Kinetics of Congo-red binding by haemin-limited and haemin-excess cells of Porphyromonas gingivalis W50

The binding of Congo red to P. gingivalis W50 grown in a chemostat under haemin-limitation and haemin-excess was quantified. Congo red bound to both haemin-excess and haemin-limited cells with similar capacity and affinity. Binding of Congo red was greater than for ferri- (haemin) or ferroprotoporphyrin IX (haem), and was not influenced by redox potential at low added ligand concentrations. Both haemin-limited and haemin-excess cells showed positive co-operativity towards Congo red binding. Pre-exposure of haemin-limited and haemin-excess cells to sub-saturating concentrations of ferriprotoporphyrin IX did not affect Congo red binding, whereas pre-exposure of haemin-excess cells to ferroprotoporphyrin IX increased binding. Iron protoporphyrin IX binding was enhanced after exposure of both haemin-excess and haemin-limited cells to Congo red, especially under reducing conditions. These results confirm that Congo red binding cannot be used as an indirect measure of haemin binding, nor can Congo red be used to inhibit haemin binding to P. gingivalis.     

Heme Mediates Cytotoxicity from Artemisinin and Serves as a General Anti-Proliferation Target

Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.     

Effect of artemisinin (qinghaosu) and chloroquine on drug-sensitive and drug-resistant strains of Plasmodium falciparum malaria: use of [2,8-3H]adenosine as an alternative to [G-3H]hypoxanthine in the assessment of in vitro antimalarial activity

Using [G-3H]hypoxanthine uptake as a radioactive indicator for the growth of malarial parasites, we measured the antimalarial activity of artemisinin (Qinghaosu, QHS) against FCMSU1/Sudan strain (chloroquine-sensitive strain) and FCB K+ strain (chloroquine-resistant strain) of Plasmodium falciparum in continuous culture in vitro. The 50% inhibitory concentrations (IC50) for QHS against FCMSU1/Sudan strain and FCB K+ strain were 2.8 X 10(-8) and 3.0 X 10(-8) M, respectively. On the contrary, the response of the two strains to chloroquine was quite different. The IC50 for chloroquine against FCMSU1/Sudan strain was 5.6 ng/ml, whereas that for the FCB K+ strain was 65.6 ng/ml. Therefore, QHS did not appear to exhibit any cross-resistance with chloroquine. If [2,8-3H]adenosine was used as a radioactive precursor instead of [G-3H]hypoxanthine for the determination of antimalarial activity, virtually identical results were obtained. Therefore, [2,8-3H]adenosine can be used as an alternative to [G-3H]hypoxanthine for the assessment of antimalarial action.     

The potentiating action of tetrandrine in combination with chloroquine or qinghaosu against chloroquine-sensitive and resistant falciparum malaria

Using chloroquine-sensitive (CS) and chloroquine-resistant (CR) strains of Plasmodium falciparum in vitro, interactions between tetrandrine (TT) and either chloroquine (CQ) or qinghaosu (QHS, artemisinin) were assessed using isobolograms. Sums of the fractional inhibitory concentration for the combination of the two drugs are less than one and therefore, we can conclude that in vitro TT and CQ or QHS act synergistically against CS and CR falciparum malaria. Remarkably, using CR malaria, TT can lower the IC50 dose of CQ as much as 40 fold. These drug combinations may impair the advantage that the development of CQ resistance conveys on the parasite.     

Simultaneous determination of artesunic acid and dihydroartemisinin in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A selective reproducible high-performance liquid chromatographic assay for the simultaneous quantitative determination of the antimalarial compound artesunic acid (ARS), dihydroartemisinin (DQHS) and artemisinin (QHS), as internal standard, is described. After extraction from plasma, ARS and DQHS were analysed using an Econosil C₈ column and a mobile phase of acetonitrile–0.05 M acetic acid (42:58, v/v) adjusted to pH 5.0 and electrochemical detection in the reductive mode. The mean recovery of ARS and DQHS over a concentration range of 50–200 ng/ml was 75.5% and 93.5%, respectively. The within-day coefficients of variation were 4.2–7.4% for ARS and 2.6–4.9% for DQHS. The day-to-day coefficients of variation were 1.6–9.6% and 0.5–8.3%, respectively. The minimum detectable concentration for ARS and DQHS in plasma was 4.0 ng/ml for both compounds. The method was found to be suitable for use in clinical pharmacological studies.     

NMR studies on novel antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin

Artemisinin and its derivatives, which have been known as antimalarial drugs, have also demonstrated their cytotoxicity against tumor cells. It has been proposed that antitumor activity depends on the lipophilicity of functional group on artemisinin derivatives. Solution structures of two artemisinin derivatives as antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin, were determined by NMR spectroscopy to elucidate structure-activity relationship. According to biological assay, antitumor efficiencies are not dependent upon lipophilicity. Instead, these compounds demonstrated their distinctive structural features of boat/chair conformation and capability to interact with receptors, as they have different efficiencies on antitumor activity. Especially, carboxypropyl moiety or carbonyl moiety in artemisinin derivatives influences the conformation and stability of ring structure. Although the detailed mechanism of antitumor activity by artemisinin derivatives has not been addressed, we suggest that antitumor activity is not determined only with lipophilicity and that artemisinin derivatives have specific target proteins in each type of cancer.     

Ligand-based and structure-based virtual screening to identify carbonic anhydrase IX inhibitors

A three-dimensional pharmacophore model of CA IX inhibitors was generated and used to screen the ZINC database of commercially available compounds. The hits were docked in a CA IX homology model. By visualizing the binding mode and score of these compounds, six derivatives were selected and evaluated for their inhibitory potency against CA IX. A highly active CA IX inhibitor was identified which may be used as a lead to design novel such derivatives.     

Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle

Modification of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin derivatives containing cyano and aryl groups showed potent antiproliferative effect in vitro against P388 and A549 cells. This activity was reflected in P388 murine leukemia by an accumulation of cells in G1 phase, and induction of apoptosis.     

Artemisinin derivatives induce iron-dependent cell death (ferroptosis) in tumor cells

BackgroundApoptosis and other forms of cell death have been intensively investigated in the past years to explain the mode of action of synthetic anticancer drugs and natural products. Recently, a new form of cell death emerged, which was termed ferroptosis, because it depends on intracellular iron. Here, the role of genes involved in iron metabolism and homeostasis for the cytotoxicity of ten artemisinin derivatives have been systematically investigated.Material and methodsLog₁₀IC₅₀ values of 10 artemisinin derivatives (artesunate, artemether, arteether, artenimol, artemisitene, arteanuin B, another monomeric artemisinin derivative and three artemisinin dimer molecules) were correlated to the microarray-based mRNA expression of 30 iron-related genes in 60 cell lines of the National Cancer Institute (NCI, USA) as determined in 218 different microarray hybridization experiments. The effect of desferoxamine and ferrostatin-1 on the cytotoxicity of artenimol of CCRF-CEM cells was determined by resazurin assays. The mRNA expression of TFRC was exemplarily validated by immunohistochemical detection of transferrin receptor protein expression.ResultsThe mRNA expression of 20 genes represented by 59 different cDNA clones significantly correlated to the log₁₀IC₅₀ values for the artemisinins, including genes encoding transferrin (TF), transferrin receptors 1 and 2 (TFRC, TFR2), cerulopasmin (CP), lactoferrin (LTF) and others. The ferroptosis inhibitor ferrostatin-1 and the iron chelator deferoxamine led to a significantly reduced cytotoxicity of artenimol, indicating ferroptosis as cell death mode.ConclusionThe numerous iron-related genes, whose expression correlated with the response to artemisinin derivatives speak in factor for the relevance of iron for the cytotoxic activity of these compounds. Treatment with ferroptosis-inducing agents such as artemisinin derivatives represents an attractive strategy for cancer therapy. Pre-therapeutic determination of iron-related genes may indicate tumor sensitivity to artemisinins. Ferroptosis induced by artemisinin-type drugs deserve further investigation for individualized tumor therapy.     

Carbonic anhydrase I,II, IV and IX inhibition with a series of 7-amino-3,4-dihydroquinolin-2(1H)-one derivatives

A series of new derivatives was prepared by derivatisation of the 7-amino moiety present in 7-amino-3,4-dihydroquinolin-2(1H)-one, a compound investigated earlier as CAI. The derivatisation was achieved by: i) reaction with arylsulfonyl isocyanates/aryl isocyanates; (ii) reaction with fluorescein isothiocyanate; (iii) condensation with substituted benzoic acids in the presence of carbodiimides; (iv) reaction with 2,4,6-trimethyl-pyrylium tetrafluoroborate; (v) reaction with methylsulfonyl chloride and (vi) reaction with maleic anhydride. The new compounds were assayed as inhibitors of four carbonic anhydrases (CA, EC 4.2.1.1) human (h) isoforms of pharmacologic relevance, the cytosolic hCA I and II, the membrane-anchored hCA IV and the transmembrane, tumour-associated hCA IX. hCA IX was the most inhibited isoform (K_Is ranging between 243.6 and 2785.6?nm) whereas hCA IV was not inhibited by these compounds. Most derivatives were weak hCA I and II inhibitors, with few of them showing K_Is?相似文献   

Rapid determination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative light scattering detector

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.     

Synthesis, antimalarial activity and cytotoxicity of 10-aminoethylether derivatives of artemisinin

In this study, a series of 11 10-aminoethylether derivatives of artemisinin were synthesised and their antimalarial activity against both the chloroquine sensitive (D10) and resistant (Dd2) strains of Plasmodium falciparum was determined. The compounds were prepared by introducing aliphatic, alicyclic and aromatic amine groups with linkers of various chain lengths through an ethyl ether bridge at C-10 of artemisinin using conventional and microwave assisted syntheses, and their structures were confirmed by NMR and HRMS. All derivatives proved to be active against both strains of the parasite. The highest overall activity was displayed by the short chain aromatic derivative 8 (IC(50)=1.44nM), containing only one nitrogen atom, while long chain polyamine derivatives were found to have the lowest activity against both strains. An interesting correlation between the IC(50), pK(a) values and resistance index (RI) was found.     

Enantiospecific syntheses of C(5)-functionalized precursors of artemisinin derivatives

Enantiomerically pure compounds as precursors for the synthesis of hydroxylated derivatives of artemisinin/arteether have been prepared from (+)-(S)-carvone and (+)-car-2-ene.     

Novel sulfonamide incorporating piperazine,aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I,II and IX inhibitory action

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with K_Is in the range of 8.5–2679.1 nM, hCA II with K_Is in the range of 4.8–380.5 nM and hCA IX with K_Is in the range of 0.4–307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5–18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.     

Plasmodium falciparum: in vitro interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine

In the scenario of drug-resistant Plasmodium falciparum malaria combination therapy represents an effective approach. Artemisinin and its derivatives are of special interest because they represent the most effective group of compounds against multidrug-resistant malaria with a rapid onset of action and a short half-life. Interactions of artemisinin with amodiaquine, pyronaridine, and chloroquine were therefore investigated against three strains of P. falciparum using a 48-h in vitro culture assay. Two of the strains were chloroquine sensitive and one was partially chloroquine resistant. Observed effective concentrations (O) of the combined compounds at different concentration ratios were calculated for different degrees of inhibition (EC50, EC90, EC99) and compared to expected calculated effective concentrations (E) using a probit method. Synergism with mean O/E EC90 values of 0.25 and 0.8 were found with the combination of artemisinin and the two Mannich bases, amodiaquine and pyronaridine, respectively, whereas chloroquine showed addition with a mean value of 1.2. Although both amodiaquine and chloroquine are 4-aminoquinolines, their interaction with artemisinin appears to be different. The combination of artemisinin with amodiaquine represents an important option for the treatment of falciparum malaria.     

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.     

Carbonic anhydrase inhibitors. Inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, IX, and XII with Schiff's bases incorporating chromone and aromatic sulfonamide moieties, and their zinc complexes

A series of Schiff's bases was prepared by reaction of 3-formyl-chromone or 6-methyl-3-formyl-chromone with aromatic sulfonamides, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide, a pyrimidinyl-substituted sulfanilamide derivative, sulfaguanidine and 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide. The zinc complexes of these sulfonamides have also been obtained. The new derivatives and their Zn(II) complexes were investigated for the inhibition of four physiologically relevant isozymes of carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms I and II, as well as the tumor-associated, transmembrane isozymes CA IX and XII. Except for the sulfaguanidine-derived compounds which were devoid of activity against all isozymes, the other sulfonamides and their metal complexes showed interesting inhibitory activity. Against isozyme CA I, the inhibition constants were in the range of 13-100 nM, against isozyme CA II in the range of 1.9-102 nM, against isozyme CA IX in the range of 6.3-48nM, and against CA XII in the range of 5.9-50nM. Generally, the formyl-chromone derived compounds were better CA inhibitors as compared to the corresponding 6-methyl-chromone derivatives, and for the simple, benzenesulfonamide derivatives activity increased with an increase of the spacer from sulfanilamide to homosulfanilamide and 4-aminoethylbenzenesulfonamide derivatives, respectively. Some of these compounds may show applications for the development of therapies targeting hypoxic tumors in which CA IX and XII are often highly overexpressed.     

Carbonic anhydrase inhibitors: the first QSAR study on inhibition of tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides

QSAR study on the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isoenzyme has been made using a large pool of distance-based topological indices : W, Sz, PI (0)chi, (1)chi, (2)chi,(0)chi(v), (1)chi(v), (2)chi(v). A combined set of 32 aromatic and heterocyclic compounds, including the six clinically used derivatives: acetazolamide, methazolamide, ethoxyzolamide, dichlorophenamide, dorzolamide, and brinzolamide are used for this purpose. The results have shown that the inhibition of the tumor-associated isoenzyme IX with aromatic and heterocyclic sulfonamides can be modeled excellently in multiparametric regression after introduction of indicator parameters. The predictive power of the models is discussed using probable error of correlation (PE), variance-inflation factor (VIF), and cross-validation parameters: PRESS, SSY, r(2) (cv) (S) PRESS, and PSE. This is the first report on QSAR study on inhibition of tumor-associated isoenzyme IX.     

Carbonic anhydrase inhibitors: synthesis and inhibition of cytosolic/tumor-associated carbonic anhydrase isozymes I, II, and IX with sulfonamides incorporating 1,2,4-triazine moieties

A series of benzenesulfonamide derivatives incorporating triazine moieties in their molecules was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide, or 4-aminoethylbenzenesulfonamide. The dichlorotriazinyl-benzenesulfonamides intermediates were subsequently derivatized by reaction with various nucleophiles, such as water, methylamine, or aliphatic alcohols (methanol and ethanol). The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumor-associated hCA IX. The new compounds reported here inhibited hCA I with K(I)s in the range of 75-136nM, hCA II with K(I)s in the range of 13-278nM, and hCA IX with K(I)s in the range of 0.12-549nM. The first hCA IX-selective inhibitors were thus detected, as the chlorotriazinyl-sulfanilamide and the bis-ethoxytriazinyl derivatives of sulfanilamide/homosulfanilamide showed selectivity ratios for CA IX over CA II inhibition in the range of 166-706. Furthermore, some of these compounds have subnanomolar affinity for hCA IX, with K(I)s in the range 0.12-0.34nM. These derivatives are interesting candidates for the development of novel unconventional anticancer strategies targeting the hypoxic areas of tumors. Clear renal cell carcinoma, which is the most lethal urologic malignancy and is both characterized by very high CA IX expression and chemotherapy unresponsiveness, could be the leading candidate of such novel therapies.