Synthesis and biological evaluation of water-soluble derivatives of chiral gossypol as HIV fusion inhibitors targeting gp41

A series of novel or known water-soluble derivatives of chiral gossypol were synthesized and screened in vitro for their anti-HIV-1 activity. (?)-gossypol derivative was more active against HIV-1 than the corresponding (+)-gossypol derivative, respectively. Among these derivatives, d-glucosamine derivative of (?)-gossypol, oligopeptide derivative of (?)-gossypol and taurine derivative of (?)-gossypol, such as compounds 1a, 3a and 14a, showed significant inhibitory activities against HIV-1 replication, HIV-1 mediated cell-cell fusion and HIV gp41 6-helix bundle formation as some amino acid derivatives of (?)-gossypol.     

The peroxidative coupling of hemigossypol to (+)- and (-)-gossypol in cottonseed extracts

Peroxidase(s) present in embryo extracts of Gossypium hirsutum cv. Texas Marker 1 catalyzed a bimolecular coupling of [4-(3)H]-hemigossypol to [4,4'-(3)H(2)]-gossypol. The reaction was dependent on the addition of H(2)O(2) and was inhibited 71-94% by 1 and 10mM sodium azide. The phenolic coupling produced 53% (+)-gossypol and 47% (-)-gossypol in close agreement to the 49% (+)-gossypol and 51% (-)-gossypol found in the intact seed. The nearly racemic mixture of (+)-and (-)-gossypol produced in these embryo extracts can be accounted for by non-enzymatic random coupling of the free radicals of hemigossypol produced by the peroxidase. In contrast, peroxidase reaction mixtures containing crude embryo extracts of G. hirsutum var. marie-galante produced 73% (+)-gossypol and 27% (-)-gossypol. These data from the marie-galante extracts and the fact that these intact seed contain 95% (+)-gossypol suggest a regio-stereoselective bimolecular coupling of hemigossypol to gossypol. The development of the peroxidative coupling of hemigossypol to gossypol in maturing seed of G. hirsutum cv. Texas Marker 1 was correlated to the formation of gossypol and suggests that peroxidative coupling of hemigossypol contributes to gossypol biosynthesis.     

Inhibition of testicular LDH-X from laboratory animals and man by gossypol and its isomers

The inhibitory effect of (+)-, (-)-, (+/-)-gossypol and (+/-)-gossypol acetic acid upon testicular cytosolic LDH-X was measured in vitro. Gossypol acetic acid (0-100 mumol/l) inhibited LDH-X prepared from the testes of the mouse greater than rabbit greater than human greater than rat greater than hamster. There was no relationship between inhibition and in-vivo antifertility activity. LDH activity measured in vitro in serum of men and hamsters was unaffected by gossypol. Gossypol and its isomers were non-competitive inhibitors of human and hamster LDH-X with respect to the coenzyme NADH, competitive inhibitors of human LDH-X and noncompetitive-competitive inhibitors of hamster LDH-X with respect to the substrate alpha-ketobutyrate. Co-incubation with human serum albumin or poly-L-lysine but not lysine protected human and hamster LDH-X from gossypol.     

Determination of the absolute configuration and solution conformation of gossypol by vibrational circular dichroism

Vibrational circular dichroism (VCD) measurements and density functional theory (DFT) calculations were used to obtain the first definitive assignment of the absolute configuration for the polyphenolic binaphpthyl dialdehyde gossypol and a determination of the solution conformation in CDCl(3). VCD spectra recorded for the two resolved enantiomers are near mirror images and excellent agreement between the observed IR and VCD spectra and intensity calculations carried out at the DFT (B3LYP/6-31G*) level establish the absolute configurations of (+)-gossypol as P and (-)-gossypol as M, with two conformations in CDCl(3) solution that differ in isopropyl group orientation.     

Synthesis and anti-H5N1 activity of chiral gossypol derivatives and its analogs implicated by a viral entry blocking mechanism

A series of chiral gossypol derivatives and its analogs were synthesized and tested in vitro for their anti-H₅N₁ activity. Interestingly, (+)-gossypol derivatives and its analogs were more active against H₅N₁ than the corresponding (?)-gossypol derivatives and its analogs. Through a simple chemical modification with amino acids, less active chiral gossypol could be converted into more active derivatives, and most of chiral gossypol derivatives were more potent against H₅N₁ than 1-adamantylamine. With regard to the mechanism of action, chiral gossypol derivatives and its analogs might impair the virus entry step of cell infection, likely targeting to HA2 protein.     

Synthesis of gossypol atropisomers and derivatives and evaluation of their anti-proliferative and anti-oxidant activity

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.     

The effect of gossypol on the frequency of DNA-strand breaks in human leukocytes in vitro

Gossypol, a human antifertility agent isolated from the cotton plant, was found to induce a dose-dependent increase in the frequency of DNA-strand breaks in human leukocytes exposed to 2-40 micrograms/ml of the drug for 1 h in serum-free medium in vitro. DNA-strand breaks were studied by alkaline elution or alkaline unwinding of DNA followed by hydroxylapatite-chromatography. No decrease of gossypol-induced DNA-strand breaks was observed after post-treatment incubation times up to 24 h, whereas X-ray-induced DNA breaks disappeared within 2 h under the same incubation conditions. Cells exposed to gossypol in the presence of 10% fetal calf serum showed no or little increase of DNA breaks, suggesting that serum proteins inhibit the DNA-damaging activity of the drug. Both optical isomers of gossypol induced DNA-strand breaks. However, the effect of (-)-gossypol was only about half of that of (+)-gossypol and the racemic form. The induction and persistence of DNA-strand breaks by gossypol, as well as the reduction of this effect in the presence of serum should be considered in the evaluation of the potential in vivo genotoxicity of the drug.     

The in vitro effect of gossypol and its interaction with salts on conidial germination and viability of Fusarium oxysporum sp. vasinfectum isolates

AIMS: To assess the effect of different concentrations of gossypol (0, 2, 4, 10 and 20 mg l(-1)) in combination with NaCl and Na(2)SO(4) (20 mS cm(-1)) on the conidial germination and viability of Fusarium oxysporum f.sp. vasinfectum (Fov). METHODS AND RESULTS: A multinomial logistic model was developed to estimate the germination probability of Fov. The inhibitory effect was markedly evident at the two highest concentrations of gossypol; it varied among the isolates tested and with time, and it was attenuated by the presence of sodium salts. The inhibition was temporary as the germination probability increased after 8 h. Fluorescent staining revealed that gossypol either killed the conidia or retarded the elongation of the germ tubes. CONCLUSION: Fov showed the ability to overcome gossypol inhibition over time, and the inhibitory effect is reduced under saline conditions. Differential responses among Fov isolates to the presence of gossypol suggest that gossypol tolerance is genetically determined in the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that selecting for high plant gossypol cultivars would have minimal effect on the overall Fov resistance of cotton. A new statistical model was developed to explore the statistical significance of plant-pathogen interactions.     

Differential binding of (+) and (-) gossypol to plasma protein and their entry into rat testis.

Concentrations of (+) and (-) gossypol were measured by high performance liquid chromatography after they were incubated with plasma proteins in vitro. The concentration of (-) gossypol decreased more than the concentration of (+) gossypol. A similar decrease in free gossypol concentrations in the blood plasma of rats was observed after intravenous infusion of gossypol enantiomers. The concentration of (-) gossypol was also found to be lower than the concentration of (+) gossypol at the blood-testis barrier. The biological effect of (-) gossypol probably results from its stereospecific binding to extra- and intracellular proteins in vivo and inhibition of the biological activity of some proteins.     

Optical stability of gossypol

The optical stability of gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimetyl-5,5'-bis(1-methylethyl)-2,2'-binaphthalene-8,8'-dicarboxaldehyde], a natural product exhibiting profoundly enantiospecific antitumor and male antifertility action, was investigated by means of computational methods and thermal racemization experiments. The calculations on gossypol and several derivatives and model compounds were carried out using the MM2 force field; energies and geometries of minimum energy conformations, as well as structures along various inversion pathways, were calculated. According to the calculations, gossypol (the dialdehyde form) and its simple analogues are not thermally racemizable (energy barriers for rotational inversion above 50 kcal/mol). By contrast, the calculations suggest that the acetal tautomer of gossypol and its dehydration product (anhydrogossypol) are thermally racemizable, although the energy barriers are still relatively high (35–40 kcal/mol). Optically pure (+)-anhydrogossypol was prepared and characterized; its racemization became rapid only at high temperatures (180–200°C). When dehydration of gossypol was hindered (in aqueous solution), no racemization of gossypol was observed after prolonged heating at 90°C. © 1992 Wiley-Liss, Inc.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

Novel O-glycosidic gossypol isomers and their bioactivities

Novel glycosidic gossypol analogs, 7,7′-gossypol diglucoside tetraacetate GS1, 6,7′-gossypol diglucoside tetraacetate GS2, 7,7′-gossypol diglycoside GS1′, 6,7′-gossypol diglycoside GS2′ were obtained by the ultrasound-assisted reaction of the potassium salt of gossypol with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide under PTC conditions and were fully characterized by 1D NMR (¹H NMR, ¹³C NMR, DEPT, 1D NOE), 2D NMR (HMBC, HMQC), FTIR, HRMS and HPLC. The anticancer activities, cytotoxic effects as well as anti-trypanosomal activities of these novel glycosidic gossypols were explored and suggest that gossypol glycosides could be used to develop new candidates for anticancer drugs and anti-trypanosomal agents.     

Differential effects of (+)- and (-)-gossypol enantiomers on LDH-C4 activity of hamster spermatogenic epithelium in vitro

Tubular fragments (spermatogenic epithelium) from immature hamsters were isolated and cultured with low doses of (+)- and (-)-gossypol enantiomers. The activity of lactate dehydrogenase isoenzyme LDH-C4 was estimated as a marker for spermatogenic cell development and alpha-ketoisovalerate was used as the substrate. In the absence of gossypol, the specific activity of LDH-C4 in the tubular fragments was increased 40% during incubation for 48 h. This developmental increase was suppressed by gossypol. The specific activity of LDH-C4 in the tubular fragments was lowered by gossypol, after 48 h of culture in the presence of low doses of racemic gossypol (1-4 microM) and 1% fetal calf serum. In this in-vitro system the (-)-enantiomer but not the (+)-enantiomer of gossypol affected the LDH-C4 activity. This is in agreement with other reports showing that only the (-)-enantiomer induces infertility. The observed action of gossypol on LDH-C4 activity in the tubular fragments may reflect gossypol-induced degeneration of spermatogenic cells. The present in-vitro system can be used to estimate the actions of gossypol derivatives, other investigational antifertility agents, and toxic agents on the spermatogenic epithelium.     

二苯胺棉酚中棉酚的含量测定

用紫外分光光度法测定二苯胺棉酚中棉酚的含量。方法回收率平均达 93.6 %以上 ,变异系数 ( CV )为1.2 %。该方法准确 ,重现性好 ,可作为二苯胺棉酚质量监控方法。     

The (+)- and (−)-gossypols potently inhibit human and rat 11β-hydroxysteroid dehydrogenase type 2

Gossypol has been proven to be a very effective male contraceptive. However, clinical trials showed that the major side effect of gossypol was hypokalemia. Gossypol occurs naturally as enantiomeric mixtures of (+)-gossypol and (−)-gossypol. The (−)-gossypol is found to be the active component of antifertility. 11β-Hydroxysteroid dehydrogenase 2 (11βHSD2) has been demonstrated to be a mineralocorticoid receptor (MR) protector by inactivating active glucocorticoids including corticosterone (CORT) in rats, and therefore mutation or suppression of 11βHSD2 causes hypokalemia and hypertension. In the present study, the potency of gossypol enantiomers was tested for the inhibition of 11βHSD1 and 2 in rat and human. Both (+) and (−)-gossypols showed a potent inhibition of 11βHSD2 with the half maximal inhibitory concentration (IC₅₀) of 0.61 and 1.33 μM for (+) and (−)-gossypols, respectively in rats and 1.05 and 1.90 μM for (+) and (−)-gossypols, respectively in human. The potency of gossypol to inhibit 11βHSD1 was far less; the IC₅₀ was ≥100 μM for racemic gossypol. The gossypol-induced hypokalemia is likely associated with its potent inhibition of kidney 11βHSD2.     

Antibacterial activity of snake, scorpion and bee venoms: a comparison with purified venom phospholipase A2 enzymes

AIMS: Venoms of snakes, scorpions, bees and purified venom phospholipase A(2) (PLA(2)) enzymes were examined to evaluate the antibacterial activity of purified venom enzymes as compared with that of the crude venoms. METHODS AND RESULTS: Thirty-four crude venoms, nine purified PLA(2)s and two L-amino acid oxidases (LAAO) were studied for antibacterial activity by disc-diffusion assay (100 microg ml(-1)). Several snake venoms (Daboia russelli russelli, Crotalus adamanteus, Naja sumatrana, Pseudechis guttata, Agkistrodon halys, Acanthophis praelongus and Daboia russelli siamensis) showed activity against two to four different pathogenic bacteria. Daboia russelli russelli and Pseudechis australis venoms exhibited the most potent activity against Staphylococcus aureus, while the rest showed only a moderate activity against one or more bacteria. The order of susceptibility of the bacteria against viperidae venoms was -S. aureus > Proteus mirabilis > Proteus vulgaris > Enterobacter aerogenes > Pseudomonas aeruginosa and Escherichia coli. The minimum inhibitory concentrations (MIC) against S. aureus was studied by dilution method (160-1.25 microg ml(-1)). A stronger effect was noted with the viperidae venoms (20 microg ml(-11)) as compared with elapidae venoms (40 microg ml(-1)). The MIC were comparable with those of the standard drugs (chloramphenicol, streptomycin and penicillin). CONCLUSION: The present findings indicate that viperidae (D. russelli russelli) and elapidae (P. australis) venoms have significant antibacterial effects against gram (+) and gram (-) bacteria, which may be the result of the primary antibacterial components of laao, and in particular, the PLA(2) enzymes. The results would be useful for further purification and characterization of antibacterial agents from snake venoms. SIGNIFICANCE AND IMPACT OF THE STUDY: The activity of LAAO and PLA(2) enzymes may be associated with the antibacterial activity of snake venoms.     

棉籽中游离棉酚的含量测定

用紫外分光光度法测定棉籽中游离棉酚的含量。脱除棉酚的棉籽乙醚提取物经石油醚和 75 %乙醇分离和纯化后 ,用 0 .5 %对氨基苯磺酸显色来检测棉籽中游离棉酚的含量 ,结果表明 ,该方法具有简便、重现性好、回收率高达 97%以上等优点 ,可作为棉籽中游离棉酚质量控制方法。     

Different pathways of cell killing by gossypol enantiomers

Gossypol, a polyphenolic, aldehyde-containing constituent of cottonseed, produced partial responses (>50% reduction in tumor size) in some patients with advanced cancer and suppressed sperm as an antifertility agent for men. This action in vivo and its novel side effect profile suggest a specific mechanism of the action of gossypol. Using the random homozygous knockout approach of Li and Cohen (1), we developed a cell line resistant to killing by gossypol, but sensitive to methotrexate and doxorubicin. It showed stereospecific resistance to killing by (-) gossypol (ED(50) 4.9 microM) compared with wild type (ED(50) 2.0 microM). The resistant and wild-type cells were equally sensitive to (+) gossypol (ED(50) 8.8 and 8.4 microM, respectively), methotrexate, and doxyrubicin. We conclude that gossypol affects cells by a stereospecific pathway for (-) gossypol, possibly related to its selective effects, and a nonstereospecific pathway for (+) gossypol and higher concentrations of (-) gossypol. Further knowledge about the stereospecific pathway may lead to new therapeutic drugs.     

Helichrysum italicum extract interferes with the production of enterotoxins by Staphylococcus aureus

AIMS: The purpose of this work was to evaluate the effect of Helichrysum italicum extract on enterotoxin (A-D) production by Staphylococcus aureus strains. METHODS AND RESULTS: The production of enterotoxins A-D in the presence or absence of H.italicum diethyl ether extract was estimated in microtiter plates using a reversed passive latex agglutination (SET-RPLA) kit (Oxoid, Basingstoke, UK). The results indicate that, in culture medium, inhibition of staphylococcal growth and enterotoxins appeared with 250-125 microg ml(-1) of the extract. Lower concentrations of the extract (62.5-31.25 microg ml(-1)) did not affect the final viable count of Staph. aureus but reduced the production of enterotoxins B and C. CONCLUSIONS: H. italicum interferes with growth and production of enterotoxins by Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: There is considerable interest in the use of natural compounds as alternative methods to control undesirable pathogenic micro-organisms.     

An improved selective isolation medium for the recovery of Listeria monocytogenes from smoked fish

AIMS: The aim of this study was to improve the selective isolation of Listeria monocytogenes from smoked haddock fillets. METHODS AND RESULTS: Listeria selective agar (LSA)--Oxford formulation was supplemented with 25 microg x ml(-1) of colistin sulphate and 30 microg x ml(-1) of nalidixic acid. Inocula from four smoked haddock fillets produced colonies (approx. 2-13 bacteria x g(-1)), identified as L. monocytogenes, on LSA supplemented with antimicrobial compounds (MLSA). Moreover, there was only negligible evidence of bacteria which were not L. monocytogenes on MLSA. In contrast, LSA supported dense bacterial growth, which was not equated with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The modified medium permitted the recovery of L. monocytogenes from smoked haddock fillets and reduced the growth of contaminating bacteria.