Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I

Kifunensine, produced by the actinomycete Kitasatosporia kifunense 9482, is an alkaloid that corresponds to a cyclic oxamide derivative of 1-amino mannojirimycin. This compound was reported to be a weak inhibitor of jack bean alpha-mannosidase (IC50 of 1.2 x 10(-4) M) (Kayakiri, H., Takese, S., Shibata, T., Okamoto, M., Terano, H., Hashimoto, M., Tada, T., and Koda, S. (1989) J. Org. Chem. 54, 4015-4016). We also found that kifunensine was a poor inhibitor of jack bean and mung bean aryl-alpha-mannosidases, but it was a very potent inhibitor of the plant glycoprotein processing enzyme, mannosidase I (IC50 of 2-5 x 10(-8) M), when [3H]mannose-labeled Man9GlcNAc was used as substrate. However, kifunensine was inactive toward the plant mannosidase II. Studies with rat liver microsomes also indicated that kifunensine inhibited the Golgi mannosidase I, but probably does not inhibit the endoplasmic reticulum mannosidase. Kifunensine was tested in cell culture by examining its ability to inhibit processing of the influenza viral glycoproteins in Madin-Darby canine kidney cells. Thus, when kifunensine was placed in the incubation medium at concentrations of 1 microgram/ml or higher, it caused a complete shift in the structure of the N-linked oligosaccharides from complex chains to Man9(GlcNAc)2 structures, in keeping with its inhibition of mannosidase I. On the other hand, even at 50 micrograms/ml, deoxymannojirimyucin did not prevent the formation of all complex chains. Thus kifunensine appears to be one of the most effective glycoprotein processing inhibitors observed thus far, and knowledge of its structure may lead to the development of potent inhibitors for other processing enzymes.     

Single-chain Fv-based anti-HIV proteins: potential and limitations

The existence of very potent, broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) offers the potential for prophylaxis against HIV-1 infection by passive immunization or gene therapy. Both routes permit the delivery of modified forms of IgGs. Smaller reagents are favored when considering ease of tissue penetration and the limited capacities of gene therapy vectors. Immunoadhesin (single-chain fragment variable [scFv]-Fc) forms of IgGs are one class of relatively small reagent that has been explored for delivery by adeno-associated virus. Here we investigated the neutralization potencies of immunoadhesins compared to those of their parent IgGs. For the antibodies VRC01, PG9, and PG16, the immunoadhesins showed modestly reduced potencies, likely reflecting reduced affinities compared to those of the parent IgG, and the VRC01 immunoadhesin formed dimers and multimers with reduced neutralization potencies. Although scFv forms of neutralizing antibodies may exhibit affinity reductions, they provide a means of building reagents with multiple activities. Attachment of the VRC01 scFv to PG16 IgG yielded a bispecific reagent whose neutralization activity combined activities from both parent antibodies. Although the neutralization activity due to each component was partially reduced, the combined reagent is attractive since fewer strains escaped neutralization.     

Glycoprotein processing and glycoprotein processing inhibitors

Considerable evidence is now accumulating from both in vivo and in vitro studies that the oligosaccharide chains of the plant N-linked glycoproteins undergo modification or processing reactions after the oligosaccharide has been transferred from its lipid-linked oligosaccharide intermediate to the protein. These processing reactions occur in the endoplasmic reticulum and Golgi apparatus of the cell and involve the removal of certain sugars and the addition of other sugars. While the processing reactions appear to be generally similar to those that occur in animal cells, there are some notable differences, such as the addition of a β-linked xylose to many of the plant glycoproteins. It will be interesting to determine the exact sequence of these reactions and how they are regulated in the cell. Recently, some very useful inhibitors have become available that act on the glycosidases that catalyze the removal of glucose and mannose. These inhibitors cause the accumulation of aberrant oligosaccharide chains on the glycoproteins. Such unusual glycoproteins should be valuable tools for studies on the role of oligosaccharide in glycoprotein function.     

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.     

Enzymes of carbohydrate metabolism as potential drug targets

The potential for chemotherapeutic exploitation of carbohydrate metabolism in the Trypanosomatidae is reviewed. This review is based largely on discussions held at a meeting of the COST B9 Action, entitled 'Bioenergetics of Protozoan Parasites'. The major questions posed were: which enzymes are the best to target; what further information is required to allow their use for rational drug development; what compounds would constitute the best inhibitors and which of the enzymes of the pentose-phosphate pathway are present inside the glycosomes, as well? Only partial answers could be obtained in many cases, but the interactive discussion between the multidisciplinary group of participants, comprising chemists, biochemists and molecular biologists, provided thought-provoking ideas and will help direct future research.     

LY227942, an inhibitor of serotonin and norepinephrine uptake: biochemical pharmacology of a potential antidepressant drug

LY227942, (+/-)-N-methyl-3-(1-naphthalenyloxy)-3-(2-thiophene)propanamine ethanedioate, is a new, competitive inhibitor of monoamine uptake in synaptosomal preparations of rat brain. LY227942 inhibits uptake of serotonin (5-hydroxytryptamine, 5HT) and norepinephrine (NE) in cortical synaptosomes and uptake of dopamine (DA) in striatal synaptosomes with inhibitor constants (Ki values) of 8.5, 45 and 300 nM, respectively. Upon administration in vivo, LY227942 lowers 5HT and NE uptake in hypothalamus homogenates to half their respective control activities (ED50) at 0.74 and 1.2 mg/kg s.c., 7 and 12 mg/kg i.p., and 12 and 22 mg/kg p.o., but LY227942 at doses up to 30 mg/kg p.o. does not change DA uptake in striatal homogenates. Lowering of 5HT and NE uptake is demonstrated after 15 min and 6 hr, but has dissipated by 16 hr after oral administration. According to radioligand binding determinations, LY227942 possesses only weak affinity for muscarinic receptors, histamine-1 receptors, adrenergic receptors, dopamine receptors and serotonin receptors. These findings suggest that LY227942 has the pharmacological profile of an antidepressant drug and is useful to study the pharmacological responses of concerted enhancement of serotonergic and noradrenergic neurotransmission.     

Thiol metabolism of the trypanosomatids as potential drug targets

Trypanosomatids produce significant amounts of four major low molecular mass thiols, trypanothione, glutathionylspermidine, glutathione, and ovothiol A. Of these, only glutathione is present in cells of the host. All four low molecular mass thiols are directly or indirectly maintained in a reduced state by trypanothione reductase. Available evidence, from gene disruption studies, indicate that this is an essential enzyme. Attempts to exploit trypanothione reductase as a chemotherapeutic target lead to the design of competitive and irreversible inhibitors of the enzyme. A promising route involves the design of redox cyclers interacting specifically with trypanothione reductase as subversive substrates. Progress in studies on the biosynthesis of ovothiol A is summarized.     

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-101), B-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [³H]concanavalin A, and a substantial decrease in the binding of [³H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [³H]leucine or [³H]proline into protein. There was, however, a significant inhibition in the incorporation of [³H]fucose, [³H]glucosamine, and [³H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.     

The effects of flavonoids on human phenolsulphotransferases: potential in drug metabolism and chemoprevention

Flavonoids are frequently found in fruits and vegetables, and are currently under investigation as potential chemopreventive agents. The present study reports the inhibitory effects of six flavonoids, quercetin, genistein, daidzein, equol, (+)-catechin and flavone, on sulphation of p-nitrophenol, a model substrate for the P-form of PST (thermostable, TS) and dopamine, a model substrate for the M-form of PST (thermolabile, TL). Kinetic studies of the PST activity and the inhibitory effects of flavonoids on the P-form of PST activity (using Hanes-Wolf and Dixon plots) show low Km and Ki values. Quercetin was found to be the most potent inhibitor and flavone was the least active inhibitor among the flavonoids. Kinetic analysis showed that the inhibition was non-competitive and Ki values were determined for each flavonoid. These observations suggest the potential for clinically important pharmacological and toxicological interactions by flavonoids.     

Synthesis of a nucleoside bioconjugate system as a potential anti-HIV agent.

Synthesis and anti-HIV data for a bioconjugate molecule incorporating a HIV protease inhibitor (A74704) and a HIV RT inhibitor (d4T) are presented.     

Withanone as an inhibitor of survivin: A potential drug candidate for cancer therapy

Survivin, the smallest inhibitor of apoptosis protein, which has been reported to be highly expressed in almost all known cancers, plays a dual role in survival as well as the proliferation of cancer cells. It inhibits apoptosis by inhibiting caspases as well as facilitating mitosis by becoming a part of chromosomal passenger complex through its BIR5 domain. Docking studies carried out with herbal ligand withanone derived from roots of Withania somnifera have shown strong binding affinity of −19.1088 kJ/mol with BIR5 domain of survivin and in turn interferes with inhibitory action against caspases and may lead to apoptosis. Binding of withanone at BIR5 domain of survivin may also interfere with chromosomal passenger complex and lead to halt the mitotic process within the cancer cell. Docking studies support various experimental outcomes and suggest withanone as a potential candidate molecule in cancer therapy.     

Synthesis of potential anti-HIV GP120 inhibitors using a lysine template

Various acylated proteins have been reported in the literature to possess anti-HIV activity. Described here is the preparation of lysine monomers, dimers and trimers acylated with various anhydrides and dioxalanones as simplified mimics of the acylated proteins. Compounds were assayed against HIV-infected C8166 cells and some showed weak anti-HIV activity.     

Dideoxycytidine,an anti-HIV drug,selectively inhibits growth but not phosphatidylcholine metabolism in neuroblastoma and glioma cells

Dideoxycytidine (ddCyd), an inhibitor of AIDS-related HIV, has been examined for effects on cell proliferation and phosphatidylcholine synthesis in tumor lines of nervous system origin. Uptake and metabolism of [³H]ddCyd, observed in all cells, was greatest in one human neuroblastoma line, HTB-10. Growth of the HTB-10 line was markedly inhibited by 40 M ddCyd, whereas growth of C₆ glioma and N1E-115 or HTB-11 neuroblastoma cells was unaltered. Phosphatidylcholine synthesis in the presence or absence of stimulation by phorbol ester was not specifically altered by ddCyd. Thus, ddCyd was incorporated and inhibited growth in a cell-specific manner but had little effect on cytidine-dependent phospholipid synthesis. This suggests that some cells derived from the nervous system may be more susceptible than others with respect to the positive and negative effects of ddCyd as a potential antiviral drug.     

Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity

G-rich oligonucleotides T30695 (or T30923), with the sequence of (GGGT)(4), and T40214, with the sequence of (GGGC)(4), have been reported to exhibit anti-HIV and anticancer activity. Here we report on the structure of a dimeric G-quadruplex adopted by a derivative of these sequences in K(+) solution. It comprises two identical propeller-type parallel-stranded G-quadruplex subunits each containing three G-tetrad layers that are stacked via the 5'-5' interface. We demonstrated control over the stacking of the two monomeric subunits by sequence modifications. Our analysis of possible structures at the stacking interface provides a general principle for stacking of G-quadruplexes, which could have implications for the assembly and recognition of higher-order G-quadruplex structures.     

Synthesis and anti-HIV activity of arylpiperazinyl fluoroquinolones: a new class of anti-HIV agents

Synthesis and anti-HIV activity of a series of novel arylpiperazinyl fluoroquinolones are reported. In the SAR study, the aryl substituents on the piperazine nitrogen were found to play an important role for the anti-HIV-1 activity. A few of the compounds exhibited potent anti-HIV activity: IC50=0.06 microM in chronically infected cells.     

Pharmacoanthropology: drug metabolism

This is a report of similarities and differences among various ethnically defined populations with respect to their capacities to metabolize the prototype drugs antipyrine, caffeine, and debrisoquine. There were equal levels of the three main metabolites of antipyrine in the urine of Caucasians and Orientals; differences in antipyrine clearance between English and Indian subjects appeared to have environmental causes. Exploration of various metabolite ratios of caffeine in the urine of Caucasians and Orientals living in Canada showed three patterns: 1) no interethnic difference occurred in the ratio thought to indicate xanthine oxidase activity; 2) products of 7-demethylation and of hydroxylation of paraxanthine , both probably produced by cytochrome P-450, showed different averages in the populations; 3) the new secondary metabolite acetylformyl -methyluracil proved to be a useful indicator of the genetically controlled acetylator status, thereby confirming the well-known population difference for acetylator gene frequency. Analysis of data on debriosquine hydroxylation suggested that interpretation of the standardized metabolic ratio may be appropriate for Caucasian and Oriental groups but is misleading for published data from Saudi Arabia, Nigeria, and Ghana; even these two closely related West African populations seem to differ in debrisoquine metabolism.     

Synthesis, drug release and anti-HIV activity of a series of PEGylated zidovudine conjugates

A series of methoxy poly(ethylene glycol)-succinyl-5'-O-zidovudine conjugates (mPEG-succinyl-AZT) with different molecular weight (M(w): 750 Da, 2, 5 or 10 kDa) of mPEG were synthesized and characterized by Fourier transform infrared (FTIR) spectroscopy, (1)H nuclear magnetic resonance ((1)H NMR) spectroscopy, and matrix-assisted laser desorption/ionization time of flight mass (MALDI TOF MS) spectrometry analysis. All conjugates showed good stability in vitro release experiments, and good anti-HIV activity and low cytotoxicity in MT-4 cells, in which, mPEG(750)-succinyl-AZT exhibited good inhibition to wild-type viruses (strains IIIB and ROD) with EC(50) values of 0.11 and 0.090 μmol/L, respectively, and it showed no cytotoxicity up to 110 μmol/L. Oral pharmacokinetic study in rats showed the half-life time (T(1/2)) of all conjugates are prolonged compared to free AZT. Especially, mPEG(750)-succinyl-AZT displayed a ~2.3-fold prolonged half-life and approximately 224% increased bioavailability of AZT.