Febrifugine is the active principal isolated 50 years ago from the Chinese herb chang shan (Dichroa febrifuga Lour), which has been used as an antimalarial in Chinese traditional medicine for more than 2,000 years. However, intensive study of the properties of febrifugine has been hindered for decades due to its side effects. We report new findings on the effects of febrifugine analogs compared with those of febrifugine extracted from the dry roots of D. febrifuga. The properties of the extracted febrifugine were comparable to those obtained from the standard febrifugine provided by our collaborators. A febrifugine structure-based computer search of the Walter Reed Chemical Information System identified 10 analogs that inhibited parasite growth in vitro, with 50% inhibitory concentrations ranging from 0.141 to 290 ng/ml. The host macrophages (J744 cells) were 50 to 100 times less sensitive to the febrifugine analogs than the parasites. Neuronal (NG108) cells were even more insensitive to these drugs (selectivity indices, >1,000), indicating that a feasible therapeutic index for humans could be established. The analogs, particularly halofuginone, notably reduced parasitemias to undetectable levels and displayed curative effects in Plasmodium berghei-infected mice. Recrudescence of the parasites after treatment with the febrifugine analogs was the key factor that caused the death of most of the mice in groups receiving an effective dose. Subcutaneous treatments with the analogs did not cause irritation of the gastrointestinal tract when the animals were treated with doses within the antimalarial dose range. In summary, these analogs appear to be promising lead antimalarial compounds that require intensive study for optimization for further down-selection and development.

Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K i of 1 to 6 M. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K i greater than 280 M). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 g/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K i values ranging from 0.05 to 0.68 M. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.Malaria, the most severe parasitic disease, infects nearly 300 million people and kills more than a million each year (28). Plasmodium falciparum and Plasmodium vivax are the two malaria species responsible for the most infections and deaths. Although several very effective antimalarial drugs have been used to control this disease, P. falciparum has developed resistance to nearly all available antimalarial drugs (27). Recently, P. vivax from Southeast Asia has developed resistance to the most widely used antimalarial drug, chloroquine. The search for novel antimalarial drugs against specific parasitic targets is thus an urgent task to pursue. In the last decade, many potential targets for new antimalarial drugs have been discovered, such as dihydropteroate synthase, hemoglobin degradation enzymes, and shikimate pathway enzymes (17). Our work focuses on the discovery of new inhibitors of hemoglobin degradation enzymes called plasmepsins.Malarial parasites invade human erythrocytes in the asexual stage of infection. While residing in erythrocytes, the parasites rely on human hemoglobin as a food source, digesting it with a series of proteases. The aspartic proteases, called plasmepsins, are critical for hemoglobin degradation and are thus logical targets for antimalarial drug development (14,19,25). At least four plasmepsins have been identified and cloned from P. falciparum (26; R. Banerjee and D. E. Goldberg, Mol. Parasite Meet., MBL, Woods Hole, Mass., 1999). Active recombinant plasmepsin II has been successfully obtained in large enough quantities (3, 10) to facilit...

Biliproteins have extended the spectral range of fluorescent proteins into the region of maximal transmission of most tissues and are favorable for multiplexing, but their application presents considerable challenges. Their fluorescence derives from open-chain tetrapyrrole chromophores which often require the introduction of dedicated reductases and lyases. In addition, their fluorescence yield generally decreases with increasing wavelengths and depends strongly on the state of the binding protein. We report fluorescent biliproteins, termed BDFPs, that are derived from the phycobilisome core subunit, ApcF2: this subunit is induced in the thermophilic cyanobacterium, Chroococcidiopsis thermalis, by far-red light and binds phycocyanobilin non-covalently. The BDFPs obtained by molecular evolution of ApcF2 bind the more readily accessible biliverdin covalently while retaining the red-shifted fluorescence in the near-infrared spectral region (~710nm). They are small monomers (~15kDa) and not only show excellent photostability, but are also thermostable up to 80°C, tolerate acid down to pH2 and high concentrations of denaturants. The result indicates far-red adapting cyanobacteria as a useful source for designing extremely red-shifted fluorescent markers. In vivo performance of BDFPs as biomarkers in conventional and super-resolution microscopy, alone or fused to target proteins, is exemplified in several mammalian cells, including, human cell lines, in the nematode, Caenorhabditis elegans and, at low pH, in Lactobacillus lactis.

No abstract

Aberrant microRNA (miRNA) expression in cancer affects the transcription of target genes, and profoundly influences cancer‑associated signaling pathways. Radiation resistance is a major problem encountered in the treatment of cancer. The present study aimed to investigate the role of miRNA (miR)‑21 in the development of radiation resistance in non‑small cell lung cancer cells. A radiation‑resistant cell line was generated from A549 cells. Significant upregulation of miR‑21 was detected in the radioresistant cancer cells, as compared with the radiosensitive cells, and overexpression of miR‑21 rendered A549 parental cells resistant to radiation. In addition, glycolysis was increased in the radioresistant cells, as compared with the sensitive cells. Furthermore, hypoxia‑inducible factor‑1α (HIF1α) was upregulated by miR‑21 in radioresistant cells, resulting in promotion of the key enzymes of glycolysis. Inhibition of HIF1α by small interfering RNA suppressed glycolysis and resensitized the cancer cells to radiation, whereas the recovery of HIF1α in miR‑21‑inhibited radioresistant cells resulted in recovery of radioresistance. In conclusion, the present study suggested that miR‑21 may modulate radioresistance through the upregulation of HIF1α. These results may provide a novel perspective on miRNA for the development of anti-radioresistance drugs.

A number of substituted 9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrones have been synthesized and their anticancer and antimalarial activities evaluated. A one-pot synthesis of 2,5,8-trimethoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4-dione (4) was achieved by heating a mixture of 1,4-dimethoxyanthracene, methoxyhydroquinone, silver oxide, and zinc iodide in toluene. Regioselective bromination of 4 and 2-methoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone (7) with N-bromosuccinimide provided 2-bromo-3,5,8-trimethoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4-dione and 2-bromo-3-methoxy-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone (1), respectively. The reactions of 1 with aliphatic primary amines and secondary amines, respectively, produced different products, a result most likely attributed to the different basicities (or nucleophilicities) and steric effects of the two kinds of amines. The structure of the displacement product, 2-bromo-3-[2-(tert-butoxycarbonyl)ethylamino]-9,10-dihydro-9,10-[1,2]benzenoanthracene-1,4,5,8-tetrone, from the reaction of 1 with tert-butyl 3-aminopropanoate was unequivocally determined by a single-crystal X-ray analysis. IC(50) values of triptycene bisquinones for the inhibition of L1210 leukemia cell viability are in the 0.11-0.27 microM range and for the inhibition of Plasmodium falciparum 3D7 are in the 4.7-8.0 microM range.

No abstract

Oxidative low-density lipoprotein (ox-LDL) is a risk factor for atherosclerosis. Ox-LDL leads to endothelial injury in the initial stage of atherosclerosis. In this study, we investigated the role of ox-LDL in endothelial injury and macrophage recruitment. We demonstrated that ox-LDL promoted a dose-dependent phosphorylation of caveolin-1 in human umbilical vein endothelial cells. Phosphorylated caveolin-1 increased ox-LDL uptake. Intracellular accumulation of ox-LDL induced NF-κB p65 phosphorylation, promoted HMGB1 translocation from nucleus to cytoplasm and cytochrome C release from mitochondria to cytoplasm, and activated caspase 3, resulting in cell apoptosis. NF-κB activation also facilitated cavolin-1 phosphorylation and HMGB1 expression. In addition, caveolin-1 phosphorylation favored HMGB1 release and nuclear translocation of EGR1. Nuclear translocation of EGR1 contributed to cytoplasmic translocation of HMGB1. The extracellular HMGB1 induced the migration of PMBC-derived macrophages toward HUVECs in a TLR4-dependent manner. Our results suggested that ox-LDL promoted HUVECs apoptosis and macrophage recruitment by regulating caveolin-1 phosphorylation.