Paul S. Wright

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes : growth inhibition in vitro

Blocking spermidine and spermine synthesis in Plasmodium falciparum-infected erythrocytes with irreversible inhibitors of S-adenosylmethionine decarboxylase (AdoMet DC; EC 4.1.1.50), prevented the growth of the parasite in vitro. The most potent of these compounds, MDL 73811, inhibited growth of chloroquine-sensitive and -resistant strains of P. falciparum equally, with an IC50 of 2-3 microM. Other structurally related compounds also inhibited parasite proliferation, but to a lesser degree, determined apparently by their potency for inhibition of AdoMet DC. The growth inhibition by MDL 73811 could be alleviated by incubating infected erythrocytes with spermidine and spermine, but not putrescine. Parasites treated with the drug were arrested at the trophozoite stage of the erythrocytic cycle and had putrescine levels which were elevated by about 3- to 4-fold. Treatment of crude extracts of purified parasites with 1 microM MDL 73811 inhibited AdoMet DC activity by greater than 90%. These biochemical changes in P. falciparum-infected cells were consistent with AdoMet DC inhibition being the primary effect of MDL 73811 treatment.

Induction of vascular cell adhesion molecule-1 expression by IL-4 in human aortic smooth muscle cells is not associated with increased nuclear NF-?B levels

Vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) are upregulated in vascular endothelial and smooth muscle cells by cytokines produced at sites of inflammation. The cytokine profile for induction of VCAM‐1, however, is different for the two cell types. Tumor necrosis factor‐α (TNF‐α) induced both VCAM‐1 and ICAM‐1 expression in human umbilical vein endothelial cells (HUVECs; ED50 ∼ 300 and 30 U/ml, respectively). TNF‐α and interleukin‐1β (IL‐1β) stimulated cell surface ICAM‐1 expression, but not VCAM‐1 expression, in human aortic smooth muscle cells (HASMCs). Conversely, IL‐4 was a potent VCAM‐1 inducer in HASMCs (ED50 ∼ 100 pg/ml) but did not induce ICAM‐1 expression. Nuclear extracts from IL‐4‐treated cells were compared with untreated cells for relative nuclear factor‐kappa B (NF‐κB) levels by using an electrophoretic mobility shift assay and surface plasmon resonance techniques. No significant increase in nuclear NF‐κB DNA binding activity was detected in IL‐4‐treated HASMCs by either method of analysis. IL‐1β and TNF‐α stimulated nuclear NF‐κB levels by about fourfold and fivefold, respectively, in HASMCs. The antioxidant pyrrolidine dithiocarbamate (PDTC) similarly inhibited VCAM‐1 upregulation in HASMCs incubated with IL‐4 and in HUVECs incubated with TNF‐α (IC50s of 25 and 40 μM, respectively). These data suggest that a significant increase in nuclear NF‐κB levels is not necessary or sufficient for VCAM‐1 upregulation in HASMCs and does not determine the relative sensitivity to inhibition of gene expression by PDTC. J. Cell. Physiol. 180:381–389, 1999.

Disruption of plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing

Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.

Depletion of estrogen receptor in human breast tumor cells by a novel substituted indole that does not bind to the hormone binding domain

Steroidal antiestrogens appear to have at least two major modes of action in breast cancer cells, direct antagonism of estrogen binding to its receptor and depletion of estrogen receptors (ER) due to inhibition of dimerization of the receptor and a resultant destabilization of the receptor protein. In a search for other classes of compounds which would act as dimerization inhibitors, a novel substituted indole (8-{2-[1-(4-chlorobenzoyl)-5-hydroxy-2-methyl-1H-indol-3-yl]-acetylamino} octanoic acid butyl-methyl amide, MDL 101,906) was synthesized. Binding of the ER to its consensus response element (ERE) was apparently decreased in nuclear extracts from MCF-7 human breast cancer cell treated with MDL 101,906. This decreased binding was found to be due to depletion of ER based on direct measurement of ER using an enzyme-linked immunoassay. Other transcription factors were apparently unaffected by MDL 101,906 treatment. Whereas depletion of ER with a steroidal antiestrogen was almost complete after 3 h of treatment of MCF-7 cells, the effect of MDL 101,906 took significantly longer to occur, suggesting a fundamental difference in the mechanisms of action of the two drugs. This was also evident in the lack of binding of MDL 101,906 to the hormone binding domain of ER. MDL 101,906 treatment also caused depletion of ER mRNA in MCF-7 cells. Depletion of ER mRNA was noted by 3 h of drug treatment and was apparently almost complete after 24 h of treatment. Depletion of ER from MCF-7 cells led to a dose-dependent decrease in the expression of luciferase by an ERE-driven luciferase reporter gene assay system. The mechanism of MDL 101,906 appears to be unique and additional studies with this chemical class seem to be warranted to assess the potential for therapeutic utility.