed Epilobium preparations are often wrongly assigned, but we should stress that the level of OeB in all tested herbs was high and always exceeded 2% in raw material.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Cytotoxic activity of secondary metabolites derived from Artemisia annua L. towards cancer cells in comparison to its designated active constituent artemisinin

Artemisia annua L. (sweet wormwood, qinhao) has traditionally been used in Chinese medicine. The isolation of artemisinin from Artemisia annua and its worldwide accepted application in malaria therapy is one of the showcase success stories of phytomedicine during the past decades. Artemisinin-type compounds are also active towards other protozoal or viral diseases as well as cancer cells in vitro and in vivo. Nowadays, Artemisia annua tea is used as a self-reliant treatment in developing countries. The unsupervised use of Artemisia annua tea has been criticized to foster the development of artemisinin resistance in malaria and cancer due to insufficient artemisinin amounts in the plant as compared to standardized tablets with isolated artemisinin or semisynthetic artemisinin derivatives. However, artemisinin is not the only bioactive compound in Artemisia annua. In the present investigation, we analyzed different Artemisia annua extracts. Dichloromethane extracts were more cytotoxic (range of IC₅₀: 1.8-14.4 μg/ml) than methanol extracts towards Trypanosoma b. brucei (TC221 cells). The range of IC₅₀ values for HeLa cancer cells was 54.1-275.5 μg/ml for dichloromethane extracts and 276.3-1540.8 μg/ml for methanol extracts. Cancer and trypanosomal cells did not reveal cross-resistance among other compounds of Artemisia annua, namely the artemisinin-related artemisitene and arteanuine B as well as the unrelated compounds, scopoletin and 1,8-cineole. This indicates that cells resistant to one compound retained sensitivity to another one. These results were also supported by microarray-based mRNA expression profiling showing that molecular determinants of sensitivity and resistance were different between artemisinin and the other phytochemicals investigated.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Determination and pharmacokinetics of gastrodin in human plasma by HPLC coupled with photodiode array detector

In present study, an HPLC method coupled with photodiode array detector (HPLC-PDA) was established for determination and pharmacokinetics of gastrodin (GAS) in human plasma after an oral administration of GAS capsule. In the method, ethanol and dichloromethane were respectively used for deproteinization and purification during the sample preparation procedure. Separation of GAS was achieved on an AichromBond-AQ C18 column (5 μm, 150 mm × 4.6 mm) with the mobile phase of methanol–0.1% phosphoric acid solution (2:98, v/v) at a flow rate of 0.8 ml/min. The wavelength was set at 220 nm and the injection volume was 20 μl. Under the conditions, the calibration curve was linear within the concentration range of 50–4000 ng/ml with the correlation coefficient (r) of 0.99554 (weight = 1/X²) and the lower limit of quantification (LLOQ) was 50 ng/ml. The inter- and intra-day precisions were less than 11% and the accuracies (%) were within the range of 95.55–103.78%. The extraction recoveries were over 65% with RSDs less than 5.50%. The GAS was proved to be stable under tested conditions. Thus, the method was valid enough to be applied for pharmacokinetic study of GAS in human plasma. The pharmacokinetic parameters of GAS in human plasma after an oral administration of 200 mg GAS capsule were described as: C_max, 1484.55 ± 285.05 ng/ml; T_max, 0.81 ± 0.16 h; t_1/2α, 3.78 ± 2.33 h; t_1/2β, 6.06 ± 3.20 h; t_1/2Ka, 0.18 ± 0.53 h; K₁₂, 0.18 ± 0.41/h; K₂₁, 0.20 ± 0.16/h; K₁₀, 4.11 ± 15.81/h; V1/F, 180.35 ± 89.44 L; CL/F, 62.50 ± 140.03 l/h; AUC_0→t, 5619.41 ± 1972.88 (ng/ml) h; and AUC_0→∞, 7210.26 ± 3472.74 (ng/ml) h, respectively. These will be useful for the clinical application of GAS.     

Cardiodepressive effect elicited by the essential oil of Alpinia speciosa is related to L-type Ca current blockade

This study was undertaken to elucidate the effect of the essential oil from Alpinia speciosa (EOAs) on cardiac contractility and the underlying mechanisms. The essential oil was obtained from Alpinia speciosa leaves and flowers and the oil was analyzed by GC-MS method. Chemical analysis revealed the presence of at least 18 components. Terpinen-4-ol and 1,8-cineole corresponded to 38% and 18% of the crude oil, respectively. The experiments were conducted on spontaneously-beating right atria and on electrically stimulated left atria isolated from adult rats. The effect of EOAs on the isometric contractions and cardiac frequency in vitro was examined. EOAs decreased rat left atrial force of contraction with an EC₅₀ of 292.2 ± 75.7 μg/ml. Nifedipine, a well known L-type Ca²⁺ blocker, inhibited in a concentration-dependent manner left atrial force of contraction with an EC₅₀ of 12.1 ± 3.5 μg/ml. Sinus rhythm was diminished by EOAs with an EC₅₀ of 595.4 ± 56.2 μg/ml. Whole-cell L-type Ca²⁺ currents were recorded by using the patch-clamp technique. EOAs at 25 μg/ml decreased I_Ca,L by 32.6 ± 9.2% and at 250 μg/ml it decreased by 89.3 ± 7.4%. Thus, inhibition of L-type Ca²⁺ channels is involved in the cardiodepressive effect elicited by the essential oil of Alpinia speciosa in rat heart.     

Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A₁ (CA1) and novobiocin (NOVO). The calculated K_d values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex: 22.2 ± 8.8 μM⁻¹ s⁻¹ and 2.7 ± 0.6 s⁻¹, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The K_d values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC₅₀ values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC₅₀ for GM was 2.80 ± 0.18 μM, and the relative IC₅₀ values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.     

Flow cytometric screening for anti-leishmanials in a human macrophage cell line

High-throughput drug screening methods against the intracellular stage of Leishmania have been facilitated by the development of in vitro models of infection. The use of cell lines rather than primary cells facilitates these methods. Peripheral blood mononuclear cell (PBMC) derived macrophages and THP-1 cells were infected with stationary phase egfp transfected Leishmania amazonensis parasites and then treated with anti-leishmanial compounds. Drug activity was measured using a flow cytometric approach, and toxicity was assessed using either the MTT assay or trypan blue dye exclusion. Calculated EC₅₀’s for amphotericin B, sodium stibogluconate, and miltefosine were 0.1445 ± 0.0005 μg/ml, 0.1203 ± 0.018 mg/ml, and 26.71 μM using THP-1 cells, and 0.179 ± 0.035 μg/ml, 0.1948 ± 0.0364 mg/ml, and 13.77 ± 10.74 μM using PBMC derived macrophages, respectively. We conclude that a flow cytometric approach using egfp transfected Leishmania species can be used to evaluate anti-leishmanial compounds against the amastigote stage of the parasite in THP-1 cells with excellent concordance to human PBMC derived macrophages.     

Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli

Irreversible inactivation of α-thrombin (T) by the serpin, heparin cofactor II (HCII), is accelerated by ternary complex formation with the glycosaminoglycans (GAGs) heparin and dermatan sulfate (DS). Low expression of human HCII in Escherichia coli was optimized by silent mutation of 27 rare codons and five secondary Shine-Dalgarno sequences in the cDNA. The inhibitory activities of recombinant HCII, and native and deglycosylated plasma HCII, and their affinities for heparin and DS were compared. Recombinant and deglycosylated HCII bound heparin with dissociation constants (K_D) of 6 ± 1 and 7 ± 1 μM, respectively, ∼6-fold tighter than plasma HCII, with K_D 40 ± 4 μM. Binding of recombinant and deglycosylated HCII to DS, both with K_D 4 ± 1 μM, was ∼4-fold tighter than for plasma HCII, with K_D 15 ± 4 μM. Recombinant HCII, lacking N-glycosylation and tyrosine sulfation, inactivated α-thrombin with a 1:1 stoichiometry, similar to plasma HCII. Second-order rate constants for thrombin inactivation by recombinant and deglycosylated HCII were comparable, at optimal GAG concentrations that were lower than those for plasma HCII, consistent with its weaker GAG binding. This weaker binding may be attributed to interference of the Asn¹⁶⁹N-glycan with the HCII heparin-binding site.     

Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate

Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H₂satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2′,3′-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2′,3′-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near − 0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~ 1.85 μ_B) are avid DNA binders giving K_b values within 1.0 × 10⁵ − 8.0 × 10⁵ M^− 1. Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC₅₀ = 8.3(± 1.0) μM) in visible light, while showing lower dark toxicity (IC₅₀ = 17.2(± 1.0) μM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC₅₀ = 30.0(± 1.0) μM in dark), while retaining its photocytotoxicity (IC₅₀ = 8.0(± 1.0) μM).     

Design of novel N-phenylnicotinamides as selective cyclooxygenase-1 inhibitors

A series of N-phenylnicotinamides (1-40) were designed and evaluated in vitro for their COX inhibitory activities. Most of the synthesized compounds were proved to be potent and selective inhibitors of COX-1. Compound 28 showed the most potent COX-1 inhibitory activity (COX-1 IC₅₀ = 0.68 ± 0.07 μM) and good selectivity (COX-2 IC₅₀ >100 μM). This compound may be useful as a lead compound for superior COX-1 inhibitors. On the basis of the biological results, structure-activity relationships for the COX-1-inhibitory activities of the synthesized N-phenylnicotinamides were discussed concisely.     

Hypericins and thioredoxin reductase: Biochemical and docking studies disclose the molecular basis for effective inhibition by naphthodianthrones

Cytosolic (TrxR1) and mitochondrial (TrxR2) thioredoxin reductases experience pronounced concentration- and time-dependent inhibition when incubated with the two naphthodianthrones hypericin and pseudohypericin. Pseudohypericin turned out to be a quite strong inhibitor of TrxR1 (IC₅₀ = 4.40 ??M) being far more effective than hypericin (IC₅₀ = 157.08 ??M). In turn, the IC₅₀ values measured toward TrxR2 were 7.45 ??M for pseudohypericin and 43.12 ??M for hypericin. When compared to pseudohypericin, the inhibition caused by hypericin usually required significantly longer times, in particular on TrxR1. These important differences in the inhibitory potencies and profiles were analysed through a molecular modeling approach. Notably, both compounds were found to accommodate in the NADPH-binding pocket of the enzyme. The binding of the two naphthodianthrones to thioredoxin reductase seems to be particularly strong as the inhibitory effects were fully retained after gel filtration. Also, we found that TrxR inhibition by hypericin and pseudohypericin does not involve the active site selenol/thiol motif as confirmed by biochemical and modeling studies. The resulting inhibition pattern is very similar to that produced by the two naphthodianthrones on glutathione reductase. As the thioredoxin system is highly overexpressed in cancer cells, its inhibition by hypericin and pseudohypericin, natural compounds showing appreciable anticancer properties, might offer new clues on their mechanism of action and open interesting perspectives for future tumor therapies.     

α-Glucosidase inhibition and antihyperglycemic activity of prenylated xanthones from Garcinia mangostana

An ethanol extract of the fruit case of Garcinia mangostan, whose most abundant chemical species are xanthones, showed potent α-glucosidase inhibitory activity (IC₅₀ = 3.2 μg/ml). A series of isolated xanthones (1-16) demonstrated modest to high inhibition of α-glucosidase with IC₅₀ values of 1.5-63.5 μM. In particular, one hitherto unknown xanthone 16 has a very rare 2-oxoethyl group on C-8. Kinetic enzymatic assays with a p-nitrophenyl glucopyranoside indicated that one of them, compound (9) exhibited the highest activity (K_i = 1.4 μM) and mixed inhibition. Using, a physiologically relevant substrate, maltose, as substrate, many compounds (6, 9, 14, and 15) also showed potent inhibition which ranged between 17.5 and 53.5 μM and thus compared favorably with deoxynojirimycin (IC₅₀ = 68.8 μM). Finally, the actual pharmacological potential of the ethanol extract was demonstrated by showing that it could elicit reduction of postprandial blood glucose levels. Furthermore, the most active α-glucosidase inhibitors (6, 9, and 14) were proven to be present in high quantities in the native seedcase by a HPLC chromatogram.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Synthesis, characterization, X-ray structure and in vitro antimycobacterial and antitumoral activities of Ru(II) phosphine/diimine complexes containing the "SpymMe2" ligand, SpymMe2=4,6-dimethyl-2-mercaptopyrimidine

The reaction of cis-[RuCl₂(dppb)(N-N)], dppb = 1,4-bis(diphenylphosphino)butane, complexes with the ligand HSpymMe₂, 4,6-dimethyl-2-mercaptopyrimidine, yielded the cationic complexes [Ru(SpymMe₂)(dppb)(N-N)]PF₆, N-N = bipy (1) and Me-bipy (2), bipy = 2,2′-bipyridine and Me-bipy = 4,4′-dimethyl-2,2′-bipyridine, which were characterized by spectroscopic and electrochemical techniques and X-ray crystallography and elemental analysis. Additionally, preliminary in vitro tests for antimycobacterial activity against Mycobacterium tuberculosis H37Rv ATCC 27264 and antitumor activity against the MDA-MB-231 human breast tumor cell line were carried out on the new complexes and also on the precursors cis-[RuCl₂(dppb)(N-N)], N-N = bipy (3) and Me-bipy (4) and the free ligands dppb, bipy, Me-bipy and SpymMe₂. The minimal inhibitory concentration (MIC) of compounds needed to kill 90% of mycobacterial cells and the IC₅₀ values for the antitumor activity were determined. Compounds 1-4 exhibited good in vitro activity against M. tuberculosis, with MIC values ranging between 0.78 and 6.25 μg/mL, compared to the free ligands (MIC of 25 to >50 μg/mL) and the drugs used to treat tuberculosis. Complexes 1 and 2 also showed promising antitumor activity, with IC₅₀ values of 0.46 ± 0.02 and 0.43 ± 0.08 μM, respectively, against MDA-MB-231 breast tumor cells.