Identification of small molecule inhibitors that distinguish between non-transferrin bound iron uptake and transferrin-mediated iron transport

Chemical genetics is an emerging field that takes advantage of combinatorial chemical and small molecule libraries to dissect complex biological processes. Here we establish a fluorescence-based assay to screen for inhibitors of iron uptake by mammalian cells. Using this approach, we screened the National Cancer Institute's Diversity Set library for inhibitors of non-transferrin bound iron uptake. This screen identified 10 novel small molecule inhibitors of iron transport with IC(50) values that ranged from 5 to 30 microM. Of these ten compounds, only two blocked uptake of iron mediated by transferrin. Thus, this study characterizes the first small molecule inhibitors that distinguish between different pathways of iron transport.     

含铁蛋白介导的铁转运分子机制

铁是生命体必需的微量元素,因为它是一些重要功能酶的协同因子。这些功能酶有着广泛的功能,从呼吸作用到核酸的复制。但是,当铁含量多于细胞稳态的时候,它将产生对机体有毒的羟基。生物体已经发展了自身的调控机制,包括铁的摄取,存储和输出来控制细胞内的铁处于平衡态。二价阳离子转运蛋白,铁输出蛋白和hephaestin参与小肠吸收,转铁蛋白和转铁蛋白受体参与铁的摄取和转运,铁蛋白可以存储铁,铁调控蛋白的功能是调节铁代谢。这篇文章综述着重阐述了含铁蛋白介导的铁传递机制。     

Development of selective inhibitors against plasma kallikrein.

Specific plasma kallikrein inhibitors were designed and synthesized and their structure-activity relationship was studied. trans-4-Aminomethylcyclohexanecarbonyl (Tra)-lysyl-4-ethoxycarbonylanilide inhibited plasma kallikrein and plasmin with IC50 values of 23 and 210 microM, respectively, indicating that this compound is fairly specific to plasma kallikrein. Tra-arginyl-4-ethoxycarbonylanilide inhibited plasma kallikrein and plasmin with IC50 values of 16 and 480 microM, respectively. Tra-homoarginyl-4-carboxyanilide inhibited plasma kallikrein and plasmin with IC50 values of 14 microM and 1 mM, respectively. Finally, Tra-Arg(Mts)-4-acetylanilide (ACA) exhibited potent and selective inhibitory activity against plasma kallikrein (IC50 value for plasma kallikrein: 2 microM and for plasmin: 42 microM).     

Cell-active dual specificity phosphatase inhibitors identified by high-content screening

Phosphorylation of extracellular signal-regulated kinase (Erk) is tightly controlled by dual specificity phosphatases (DSPases), but few inhibitors of Erk dephosphorylation have been identified. Using a high-content, fluorescence-based cellular assay and the National Cancer Institute's 1990 agent Diversity Set, we identified ten compounds (0.5%) that significantly increased phospho-Erk cytonuclear differences in intact cells. Three of the ten positive compounds inhibited the mitogen-activated protein kinase phosphatase-3 (MKP-3/PYST-1) in vitro without affecting VHR or PTP1B phosphatases. The most potent inhibitor of MKP-3 had an IC(50) of <10 microM and inhibited MKP-3 in a novel, fluorescence-based multiparameter chemical complementation assay. These results suggest that the phospho-Erk nuclear accumulation assay may be a useful tool to discover DSPase inhibitors with biological activity.     

Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.     

Inhibitory effects of 3,3',4,5'-tetrahydroxystilbene and 3,3',4,5'-tetrahydroxybibenzyl, the constituents of Cassia garrettiana on antigen-induced histamine release in vitro.

3,3',4,5'-Tetrahydroxystilbene (I) and 3,3',4,5'-tetrahydroxybibenzyl (II), isolated from the heartwood of Cassia garrettiana Craib (Leguminosae), showed inhibitory effects on antigen-induced histamine release from rat peritoneal mast cells in vitro. The inhibitory effect of I (IC50 = 30.2 microM) was much stronger than that of II (greater than 100 microM). Compound II, as well as I (IC50 = 7.3 microM) reported previously, also inhibited the histamine release from human peripheral basophils induced by anti-immunoglobulin E (IgE) in vitro, and its IC50-value was 68.0 microM. These results suggest that the trans-olefin structure in the molecule may be necessary for I to have an inhibitory effect on histamine release. Considering that disodium cromoglycate did not show any significant inhibitory effect on anti-IgE-induced histamine release from human basophils, the strong inhibitory effects of I in both tests are of considerable interest.     

Superoxide dismutase activity of iron(II)TPEN complex and its derivatives

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.     

Studies on Fe complexes produced by yeast. IV. Mechanism of Fe transport from an Fe(II)-oligosaccharide complex across the mucosal membrane of the rat intestine

The mechanism of Fe transport across the rat duodenal membrane from an Fe(II)-oligosaccharide complex (designated B1-c) produced in wine by yeast and showing high hematopoietic activity in rats was examined. The Fe uptake from B1-c by brush border membrane (BBM) vesicles isolated from the rat intestine was based mainly on the Fe binding to membrane components which were suggested to be inside the vesicles. Evidence that Fe was transported into the vesicles by a special transport system other than simple diffusion was obtained by observing saturation kinetics under conditions of isotope exchange, and temperature and pH dependence. This Fe uptake was not inhibited by any metal ions tested, including inorganic Fe(II), and the BBM vesicles from the duodenum had a higher Fe uptake than those from the other parts of the small intestine. Furthermore, the BBM vesicles isolated from rats with Fe deficiency showed a significantly increased Fe uptake. The Km value for B1-c uptake was 0.16 mM, lower than the values for FeSO4 and ferrous ascorbate. These results suggest that a special transport system selective for B1-c may be present on the mucosal membrane. B1-c was taken up by the BBM vesicles in the form of Fe-oligosaccharide complex. From the preloaded vesicles, B1-c was released temperature- and pH-dependently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity relationship of photocytotoxic iron(III) complexes of modified dipyridophenazine ligands

Iron(III) complexes [FeL(B)] (1-5) of a tetradentate trianionic phenolate-based ligand (L) and modified dipyridophenazine bases (B), namely, dipyrido-6,7,8,9-tetrahydrophenazine (dpqC in 1), dipyrido[3,2-a:2',3'-c]phenazine-2-carboxylic acid (dppzc in 2), dipyrido[3,2-a:2',3'-c]phenazine-11-sulfonic acid (dppzs in 3), 7-aminodipyrido[3,2-a:2',3'-c]phenazine (dppza in 4) and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine (dppn in 5), have been synthesized and their photocytotoxic properties studied along with their dipyridophenazine analogue (6). The complexes have a five electron paramagnetic iron(III) center, and the Fe(III)/Fe(II) redox couple appears at about -0.69 V versus SCE in DMF-0.1 M TBAP. The physicochemical data also suggest that the complexes possess similar structural features as that of its parent complex [FeL(dppz)] with FeO3N3 coordination in a distorted octahedral geometry. The DNA-complex and protein-complex interaction studies have revealed that the complexes interact favorably with the biomolecules, the degree of which depends on the nature of the substituents present on the dipyridophenazine ring. Photocleavage of pUC19 DNA by the complexes has been studied using visible light of 476, 530, and 647 nm wavelengths. Mechanistic investigations with inhibitors show formation of HO(?) radicals via a photoredox pathway. Photocytotoxicity study of the complexes in HeLa cells has shown that the dppn complex (5) is highly active in causing cell death in visible light with sub micromolar IC(50) value. The effect of substitutions and the planarity of the phenazine moiety on the cellular uptake are quantified by determining the total cellular iron content using the inductively coupled plasma-optical emission spectrometry (ICP-OES) technique. The cellular uptake increases marginally with an increase in the hydrophobicity of the dipyridophenazine ligands whereas complex 3 with dppzs shows very high uptake. Insights into the cell death mechanism by the dppn complex 5, obtained through DAPI nuclear staining in HeLa cells, reveal a rapid programmed cell death mechanism following photoactivation of complex 5 with visible light. The effect of substituent on the DNA photocleavage activity of the complexes has been rationalized from the theoretical studies.     

Arteminolides B,C, and D,new inhibitors of farnesyl protein transferase from Artemisia argyi

Arteminolides B-D (2-4), new farnesyl protein transferase inhibitors, were isolated together with a known arteminolide A (1) and new regioisomers (5-7) of the compounds from the aerial parts of Artemisia argyi. Structures of these compounds were elucidated by spectroscopic methods and chemical conversion. Arteminolides inhibited the farnesyl protein transferase with IC(50) values of 0.7-1 microM, while the regioisomers 5-7 were inactive. In addition, it was proved that the exocyclic double bond of sesquiterpene lactone did not affect the inhibitory activity of arteminolide. The effects of compound 2 on H-Ras processing and cellular growth in H-ras-transformed cells were also evaluated.     

Predicting how polyphenol antioxidants prevent DNA damage by binding to iron

Prevention of oxidative DNA damage due to hydroxyl radical is important for the prevention and treatment of disease. Because of their widely recognized antioxidant ability, 12 polyphenolic compounds were assayed by gel electrophoresis to directly quantify the inhibition of DNA damage by polyphenols with Fe(2+) and H2O2. All of the polyphenol compounds have IC50 values ranging from 1-59 microM and inhibit 100% of DNA damage at 50-500 microM concentrations. Gel electrophoresis results with iron(II)EDTA and UV-vis spectroscopy experiments confirm that binding of the polyphenol to iron is essential for antioxidant activity. Furthermore, antioxidant potency of polyphenol compounds correlates to the pKa of the first phenolic hydrogen, representing the first predictive model of antioxidant potency based on metal-binding. Understanding this iron-coordination mechanism for polyphenol antioxidant activity will aid in the design of more-potent antioxidants to treat and prevent diseases caused by oxidative stress, and help develop structure-activity relationships for these compounds.     

Influence of organic and inorganic arsenicals on glucose uptake in Madin-Darby canine kidney (MDCK) cells.

The effect of organic (oxophenylarsine; PhAsO) and inorganic (arsenite) arsenicals on the availability of glucose to Madin-Darby canine kidney (MDCK) cells was investigated. The MDCK cells revealed stereospecific D-glucose uptake which was inhibited by both arsenicals in a time- and concentration-dependent manner. After 10 min (37 degrees C), the effects on D-glucose and 2-deoxy-D-glucose accumulation were analogous, suggesting an impaired hexose uptake. With arsenite, 0.5-1 mmol dm-3 were required for half-maximum inhibition (IC50), whereas PhAsO inhibited glucose uptake in the micromolar range (IC50 5-30 mumol dm-3). Under these conditions neither cell morphology nor cellular viability was affected. After 60 min, however, the inhibition of glucose utilization was paralleled by the formation of blebs, detachment of the monolayer and a loss of cellular viability as confirmed by dye exclusion, lactate dehydrogenase and potassium release. It is concluded that inhibition of glucose uptake may contribute to the acute toxicity, especially of organic arsenicals, by further aggravating the depletion of intracellular carbohydrates.     

Syntheses and biological activity of amamistatin B and analogs

Amamistatins A and B, natural products isolated from a strain of Nocardia, showed growth inhibition against three human tumor cell lines (IC(50) 0.24-0.56 microM). Structurally related mycobactins affect the growth of both mycobacterial and human cells through interference with iron chelation. To further probe the biological activity of this class of compounds, the total syntheses of amamistatin B and two analogs were completed, and the synthetic samples were screened for tumor cell growth inhibition, HDAC inhibition, and Mycobacterium tuberculosis growth inhibition. Amamistatin B (15) and diastereomer 18 were both active against MCF-7 cells (IC(50) 0.12-0.20 microM), and less so against PC-3 cells (IC(50) 8-13 microM). Amamistatin B only moderately inhibited the growth of M. tuberculosis (MIC 47 microM) but showed growth promotion of Mycobacterium smegmatis and other bacteria.     

Analysis of iron(II)/iron(III) phytosiderophore complexes by nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nano-ESI-FTICRMS) was employed for the analysis of the phytosiderophore 2'-deoxymugineic acid (DMA) and the candidate ligand for the intracellular iron transport in plants nicotianamine (NA). Due to the zwitterionic nature of NA and DMA, complementary mass spectra were obtained in positive and negative ionization modes. The technique was also used for speciation of their complexes with Fe(II) and Fe(III), respectively. The species observed at pH 7.3 are the 1:1 Fe-ligand complexes and no evidence for the existence of dimeric complexes was observed. NA and DMA differ only by one mass unit. Consequently, in the system NA + DMA + Fe(II)/Fe(III), there are pairs of iron species (i.e. NA-Fe(II) and DMA-Fe(III)) with the same nominal mass, which differ only by approximately 0.02 mass units. It is shown that high-resolution MS accompanied by accurate mass data analysis allows the unequivocal identification of all four iron species (NA-Fe(II), NA-Fe(III), DMA-Fe(II), DMA-Fe(III)) in one solution without separation. We also addressed the possible alteration of the oxidation state of chelated iron under nano-ESI conditions, but no redox reactions were observed under optimized conditions.     

Metal-dependent self-assembly of a microbial surfactant

Small-angle neutron scattering (SANS), cryogenic transmission electron microscopy (cryo-TEM), and dynamic light scattering (DLS) were used to study the metal-dependent phase behavior of microbially produced surfactants-marinobactins B, D, and E (MB, MD, and ME). Marinobactins A-E are siderophores that facilitate Fe(III) acquisition by the source bacterium through the coordination of Fe(III) by the peptidic headgroup. All of the marinobactins have the same six amino acid headgroup but differ in the length and saturation of the monoalkyl fatty acid tail. Fe(III) coordinated to ME (Fe(III)-ME) was found to form micelles with a diameter of approximately 3.5 nm that underwent a supramolecular transformation to produce a monodisperse population of vesicles with an average diameter ranging from approximately 90 to 190 nm upon addition of Cd(II), Zn(II), or La(III). SANS profiles of the transition-metal-induced phase exhibit a Bragg peak at QB approximately 0.11-0.12 A-1 and were fit to a SANS model for multilamellar vesicles that have an interbilayer repeat distance of 2pi/QB approximately 5.6-5.0 nm. Cryo-TEM images of the Zn(II)-induced phase reveals the presence of approximately 100 nm diameter approximately spherical aggregates of uniform electron density. The temperature dependence of the Zn(II)-induced transformation was also investigated as a function of the length and degree of unsaturation of the Fe(III)-marinobactin fatty acid tail. The Cd(II)-, Zn(II)-, and La(III)-induced phase changes have features that are similar to those of the previously reported Fe(III)-induced micelle-to-vesicle transition, and this observation has opened questions regarding the role that Cd(II) and Zn(II) may play in bacterial iron uptake.     

Biflavonoids inhibited phosphatase of regenerating liver-3 (PRL-3)

Two biflavonoids, ginkgetin (1) and sciadopitysin (2), were isolated from the MeOH extract of the young branches of Taxus cuspidata, which inhibited phosphatase of regenerating liver-3 (PRL-3) with IC50 values of 25.8 and 46.2 microM, respectively. This is the first report on PRL-3 inhibitors, isolated from natural sources.     

Synthesis and evaluation of novel pyrimido-acridone, -phenoxadine, and -carbazole as topoisomerase II inhibitors

As part of a series of studies to discover new topoisomerase II inhibitors, novel pyrimidoacridones, pyrimidophenoxadines, and pyrimidocarbazoles were synthesized, and in vitro and in vivo antitumor activities and DNA-protein and/or DNA-topoisomerase II cross-linking activity as an indicator of topoisomerase II-DNA cleavable complex formation were evaluated. The pyrimidocarbazoles possessed high in vitro and in vivo potencies. Compound 26 (ER-37326), 8-acetyl-2-[2-(dimethylamino)ethyl]-1H-pyrimido[5,6,1-jk]carbazole-1,3(2H)-dione, showed in vitro growth inhibitory activity with respective IC(50) values of 0.049 microM and 0.35 microM against mouse leukemia P388 and human oral cancer KB. In vivo, this compound inhibited the tumor growth of mouse sarcoma M5076 implanted into mice with T/C values of 42% and 13% at 3.13 and 6.25 mg/kg/d respectively without significantly affecting the body weight. In addition, compound 26 (ER-37326) increased the formation of DNA-topoisomerase II cross-linking in P388 cells.     

CCR5 antagonists as anti-HIV-1 agents. 1. Synthesis and biological evaluation of 5-oxopyrrolidine-3-carboxamide derivatives

A novel lead compound, N-(3-[4-(4-fluorobenzoyl)piperidin-1-yl]propyl)-1-methyl-5-oxo-N-phenylpyrrolidine-3-carboxamide (1), was identified as a CCR5 antagonist by high-throughput screening using [(125)I]RANTES and CCR5-expressing CHO cells. The IC(50) value of 1 was 1.9 microM. In an effort to improve the binding affinity of 1, a series of 5-oxopyrrolidine-3-carboxamides was synthesized. Introduction of 3,4-dichloro substituents to the central phenyl ring (10i, IC(50)=0.057 microM; 11b, IC(50)=0.050 microM) or replacing the 1-methyl group of the 5-oxopyrrolidine moiety with a 1-benzyl group (12e, IC(50)=0.038 microM) was found to be effective for improving CCR5 affinity. Compound 10i, 11b, and 12e also inhibited CCR5-using HIV-1 envelope-mediated membrane fusion with IC(50) values of 0.44, 0.19, and 0.49 microM, respectively.     

Stimuli-responsive polyguanidino-oxanorbornene membrane transporters as multicomponent sensors in complex matrices

We introduce guanidinium-containing synthetic polymers based on polyguanidino-oxanorbornenes (PGONs) as anion transporters in lipid bilayers that can be activated and inactivated by chemical stimulation. According to fluorogenic anion export experiments with vesicles, PGON transporters are most active in neutral bilayers near their phase transition, with EC50's in the nanomolar range. Six times higher effective transporter concentrations were measured with aminonaphthalene-1,3,6-trisulfonate than with 5(6)-carboxyfluorescein, demonstrating the importance of anion binding for transport and excluding nonspecific efflux. Negative surface potentials efficiently annihilate transport activity, while inside-negative membrane potentials slightly increase it. These trends demonstrate the functional importance of counterions to hinder the binding of hydrophilic counterions and to minimize the global positive charge of the transporter-counterion complexes. Strong, nonlinear increases in activity with polymer length reveal a significant polymer effect. Overall, the characteristics of PGONs do not match those of similar systems (for example, polyarginine) and hint toward an interesting mode of action, clearly different from nonspecific leakage caused by detergents. The activity of PGONs increases in the presence of amphiphilic anions such as pyrenebutyrate (EC50 = 70 microM), while several other amphiphilic anions tested were inactive. PGONs are efficiently inactivated by numerous hydrophilic anions including ATP (IC 50 = 150 microM), ADP (IC50 = 460 microM), heparin (IC50 = 1.0 microM), phytate (IC50 = 0.4 microM), and CB hydrazide (IC50 = 26 microM). The compatibility of this broad responsiveness with multicomponent sensing in complex matrices is discussed and illustrated with lactate sensing in sour milk. The PGON lactate sensor operates together with lactate oxidase as a specific signal generator and CB hydrazide as an amplifier for covalent capture of the pyruvate product as CB hydrazone (IC50 = 1.5 microM).     

Xanthine oxidase inhibitors from the heartwood of Vietnamese Caesalpinia sappan

From the MeOH extract of Vietnamese Caesalpinia sappan, a novel biogenetically exclusive benzindenopyran, with a new carbon framework, neoprotosappanin (1), and a new compound, protosappanin A dimethyl acetal (3), were isolated together with protosappanin E-2 (2), neosappanone A (4), and 13 previously reported phenolic compounds (5-17). Their structures were elucidated on the basis of spectroscopic data. Compounds 1-4, 7, 13, and 15-17 showed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and sappanchalcone (17) showed the most potent activity with an IC50 value of 3.9 microM, comparable to that of positive control allopurinol (IC50, 2.5 microM). The kinetic study of these inhibitors indicated that they are competitive inhibitors, the same as allopurinol, except for 1 and 16 which are noncompetitive inhibitors.