Carl P. Schaffner

Ezetimibe Is an Inhibitor of Tumor Angiogenesis

Epidemiological and preclinical observations have suggested a role for one or more products of the mevalonate/cholesterol biosynthesis pathway in the progression of prostate cancer. In this study, we used ezetimibe (Zetia), a specific, FDA-approved, cholesterol uptake-blocking drug, in combination with either a hyper- or hypocholesterolemic diet, to show that elevated circulating cholesterol levels promote, whereas a reduction in circulating cholesterol levels retard, the growth of human prostate cancer xenograft tumors in mice. Circulating cholesterol levels also modified tumor angiogenesis; higher cholesterol levels increased microvessel density and other indicators of vascularity. Consistent with these data, the reduction of cholesterol levels also increased the levels of the angiogenesis inhibitor thrombospondin-1 in the xenografts. Our results thus suggest that hypercholesterolemia directly accelerates the growth of prostate carcinomas, and that the pharmacological reduction of serum cholesterol levels may retard prostate cancer growth by inhibiting tumor angiogenesis.

Factors Affecting the Production of Candicidin

Factors affecting candicidin synthesis and mycelial growth of Streptomyces griseus IMRU 3570 were studied. Inorganic phosphate was found to inhibit candicidin synthesis but to stimulate mycelial growth. Zinc, iron, and magnesium ions stimulated candicidin synthesis at relatively high concentrations in a complex medium but not in a synthetic medium. No other factors studied, such as temperature, oxygen absorption rate, and sugar concentration, were found to differentially affect antibiotic synthesis and mycelial growth. Optimum concentration of inorganic phosphate for candicidin synthesis in a chemically defined medium was found to be between 5 × 10−5 and 5 × 10−4 M. The culture in idiophase stage can be reverted to typical trophophase growth by the addition of inorganic phosphate, suggesting the controlling role of inorganic phosphate in repression and derepression of secondary metabolic and primary metabolic activity of the culture. With a soya peptone-glucose medium, the maximum rate of candicidin production could be maintained and extended for a considerable length of time by controlling the culture pH at 8.0, using glucose to adjust the pH during the later stages of a batch fermentation. Carrying out fermentations in this way has given candicidin yields up to 4 g/liter.

Inhibition of HIV-1 Replication by Amphotericin B Methyl Ester SELECTION FOR RESISTANT VARIANTS

Membrane cholesterol plays an important role in human immunodeficiency virus type 1 (HIV-1) particle production and infectivity. Here, we have investigated the target and mechanism of action of a cholesterol-binding compound, the polyene antifungal antibiotic amphotericin B methyl ester (AME). We found that AME potently inhibited the replication of a highly divergent panel of HIV-1 isolates in various T-cell lines and primary cells irrespective of clade or target cell tropism. The defects in HIV-1 replication caused by AME were due to profoundly impaired viral infectivity as well as a defect in viral particle production. To elucidate further the mechanism of action of AME, we selected for and characterized AME-resistant HIV-1 variants. Mutations responsible for AME resistance mapped to a highly conserved and functionally important endocytosis motif in the cytoplasmic tail of the transmembrane glycoprotein gp41. Interestingly, truncation of the gp41 cytoplasmic tail in the context of either HIV-1 or rhesus macaque simian immunodeficiency virus also conferred resistance to AME. The infectivity of HIV-1 virions bearing murine leukemia virus or vesicular stomatitis virus glycoproteins was unaffected by AME. Our data define the target and mechanism of action of AME and provide support for the concept that cholesterol-binding compounds should be pursued as antiretroviral drugs to disrupt HIV-1 replication.

Inhibition of Human Immunodeficiency Virus Type 1 Assembly and Release by the Cholesterol-Binding Compound Amphotericin B Methyl Ester: Evidence for Vpu Dependence

ABSTRACT We investigated the mechanism by which the cholesterol-binding compound amphotericin B methyl ester (AME) inhibits human immunodeficiency virus type 1 (HIV-1) particle production. We observed no significant effect of AME on Gag binding to the plasma membrane, Gag association with lipid rafts, or Gag multimerization, indicating that the mechanism of inhibition by AME is distinct from that by cholesterol depletion. Electron microscopy analysis indicated that AME significantly disrupts virion morphology. Interestingly, we found that AME does not inhibit the release of Vpu-defective HIV-1 or Vpu− retroviruses such as murine leukemia virus and simian immunodeficiency virus. We demonstrated that the ability of Vpu to counter the activity of CD317/BST-2/tetherin is markedly reduced by AME. These results indicate that AME interferes with the anti-CD317/BST-2/tetherin function of Vpu.

Separation of polyene antifungal antibiotics by high-speed liquid chromatography

Abstract High-speed liquid chromatography was applied to separate the components of different classes of polyene macrolides. The instrument contained at UV detector and VYDAC RP packing and a solvent mixture of water methanol tetrahydrofuran (420:90:45-90, v/v) was employed. Optimum conditions for the separation of different antibiotics were achieved by changing the amount of tetrahydrofuran in the solvent mixture. Eurdocidin separated completely in 5 min into two components, which were quantitated from peak height measurements. In less than 20 min. candicidin separated into five components with better resolution than that obtained after 600 transfer with counter-current distribution separation. The reproducibility of retention times was very good. It was observed that antibiotics with longer retention times, as measured under identical conditions, were also more active as antifungal agents. The heptaenes appeared to form three distinct groups with characteristic similar ranges of retention time and levels of antifungal activity.

The separatin and differentiation of the gentamicin complex

Abstract Column chromatography on a strongly basic ion-exchange resin, with water as eluent, fractionated the gentamicin antibiotic complex; each fraction, representing different mixtures of antibiotics, further separated upon thin-layer chromatography. Sixteen antibiotics were clearly differentiated. Paper chromatography of the N-acetyl derivatives supplemted thin-layer chromatography, detecting many additional constituents of the complex. A number of important, basic, water-soluble antibiotics were chromatographically compared with the major gentamicin antibiotics.

HIV-1 escape from the entry-inhibiting effects of a cholesterol-binding compound via cleavage of gp41 by the viral protease

HIV-1 virions are highly enriched in cholesterol relative to the cellular plasma membrane. We recently reported that a cholesterol-binding compound, amphotericin B methyl ester (AME), blocks HIV-1 entry and that single amino acid substitutions in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 confer resistance to AME. In this study, we defined the mechanism of resistance to AME. We observed that the gp41 in AME-resistant virions is substantially smaller than wild-type gp41. Remarkably, we found that this shift in gp41 size is due to cleavage of the gp41 cytoplasmic tail by the viral protease. We mapped the protease-mediated cleavage to two sites in the cytoplasmic tail and showed that gp41 truncations in this region also confer AME resistance. Thus, to escape the inhibitory effects of AME, HIV-1 evolved a mechanism of protease-mediated envelope glycoprotein cleavage used by several other retroviruses to activate envelope glycoprotein fusogenicity. In contrast to the mechanism of AME resistance observed for HIV-1, we demonstrate that simian immunodeficiency virus can escape from AME via the introduction of premature termination codons in the gp41 cytoplasmic tail coding region. These findings demonstrate that in human T cell lines, HIV-1 and simian immunodeficiency virus can evolve distinct strategies for evading AME, reflecting their differential requirements for the gp41 cytoplasmic tail in virus replication. These data reveal that HIV-1 can escape from an inhibitor of viral entry by acquiring mutations that cause the cytoplasmic tail of gp41 to be cleaved by the viral protease.

Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

SummaryThe comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone2 (FZ), toward five cell lines has been determined as measured by early membrane damage (51Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems.

Hypercholesterolemia induces angiogenesis and accelerates growth of breast tumors in vivo.

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.

In vitro antiherpetic activity of water-soluble amphotericin B methyl ester

SummaryAntiherpetic activity of AME was demonstrated inin vitro inactivation tests and in cell culture systems against herpes zoster and five strains of herpes simplex virus.