Potent inhibition of influenza sialidase by a benzoic acid containing a 2-pyrrolidinone substituent.

On the basis of the lead compound 4-(N-acetylamino)-3-guanidinobenzoic acid (BANA 113), which inhibits influenza A sialidase with a Ki of 2.5 microM, several novel aromatic inhibitors of influenza sialidases were designed. In this study the N-acetyl group of BANA 113 was replaced with a 2-pyrrolidinone ring, which was designed in part to offer opportunities for introduction of spatially directed side chains that could potentially interact with the 4-, 5-, and/or 6-subsites of sialidase. While the parent structure 1-(4-carboxy-2-guanidinophenyl)pyrrolidin-2-one (8) was only a modest inhibitor of sialidase, the introduction of a hydroxymethyl or bis(hydroxymethyl) substituent at the C5' position of the 2-pyrrolidinone ring resulted in inhibitors (9 and 12, respectively) with low micromolar activity. Crystal structures of these inhibitors in complex with sialidase demonstrated that the substituents at the 5'-position of the 2-pyrrolidinone ring interact in the 4- and/or 5-subsites of the enzyme. Replacement of the guanidine in 12 with a hydrophobic 3-pentylamino group resulted in a large enhancement in binding to produce an inhibitor (14) with an IC50 of about 50 nM against influenza A sialidase, although the inhibition of influenza B sialidase was 2000-fold less. This represents the first reported example of a simple, achiral benzoic acid with potent (low nanomolar) activity as an inhibitor of influenza sialidase.

Hydrophobic benzoic acids as inhibitors of influenza neuraminidase.

Neuraminidase (NA) plays a critical role in the life cycle of influenza virus and is a target for new therapeutic agents. A new benzoic acid inhibitor (11) containing a lipophilic side chain at C-3 and a guanidine at C-5 was synthesized. The X-ray structure of 4-(N-acetylamino)-5-guanidino-3-(3-pentyloxy)benzoic acid in complex with NA revealed that the lipophilic side chain binds in a newly created hydrophobic pocket formed by the movement of Glu 278 to interact with Arg 226, whereas the guanidine of 11 interacts in a negatively charged pocket created by Asp 152, Glu 120 and Glu 229. Compound 11 was highly selective for type A (H2N2) influenza NA (IC50 1 microM) over type B (B/Lee/40) influenza NA (IC50 500 microM).

Glutathione S-transferases and P-glycoprotein in normal rat hepatocytes and hepatoma cells: analysis using flow cytometry.

Using indirect immunofluorescence with fluorescein isothiocyanate-conjugated antibodies, in combination with flow cytometry (FCM), we have developed a technique to detect the alpha, mu and pi isozymes of GST in cell suspensions from normal rat liver, and in H4IIE cells, a rat hepatoma cell line. Cell suspensions fixed in 1% paraformaldehyde were observed to require cell membrane permeation with lysolecithin to allow access and binding of antibodies to immunoreactive proteins within the cytoplasm. FCM analysis indicated normal rat hepatocytes to be positive for GST alpha and mu, but not GST pi, and the H4IIE cells to be positive for all three GST isozymes. Further analysis by FCM for the expression of P-glycoprotein (mdr), a membrane-associated protein product of the multidrug resistance gene, showed an association between the presence of GST pi and mdr in the two cell types. Thus, mdr was detected in significant amounts in H4IIE cells, but not in rat hepatocytes. The method described here has potential applications in screening, sorting and further characterisation for GST pi-positive hepatocytes for mechanistic studies during sequential rat liver carcinogenesis, as well as for characterisation of human tumors for the expression of different GST isozymes and P-glycoprotein during therapeutic management.

Acute hepatic response to aflatoxin B1 in rats fed a methyl-deficient, amino acid-defined diet.

In order to evaluate the relative contribution of aflatoxin B1 (AFB1)-induced toxicity towards a methyl-deficient diet influenced AFB1 carcinogenesis, a no-observed-effect-level (NOEL) for AFB1, with reference to liver damage, was determined in rats fed a nutritionally complete amino acid-defined basal (CMS) diet or a choline-methionine-deficient (CMD) diet. After 3 weeks of dietary treatment, male Fischer 344 rats received a single, oral dose of AFB1 in the range of 100-600 pg/kg body weight. At 24, 48 and 72 h after AFB1 treatment, six serum biochemical parameters were analysed in parallel with histological examination of liver sections. In rats fed the CMS diet and receiving 250-600 micrograms/kg AFB1, serum levels of glutamyl oxalo-transaminase (SGOT), glutamyl pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin increased, glucose levels decreased and gamma glutamyl transpeptidase (GGT) levels remained unchanged over the 72-h period following mycotoxin treatment. However, at 100 micrograms/kg AFB1, these serum parameters remained at control levels. Pathological examination of liver sections indicated no significant lesions at 100 micrograms/kg AFB1 confirming this as the non-necrogenic dose or NOEL in CMS diet group rats. In contrast, in CMD diet fed rats, serum or pathology data showed no obvious time- or dose-response to mycotoxin treatment, extensive hepatic lipidosis in response to dietary treatment being the only predominant lesion in this diet group. The milder response of CMD rat livers to a single dose of AFB1 suggest a possible reduction in the susceptibility of these livers to AFB1 toxicity.

Liver DNA adducts in methyl-deficient rats administered a single dose of aflatoxin B1.

Using an 8 week Solt-Farber protocol with selection pressure (2-acetylaminofluorene/partial hepatectomy) applied during weeks 6 and 7, we have observed that a single oral administration of aflatoxin B1 (AFB1) to Fischer 344 rats on day 1 of the study, followed by a 3 week feeding regimen of either a methyl-deficient (CMD) or a basal (CMS) diet, results in a relative increase in hepatic preneoplastic lesions in CMD diet fed rats. It has previously been shown that a multiple dosing regimen with AFB1, started after 3 weeks of CMD diet, enhances tumor incidence. In the present study, the role of metabolic activation in the induction of preneoplastic lesions, and liver DNA adduct levels after the first dose of AFB1 in the tumorigenesis model have been investigated. AFB1-DNA adducts were determined at 2-168 h following a single non-necrogenic (100 micrograms/kg body wt) or necrogenic (600 micrograms/kg body wt) dose of AFB1 on day 1 or day 21 of a 3 week treatment with a complete basal or CMD diet. In all rats irrespective of dose, dietary treatment or time of AFB1 dosing, the patterns of adduct formation and repair did not change. In rats receiving AFB1 on day 1, total DNA adduct levels between the diet or dose groups were not significantly different, and quantitatively did not correlate with the observed increase in preneoplastic lesions, suggesting a contribution by additional factors in the initiation of these lesions. Administration of AFB1 on day 21, however, resulted in significantly reduced levels of total adducts at both dose levels in CMD diet fed rats compared to controls. Serum biochemistry data suggest that a prolonged exposure to CMD diet may cause pathological and/or biochemical alterations in hepatocytes with a resultant decrease in metabolic activation of AFB1, thus making it difficult to evaluate whether DNA damage is directly related to tumorigenesis.

Hexachlorobenzene residues and effects on esterase activities in pre-weanling rats after a reciprocal transfer between HCB-treated and control dams.

Female Wistar rats were divided into two groups. One group was fed ad libitum powdered rat chow containing 80 ppm hexachlorobenzene (HCB) and the other was fed HCB-free diet 2 weeks before mating until the termination of the experiment. Immediately after parturition, the pups were randomly culled to 10 per litter and 5 pups were reciprocally transferred between HCB and control dams. All pups within same paired litters were sacrificed on the 16th to 17th day and on the 20th to 23rd day after birth. Liver esterase activity toward indophenyl, thiophenyl, and p-nitrophenyl acetates was determined. The liver to body weight ratio, liver esterase activity and HCB residues were significantly higher in pups nursed by HCB-fed dams than in those nursed by control dams. The HCB residue in the liver, kidney, spleen, heart and brain was analyzed by gas-liquid chromatography. At about the weaning time, the HCB residue levels in pups transferred after parturition from HCB fed to control dams were similar to those in pups which remained with the control dams. The results demonstrated that HCB in tissues at about weaning time was obtained largely through milk. In addition, transmission of HCB through milk had greater effects on esterase activities in suckling pups than placental transmission. The pups born to HCB-treated dams and nursed by the control dams were similar to the pups born and nursed by the same control dams.