A Novel Sulfated Holostane Glycoside from Sea Cucumber Holothuria leucospilota

A new sulfated holostane glycoside, leucospilotaside B ( 1 ), together with the two related structurally known compounds holothurin B₂ ( 2 ) and holothurin B ( 3 ), was isolated from sea cucumber Holothuria leucospilota collected from the South China Sea. The structure of 1 was elucidated by spectral analysis (¹H‐, ¹³C‐, and 2D‐NMR, ESI‐MS, and HR‐ESI‐MS) and chemical methods. The compounds 1 – 3 possess the same disaccharide moiety, but were different in the side chains of the triterpene aglycone. Compound 1 showed significant cytotoxicities against four human tumor cell lines, HL‐60, MOLT‐4, A‐549, and BEL‐7402.     

Formation of the complex of the triterpene glycoside holothurin A with cholesterol in liposomal membranes

Interaction between holothurin A triterpene glycoside and lipid-cholesterol liposomes was studied by differential scanning microcalorimetry. Partial restoration of the peak of basic phase transition of dipalmitoyl phosphatidyl choline was shown to be related to the formation of holothurin A (in the membrane)-cholesterol complex. The data obtained are in favor of "sterol" hypothesis of the mechanism of membrane-tropic action of holothurin A.     

Effect of Holothurin on Trypanosoma musculi infection in Three Strains of Mice1

The effect of holothurin (a marine biotoxin) on the resistance of mice to Trypanosoma musculi was measured by studying changes in the parasite population in vivo. Swiss Webster (SW), Beige (BG), and Black (BL) mice treated with holothurin prior to and simultaneously with infection of trypanosomes had lower parasitemias than controls. Higher levels of parasitemia were observed in mice treated after infection with trypanosomes. The timing of administration of holothurin appeared to be an important factor in the observed effect. The minor variations in the parasitemia seemed to be related to the mouse strain.     

Effect of Holothurin on Trypanosoma lewisi Infections in Rats

SYNOPSIS. Crude holothurin from the Bahamian sea cucumber Actinopyga agassiz , inoculated intraperitoneally into rats with Trypanosoma lewisi infection, altered the host-parasite relationship. The effect was measured by a study of parasite population during infections. Rats treated with holothurin prior to and simultaneously with an infection of trypanosomes had lower parasitemias than controls. A higher level of parasitemia was observed in treated after infection with trypanosomes. Both dosage and timing appeared to have been important parameters of the observed effect. The reticuloendothelial system is suspected to play a role in these findings.     

Interaction of sea cucumber saponins with multilamellar liposomes

The effect of three sea cucumber saponins, echinoside A, bivittoside D and holothurin A, on multilamellar liposomes was investigated. An ideal osmotic behavior of liposomes was described as a linear relationship between the reciprocal $\text{3}\text{2}\text{s}$ power of absorbance at 450 nm and the osmotic gradient across the membrane. Sea cucumber saponins at concentrations below critical micelle concentration (CMC) disturbed this linear relationship in liposomes composed of egg phosphatidylcholine, phosphatidic acid and cholesterol. Cholesterol-free liposomes were not susceptible to these saponins. Results of optical measurements were consistent with those of transmission electron microscopy, which showed saponin-induced changes in liposomal structure. The lytic activity of sea cucumber saponins on liposomes depended on their chemical structure.These results suggest that sea cucumber saponins as monomers can interact with liposomes and that cholesterol serve as a principal binding site for the sea cucumber saponins.     

Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).     

Synthesis, stereochemistry and in vitro antimicrobial evaluation of novel 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles

2-[(2,4-Diaryl-3-azabicyclo[3.3.1]nonan-9-ylidene)hydrazono]-4-phenyl-2,3-dihydrothiazoles (3a-3k) have been synthesized by the cyclization of 2-[(2,4-diaryl-3-azabicyclo[3.3.1]nonan-9-one thiosemicarbazones with phenacyl bromide and characterized by analytical (melting point and elemental analysis) and spectral (IR, (1)H NMR, (13)C NMR, D(2)O exchange, NOESY and mass) techniques. The novel Hantzsch products (3a-3k) were screened for their in vitro antibacterial and antifungal activities against some selected microorganisms. Structure activity relationship (SAR) for the reported compounds was studied by comparing their MIC values with standard drugs (Streptomycin and Amphotericin B). The results show that 3e against Escherichia coli and Cryptococcus neoformans3i against Bacillus Subtilis, 3b against Aspergillus flavus, and 3k against Rhizopus sp. were found to show significant growth inhibition.     

Synthesis and biological activity of tricyclic analogues of 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine

The base moiety of the potent antiherpetic agent 9-[[cis-1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine 3 was transformed into that of the tricyclic 3,9-dihydro-9-oxo-6-R-5H-imidazo[1,2-a]purine system. The tricyclic analogues 5a-d were evaluated for their activity against herpes viruses as well as for cytostatic activity against HSV-1 thymidine kinase (TK) gene-transduced human osteosarcoma tumor cells. Marked activity was found against VZV. The 6-phenyl-substituted fluorescent analogues 5c and d were comparable to that of parent 3 in activity against the VZV strain YS and were 3-fold less active against the VZV strain OKA. The compounds 5a-d also showed marked activity against HSV-1 (KOS) and HSV-2 (G)-against the former generally approximately comparable to that of acyclovir 1a and one order of magnitude lower than 3; against the latter comparable to that of 1a and approximately 6- to 30-fold lower than that of 3. The most pronounced cytostatic activity (5-fold lower than that of 3) was exhibited by compounds 5c and d. Tricyclic analogues with pseudosugar moieties are intrinsically bio-active.     

Cysteine chloromethyl and diazomethyl ketone derivatives with potent anti-leukemic activity

A series of cysteine diazomethyl- and chloromethyl ketone derivatives has been synthesized and evaluated against human B-lineage (Nalm-6) and T-lineage (Molt-3) acute lymphoblastic leukemia cell lines. The chloromethyl ketone compounds showed potent cytotoxicity against these cell lines, with IC50 values in the low micromolar range. The best compounds were N-acetyl-S-dodecyl-Cys chloromethyl ketone (IC50 = 2.0 microM against Nalm-6, 2.3 microM against Molt-3) and N-acetyl-S-trans,trans-farnesyl-Cys chloromethyl ketone (IC50 = 3.0 microM against Nalm-6 and 1.4 microM against Molt-3).     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

Synthesis and bioactivity of 4-alkyl(aryl)thioquinazoline derivatives

Some S'-substituted 4-alkyl(aryl)thioquinazoline derivatives were synthesized through thioetherification reaction of 4-chloroquinazolines 2 and thiol compounds 1 refluxed in acetone in the presence of K(2)CO(3). Their structures were verified by elemental analysis, IR, (1)H NMR, and (13)C NMR. The compounds were evaluated for their anti-proliferative activities against some cancer cells in vitro by MTT method. Among them, 3c, 3a, 3d, 3f, and 3l were highly effective against PC3 cells and 3a-3m showed weak activities against Bcap37 and BGC823 cells. The IC(50) value of 3c, 3a, 3d, 3f, and3l against PC3 cell was 1.8, 5.6, 8.1, 8.7, and 8.9 microM, respectively.     

Preliminary in vitro studies on two potent, water-soluble trimethoprim analogues with exceptional species selectivity against dihydrofolate reductase from Pneumocystis carinii and Mycobacterium avium

2,4-Diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (6) and 2,4-diamino-5-[3',4'-dimethoxy-5'-(4-carboxyphenylethynyl)benzylpyrimidine (7) were synthesized from 2,4-diamino-5-(5'-iodo-3',4'-dimethoxybenzyl)pyrimidine (9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC(50)) against rat DHFR by its IC(50) against Pc, Tg, or Ma DHFR. The IC(50) of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC(50) of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC(50) of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure-activity optimization.     

Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis

LpxD catalyzes the third step of lipid A biosynthesis, the (R)-3-hydroxymyristoyl-acyl carrier protein ( R-3-OHC14-ACP)-dependent N-acylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-alpha-D-glucosamine [UDP-3-O-(R-3-OHC14)-GlcN]. We have now overexpressed and purified Escherichia coli LpxD to homogeneity. Steady-state kinetics suggest a compulsory ordered mechanism in which R-3-OHC14-ACP binds prior to UDP-3-O-(R-3-OHC14)-GlcN. The product, UDP-2,3-diacylglucosamine, dissociates prior to ACP; the latter is a competitive inhibitor against R-3-OHC14-ACP and a noncompetitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN. UDP-2-N-[(R)-3-Hydroxymyristoyl]-alpha-D-glucosamine, obtained by mild base hydrolysis of UDP-2,3-diacylglucosamine, is a noncompetitive inhibitor against both substrates. Synthetic (R)-3-hydroxylauroyl-methylphosphopantetheine is an uncompetitive inhibitor against R-3-OHC14-ACP and a competitive inhibitor against UDP-3-O-(R-3-OHC14)-GlcN, but (R)-3-hydroxylauroyl-methylphosphopantetheine is also a very poor substrate. A compulsory ordered mechanism is consistent with the fact that R-3-OHC14-ACP has a high binding affinity for free LpxD whereas UDP-3-O-(R-3-OHC14)-GlcN does not. Divalent cations inhibit R-3-OHC14-ACP-dependent acylation but not (R)-3-hydroxylauroyl-methylphosphopantetheine-dependent acylation, indicating that the acidic recognition helix of R-3-OHC14-ACP contributes to binding. The F41A mutation increases the K(M) for UDP-3-O-(R-3-OHC14)-GlcN 30-fold, consistent with aromatic stacking of the corresponding F43 side chain against the uracil moiety of bound UDP-GlcNAc in the X-ray structure of Chlamydia trachomatis LpxD. Mutagenesis implicates E. coli H239 but excludes H276 as the catalytic base, and neither residue is likely to stabilize the oxyanion intermediate.     

Trypanocidal properties, structure-activity relationship and computational studies of quinoxaline 1,4-di-N-oxide derivatives

Pyrazole and propenone quinoxaline derivatives were tested against intracellular forms of Leishmania peruviana and Trypanosoma cruzi. Both series were tested for toxicity against proliferative and non-proliferative cells. The pyrazole quinoxaline series was quite inactive against T. cruzi; however, the compound 2,6-dimethyl-3-f-quinoxaline 1,4-dioxide was found to inhibit 50% of Leishmania growth at 8.9 μM, with no impact against proliferative kidney cells and with low toxicity against THP-1 cells and murine macrophages. The compounds belonging to the propenone quinoxaline series were moderately active against T. cruzi. Among these compounds, two were particularly interesting, (2E)-1-(7-fluoro-3-methyl-quinoxalin-2-yl)-3-(3,4,5-trimethoxy-phenyl)-propenone and (2E)-3-(3,4,5-trimethoxy-phenyl)-1-(3,6,7-trimethyl-quinoxalin-2-yl)-propenone. The former possessed selective activity against proliferative cells (cancer and parasites) and was inactive against murine peritoneal macrophages; the latter was active against Leishmania and inactive against the other tested cells. Furthermore, insilico studies showed that both series respected Lipinski’s rules and that they confirmed a linear correlation between trypanocidal activities and LogP. Docking studies revealed that compounds of the second series could interact with the poly (ADP-ribose) polymerase protein of Trypanosoma cruzi.     

Antibody responses to intradermal or intramuscular MF59-adjuvanted influenza vaccines as evaluated in elderly institutionalized volunteers during a season of partial mismatching between vaccine and circulating A(H3N2) strains

Background

The age-related weakening of the immune system makes elderly subjects less responsive to influenza vaccination. In the last years, two “enhanced vaccines” were licensed for individuals aged ≥65 years, one being a subunit vaccine (Fluad®) containing the MF59 adjuvant administered intramuscularly (IM-MF59) and the other one a split non-adjuvanted vaccine administered intradermally (Intanza® 15mcg) (ID). In the present study, we evaluated and compared the antibody responses against the three vaccine antigens and heterovariant A(H3N2) circulating viruses induced by IM-MF59 and ID influenza vaccines in 80 elderly institutionalized volunteers (40 per group) during the Winter season 2011–2012.

Results

Hemagglutination inhibiting (HI) antibody titers were assessed in blood samples collected before, 1 and 6 months after vaccination. One month after vaccination both the IM-MF59 and ID vaccines induced increases in HI titers against all the three vaccine strains. The results in the two groups were similar against the A(H3N2) and A(H1N1) strains. Responses against the B strain typically tended to be higher after ID than IM-MF59, yet both vaccines stimulated lower responses against the B strain than against the two A strains. The two vaccines induced favorable results also against four epidemic drifted A(H3N2) viruses circulating in Winter 2011–2012. Six months after vaccination, the HI titers decreased in both groups.

Conclusion

The responses induced by IM-MF59 and ID vaccines in institutionalized elderly people were similar against the A(H3N2) and A(H1N1) strains but frequently higher, for the ID, against the B strain. The two vaccines induced positive responses against drifted A(H3N2) circulating viruses.

     

Passive protection against rotavirus-induced diarrhea by monoclonal antibodies to surface proteins vp3 and vp7.

Monoclonal antibodies directed against two rotavirus surface proteins (vp3 and vp7) as well as a rotavirus inner capsid protein (vp6) were tested for their ability to protect suckling mice against virulent rotavirus challenge. Monoclonal antibodies to two distinct epitopes of vp7 of simian rotavirus strain RRV neutralized RRV in vitro and passively protected suckling mice against RRV challenge. A monoclonal antibody directed against vp3 of porcine rotavirus strain OSU neutralized three distinct serotypes in vitro (OSU, RRV, and UK) and passively protected suckling mice against OSU, RRV, and UK virus-induced diarrhea. The role of vp3 in eliciting protection against heterotypic rotavirus challenge should be considered when developing a vaccine with cloned rotavirus genes. Alternatively, immunization with a reassortant rotavirus containing vp3 and vp7 from two antigenically distinct rotavirus parents might protect against diarrhea induced by two or more rotavirus serotypes.     

3-bromohomofascaplysin A, a fascaplysin analogue from a Fijian Didemnum sp. ascidian

A new fascaplysin analogue, 3-bromohomofascaplysin A (1), along with two known analogues, homofascaplysin A (2) and fascaplysin (3), were isolated from a Fijian Didemnum sp. ascidian. The absolute configurations of 3-bromohomofascaplysin A (1) and homofascaplysin A (2) were determined via experimental and theoretically calculated ECD spectra. The differential activities of 1-3 against different blood-borne life stages of the malaria pathogen Plasmodium falciparum were assessed. Homofascaplysin A (2) displayed an IC(50) of 0.55±0.11 nM against ring stage parasites and 105±38 nM against all live parasites. Given the stronger resistance of ring stage parasites against most current antimalarials relative to the other blood stages, homofascaplysin A (2) represents a promising agent for treatment of drug resistant malaria.     

Camphorsulfonic acid catalysed facile tandem double Friedlander annulation protocol for the synthesis of phenoxy linked bisquinoline derivatives and discovery of antitubercular agents

A series of phenoxy linked bisquinoline derivatives were synthesised from the Friedlander annulation of 2-(4-acetylphenoxy)-1-aryl-1-ethanones with 2-aminobenzophenone in good yields using (±)-camphor-10-sulfonic acid (CSA) as the catalyst. These compounds were screened for their in vitro activity against Mycobacterium tuberculosis H37Rv (MTB) and among the 23 compounds screened, 2-(3-bromophenyl)-6-chloro-3-[4-(6-chloro-4-phenyl-2-quinolyl)phenoxy]-4-phenylquinoline (3q) and 2-(4-bromophenyl)-6-chloro-3-[4-(6-chloro-4-phenyl-2-quinolyl)phenoxy]-4-phenylquinoline (3o) were found to be the most active compounds with MIC of 1.1 and 2.2 μM against MTB. The cytotoxic effects against mouse fibroblasts (NIH 3T3) in vitro were evaluated for 3o and 3q, which displayed no toxic effects against mouse fibroblast cell line NIH 3T3.     

Exploration of biguanido–oxovanadium complexes as potent and selective inhibitors of protein tyrosine phosphatases

The inhibitory effects of three biguanido-oxovanadium complexes ([VO(L(1-3))(2)]·nH(2)O: HL(1) = metformin, HL(2) = phenformin, HL(3) = moroxydine) against four protein tyrosine phosphatases (PTPs) and an alkaline phosphatase (ALP) were investigated. The complexes display strong inhibition against PTP1B and TCPTP (IC(50), 80-160 nM), a bit weaker inhibition against HePTP (IC(50), 190-410 nM) and SHP-1(IC(50), 0.8-3.3 μM) and much weaker inhibition against ALP (IC(50), 17-35 μM). Complex 3 is about twofold less potent against PTP1B, TCPTP and HePTP than complexes 1 and 2, while complex 2 inhibits SHP-1 more strongly (about three to fourfold) than the other two complexes. These results suggest that the structures of the ligands slightly influence the potency and selectivity against PTPs. The complexes inhibit PTP1B and ALP with a typical competitive type.     

Synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a range of phenyl alkyl imidazole-based compounds as potent inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. We report the synthesis, biochemical evaluation and rationalisation of the inhibitory activity of a number of azole-based compounds as inhibitors of the two components of P450(17alpha), i.e., 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results suggest that the imidazole-based compounds are highly potent inhibitors of both components, with N-7-phenyl heptyl imidazole (21) (IC(50)=0.32 microM against 17alpha-OHase and IC(50)=0.10 microM against lyase) and N-8-phenyl octyl imidazole (23) (IC(50)=0.25 microM against 17alpha-OHase and IC(50)=0.21 microM against lyase) being the two most potent compounds within the current study, in comparison to ketoconazole (KTZ) (IC(50)=3.76 microM against 17alpha-OHase and IC(50)=1.66 microM against lyase). Furthermore, consideration of the inhibitory activity against the two components show that the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha). Structure-activity relationship determination of the range of compounds synthesised suggests that logP (log of the partition coefficient) is a key physicochemical factor in determining the overall inhibitory activity. In an effort to determine the viability of these compounds becoming potential drug candidates as well as to show specificity of these compounds, we undertook the biochemical evaluation of the synthesised compounds against two isozymes of 17beta-hydroxysteroid dehydrogenase [namely type 1 (17beta-HSD1) and type 3 (17beta-HSD3)] and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Consideration of the inhibitory activity possessed by the compounds considered within the current study against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 shows that there is no clear structure-activity relationship and that the compounds appear to possess similar inhibitory activity against both 3beta-HSD and 17beta-HSD3 whilst against 17beta-HSD1, the compounds appear to possess poor inhibitory activity at [I]=100 microM. Indeed, two of the most potent inhibitors of P450(17alpha), (compounds 21 and 23), were found to possess relatively good levels of inhibition against the three enzymes-compound 21 was found to possess approximately 32%, approximately 21% and approximately 37% inhibition whilst compound 23 was found to possess approximately 38%, approximately 30% and approximately 28% inhibition against 3beta-HSD, 17beta-HSD1 and 17beta-HSD3 respectively. We therefore concluded that the azole-based compounds synthesised within the current study are not suitable for further consideration as potential drug candidates due to their lack of specificity.