Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia. Interaction with tubulin and effects of cellular microtubules.

Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. We have compared dolastatin 15 with dolastatin 10 for its effects on cells grown in culture and on biochemical properties of tubulin. The IC50 values for cell growth were obtained for dolastatin 15 with L1210 murine leukemia cells, human Burkitt lymphoma cells, and Chinese hamster ovary (CHO) cells (3, 3, and 5 nM with the three cell lines, respectively). For dolastatin 10, IC50 values of 0.4 and 0.5 nM were obtained with the L1210 and CHO cells, respectively. At toxic concentrations dolastatin 15 caused the leukemia and lymphoma cells to arrest in mitosis. In the CHO cells both dolastatin 15 and dolastatin 10 caused moderate loss of microtubules at the IC50 values and complete disappearance of microtubules at concentrations 10-fold higher. Despite its potency and the loss of microtubules in treated cells, the interaction of dolastatin 15 with tubulin in vitro was weak. Its IC50 value for inhibition of glutamate-induced polymerization of tubulin was 23 microM, as compared to values of 1.2 microM for dolastatin 10 and 1.5 microM for vinblastine. Dolastatin 10 noncompetitively inhibits the binding of vincristine to tubulin, inhibits nucleotide exchange, stabilizes the colchicine binding activity of tubulin, and inhibits tubulin-dependent GTP hydrolysis (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990; Bai et al. J Biol Chem 265: 17141-17149, 1990). Only the latter reaction was inhibited by dolastatin 15. Nevertheless, its structural similarity to dolastatin 10 indicates that dolastatin 15 may bind weakly in the "vinca domain" of tubulin (a region of the protein we postulate to be physically close to but not identical with the specific binding site of vinca alkaloids and maytansinoids), presumably in the same site as dolastatin 10 (the "peptide site").     

Intracellular activation and deactivation of tasidotin, an analog of dolastatin 15: correlation with cytotoxicity

Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [(3)H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [(3)H]P5 was not the end product of tasidotin metabolism. Large amounts of [(3)H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC(50) of tasidotin and a marked increase in intracellular [(3)H]tasidotin. BCPP also caused a 4-fold increase in the IC(50) of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC(50) obtained for P5 in the presence of BCPP.     

Sustained intracellular retention of dolastatin 10 causes its potent antimitotic activity

Dolastatin 10 is a highly cytotoxic antimitotic peptide in phase II clinical trials. Its cytotoxicity has been as much as 50-fold greater than that of vinblastine, despite quantitatively similar effects of the two drugs on tubulin polymerization. We compared uptake and efflux of radiolabeled dolastatin 10 and vinblastine in human Burkitt lymphoma CA46 cells to gain an understanding of the greater cytotoxicity of the peptide. In the Burkitt cells, dolastatin 10 was 20-fold more cytotoxic than vinblastine (IC(50) values, 50 pM and 1.0 nM). When drug uptake at 24 h was compared at IC(50) values of the two drugs, the intracellular concentrations were almost identical (50-100 nM). The accumulation factor observed for dolastatin 10 was 900 to 1800 versus 60 to 100 for vinblastine. The two drugs showed very divergent uptake kinetics, however. Vinblastine and dolastatin 10 reached maximum intracellular concentrations after 20 min and 6 h, respectively. Depletion of cellular ATP content did not alter the uptake of either drug, indicating passive uptake of both. When drug-preloaded cells were transferred to drug-free medium, there was no loss of dolastatin 10 for at least 2 h, whereas vinblastine exited the cells rapidly (approximate intracellular half-life, 10 min), with less than 10% of the initial drug remaining in the cells after the 2-h incubation. The potency of dolastatin 10 probably derives from its tenacious binding to tubulin, a property that in cells becomes translated into prolonged intracellular retention of the drug. Optimal clinical use of dolastatin 10 may require administration by infusion rather than by bolus.     

Diazonamide A and a synthetic structural analog: disruptive effects on mitosis and cellular microtubules and analysis of their interactions with tubulin

The marine ascidian Diazona angulata was the source organism for the complex cytotoxic peptide diazonamide A. The molecular structure of this peptide was recently revised after synthesis of a biologically active analog of diazonamide A in which a single nitrogen atom was replaced by an oxygen atom. Diazonamide A causes cells to arrest in mitosis, and, after exposure to the drug, treated cells lose both interphase and spindle microtubules. Both diazonamide A and the oxygen analog are potent inhibitors of microtubule assembly, equivalent in activity to dolastatin 10 and therefore far more potent than dolastatin 15. This inhibition of microtubule assembly is accompanied by potent inhibition of tubulin-dependent GTP hydrolysis, also comparable with the effects observed with dolastatin 10. However, the remaining biochemical properties of diazonamide A and its analog differ markedly from those of dolastatin 10 and closely resemble the properties of dolastatin 15. Neither diazonamide A nor the analog inhibited the binding of [3H]vinblastine, [3H]dolastatin 10, or [8-14C]GTP to tubulin. Nor were they able to stabilize the colchicine binding activity of tubulin. These observations indicate either that diazonamide A and the analog have a unique binding site on tubulin differing from the vinca alkaloid and dolastatin 10 binding sites, or that diazonamide A and the analog bind weakly to unpolymerized tubulin but strongly to microtubule ends. If the latter is correct, diazonamide A and its oxygen analog should have uniquely potent inhibitory effects on the dynamic properties of microtubules.     

Novel cyano- and amidino-substituted derivatives of styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines. Synthesis, photochemical synthesis, DNA binding, and antitumor evaluation, part 3

Synthesis of novel cyano- and amidino-substituted styryl-2-benzimidazoles and benzimidazo[1,2-a]quinolines by condensation reactions and photochemical dehydrocyclization and dehydrohalogenation cyclization is described. Thermal denaturation experiments reveal that cyclic derivatives considerably stabilize DNA double helix, while the effect of their acyclic analogues is negligible. According to the spectroscopic study of the interaction of cyclic derivative 19, we propose intercalation of benzimidazo[1,2-a]quinoline moiety into ct-DNA as a dominant interaction underlying biologically relevant effects of this compound, whereas for its acyclic derivative 11, we propose binding into the minor groove of DNA. All compounds show noticeable antiproliferative effect. Morpholino- and chloro-substituted compound 9 is the most active among all acyclic derivatives. All cyclic compounds were 2- to 10-fold more potent, which is correlated with their property to intercalate into DNA. The most active imidazolyl-substituted compound 19 inhibits topoisomerase II and induces strong G2/M cell cycle arrest, pointing to the impairment in mitotic progression. Its pronounced selectivity toward colon carcinoma cells encourages further development of this compound as a lead.     

Decreased sensitivity of isolated hepatocytes from baby rats,from regenerating and from poisoned livers to phalloidin

Summary Isolated hepatocytes, prepared from 5 day old rats, from regenerating livers or from livers after poisoning with carbon tetrachloride, are less sensitive to phalloidin in vitro than hepatocytes from untreated adult controls. The time course of the reduced susceptibility to phalloidin was compared with the ability of hepatocytes to take up bile acids under various conditions. SDS-electrophoresis of cell lysates gave no evidence for decreased levels of actin in cells with reduced sensitivity to phalloidin. In contrast, there was a good relationship between the active uptake of bile acids and the sensitivity of hepatocytes to phalloidin. The decreased response of hepatocytes from baby rats, from regenerating livers or from poisoned livers to phalloidin is more probably related to differences in phalloidin uptake than to a reduced endowment with microfilamentous structures.This work is supported by the Deutsche Forschungsgemeinschaft     

Trypsin protection of hepatocytes against phalloidin

Summary The most typical effect of phalloidin in vitro is the development of protrusions on the surface of hepatocytes within 10–20 min. This mode of cell deformation corresponds to the invaginations of the plasma membrane with subsequent vacuolization, seen in whole livers during phalloidin poisoning. Low concentrations of trypsin delay or prevent this typical response of hepatocytes to phalloidin in a dose dependent manner. The protective effect of trypsin depends on the concentrations of the enzyme and of the toxin, but not markedly on the duration of the preincubation with the enzyme.The protective action of trypsin needs intactness of its active center; it is fully reversible after removal of the enzyme by washing. Therefore protection of hepatocytes by trypsin is not due to solubilization of binding sites for phalloidin.The interpretation of the experimental data suggests that trypsin might induce conformational changes in the membrane structure which prevent or delay the first step of interaction between phalloidin and the membrane surface. Chymotrypsin and elastase were found to be less active in comparison with trypsin. The phalloidin acceptor on the surface of liver cells is unlike actin or myosin, because antiactin and also antimyosin do not influence the response of liver cells to phalloidin treatment.     

Characterization of the activities of actin-affecting drugs on tumor cell migration

Metastases kill 90% of cancer patients. It is thus a major challenge in cancer therapy to inhibit the spreading of tumor cells from primary tumor sites to those particular organs where metastases are likely to occur. Whereas the actin cytoskeleton is a key component involved in cell migration, agents targeting actin dynamics have been relatively poorly investigated. Consequently, valuable in vitro pharmacological tools are needed to selectively identify this type of agent. In response to the absence of any standardized process, the present work aims to develop a multi-assay strategy for screening actin-affecting drugs with anti-migratory potentials. To validate our approach, we used two cancer cell lines (MCF7 and A549) and three actin-affecting drugs (cytochalasin D, latrunculin A, and jasplakinolide). We quantified the effects of these drugs on the kinetics of actin polymerization in tubes (by means of spectrofluorimetry) and on the dynamics of actin cytoskeletons within whole cells (by means of fluorescence microscopy). Using quantitative videomicroscopy, we investigated the actual effects of the drugs on cell motility. Finally, the combined drug effects on cell motility and cell growth were evaluated by means of a scratch-wound assay. While our results showed concordant drug-induced effects on actin polymerization occurring in vitro in test tubes and within whole cells, the whole cell assay appeared more sensitive than the tube assay. The inhibition of actin polymerization induced by cytochalasin D was paralleled by a decrease in cell motility for both cell types. In the case of jasplakinolide, which induces actin polymerization, while it significantly enhanced the locomotion of the A549 cells, it significantly inhibited that of the MCF-7 ones. All these effects were confirmed by means of the scratch-wound assay except of the jasplakinolide-induced effects on MCF-7 cell motility. These later seemed compensated by an additional effect occurring during wound recolonization (possibly acting on the cell growth features). In conclusion, the use of multi-assays with different levels of sophistication and biological relevance is recommended in the screening of new actin-affecting drugs with potentially anti-migratory effects.     

Ca2+ channel inhibition by a new dihydropyridine derivative, S11568, and its enantiomers S12967 and S12968.

Biochemical and electrophysiological techniques were used to describe the Ca2+ channel blocking properties of a new dihydropyridine derivative, S11568 (+/-)- ([(amino-2-ethoxy)-2-ethoxy]methyl)-2-(dichloro-2',3'-phenyl)-4- ethoxy-carbonyl-3-methoxycarbonyl-5-methyl-6-dihydro-1,4-pyridine and its enantiomers S12967 ((+)-S11568) and S12968 ((-)-S11568). In binding studies, S11568 and S12968 displaced specifically bound [3H]PN 200-110 from cardiac and vascular smooth muscle preparations with potencies of 5.6-51 nM, respectively. S12967 was 6- to 18-fold less potent than S12968. A good correlation was found between the IC50 value for the inhibition of 45Ca2+ uptake by A7r5 aortic smooth muscle cells and binding data. Whole-cell patch clamp studies in both guinea-pig ventricular myocytes and A7r5 cells yielded similar results. At holding potential (VH) -50 mV, S12968 inhibited L-type Ca2+ current with an IC50 value near 70 nM, 2- to 3-fold more potently than S11568 and 30-fold more potently than S12967. With VH -100 mV, all three compounds were less potent, with IC50 values ranging from 500 nM to 3 microM. These results demonstrate conclusively that S12968 is the more active enantiomer. Furthermore, the pronounced voltage dependence of its actions in vitro suggests that in vivo it could exhibit good selectivity for vascular smooth muscle over cardiac muscle.     

Structure-activity studies with chiral isomers and with segments of the antimitotic marine peptide dolastatin 10

Eighteen configurational isomers of the antimitotic peptide dolastatin 10 (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990) derived from Dolabella auricularia, together with segments obtained as precursors in its synthesis (Pettit et al., J Am Chem Soc 111: 5463-5465, 1989), were examined as inhibitors of tubulin polymerization and as inhibitors of growth of L1210 murine leukemia cells in culture. Dolastatin 10 consists of four amino acids (in order from the amino terminus: dolavaline, valine, dolaisoleucine, and dolaproine), three unique to D. auricularia, linked to an unusual primary amine (dolaphenine, probably derived from phenylalanine) at what would otherwise be its carboxyl terminus. Dolastatin 10 has nine asymmetric carbon atoms, and available isomers included alternate configurations at five positions (positions 9 and 10 in the dolaproine moiety and positions 18, 19 and 19a in the dolaisoleucine moiety). For tubulin polymerization, only alterations at positions 18 and 19 resulted in loss of inhibitory activity of the isomer. In addition, a tripeptide containing dolavaline, valine and dolaisoleucine with all asymmetric carbons identical configurationally to those in dolastatin 10 was found to be about 30% as effective as dolastatin 10 in inhibiting tubulin polymerization. Cytotoxic effects were much more sensitive to alterations in the dolastatin 10 structure. The only modification which did not lead to reduced cytotoxicity was reversal of configuration at position 19a in the dolaisoleucine moiety. Both this isomer and dolastatin 10 had IC50 values of less than 1 nM. Several other isomers had IC50 values with the L1210 cells in the range of 30-90 nM, but these did not correlate well with their inhibitory effects on tubulin polymerization. The tripeptide effective as an inhibitor of tubulin polymerization had no activity against the L1210 cells.     

Design, synthesis, molecular modeling studies, and calpain inhibitory activity of novel alpha-ketoamides incorporating polar residues at the P1'-position

A series of novel alpha-ketoamides incorporating stereoisomeric residues with different electronic properties at the P(1)'-position were synthesized to study the electronic requirements for inhibitor binding to the S(1)'-subsite of calpain I. The results of the study suggested the presence of an acidic amino acid residue at the S(1)'-subsite of calpain I. For example, ester 1a (Cbz-l-Leu-l-Phe-CO-d-Phe-OMe) was over 450-fold more potent than its carboxylic acid derivative 2a (Cbz-l-Leu-l-Phe-CO-d-Phe-OH). Additionally, amidino derivative 3a (Cbz-l-Leu-l-Phe-CONH-d-CH[C(NH)NH(2)]Bn) was about 6000-fold more potent than 2a. Furthermore, 4a (Cbz-l-Leu-l-Phe-CONHCH(2)Bn) was 12-fold less potent than its aza analogue 4b (Cbz-l-Leu-l-Phe-CONHNHBn). The results are consistent with the presence of an acidic amino acid residue at the S(1)'-subsite of calpain I. The acidic amino acid residue was found to be Glu261 via molecular modeling studies.     

Dolastatin 10, a powerful cytostatic peptide derived from a marine animal. Inhibition of tubulin polymerization mediated through the vinca alkaloid binding domain

Dolastatin 10, a cytostatic peptide containing several unique amino acid subunits, was isolated from the marine shell-less mollusk Dolabella auricularia (Pettit GR, Kamano Y, Herald CL, Tuinman AA, Boettner FE, Kizu H, Schmidt JM, Baczynskyj L, Tomer KB and Bontems RJ, J Am Chem Soc 109: 6883-6885, 1987). Since our preliminary studies demonstrated that dolastatin 10 inhibited tubulin polymerization and the binding of radiolabeled vinblastine to tubulin, an initial characterization of the properties of dolastatin 10 included a comparison to other antimitotic drugs interfering with vinca alkaloid binding to tubulin (vinblastine, maytansine, rhizoxin, and phomopsin A). Dolastatin 10 inhibited the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 microM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 microM for dolastatin 10, 1.4 microM for phomopsin A, 1.5 microM for vinblastine, 3.5 microM for maytansine, and 6.8 microM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule-associated proteins. Preliminary studies indicated that dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. We confirmed that rhizoxin, phomopsin A, and maytansine also inhibit the binding of radiolabeled vinblastine and vincristine to tubulin. Dolastatin 10 and phomopsin A were the strongest inhibitors of these reactions, and rhizoxin the weakest. Dolastatin 10, phomopsin A, maytansine, vinblastine, and rhizoxin all inhibited tubulin-dependent GTP hydrolysis. The greatest inhibition of hydrolysis was observed with dolastatin 10 and phomopsin A, and the least inhibition with rhizoxin.     

An antimicrotubule agent, TZT-1027, does not induce neuropathologic alterations which are detected after administration of vincristine or paclitaxel in animal models

One of the major dose-limiting toxicities induced by antimicrotubule antitumor agents such as vinca alkaloids and taxanes is peripheral neuropathy. The neurotoxicity of TZT-1027 (a dolastatin 10 derivative antimicrotubule agent) was thus assessed using the animal models for antimicrotubule agent-induced neurotoxicity. Rabbits were intravenously given TZT-1027 or vincristine weekly for 5 weeks. In the mouse study, TZT-1027, vincristine or paclitaxel was intravenously given every 2 days and/or weekly. Despite the neuropathologic evidence such as myelinated axonal and fiber degeneration in the peripheral nerves and in the sensory tracts of the spinal cord following the treatment with vincristine or paclitaxel, no drug-induced alteration was observed in the TZT-1027 groups. Although there are reports that some other dolastatin derivatives with antimicrotubule activity showed no neurotoxic potential in humans, the present study represents the first demonstration in experimental animals that a dolastatin derivative has no, or at least a lower, neurotoxic potential compared to other antimicrotubule agents.     

Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluorocarbocyclic 2'-deoxy guanosines.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.     

Activity of a novel antitumor agent, TZT-1027

We studied the antitumor activity of newly synthesized dolastatin 10 analogs. TZT-1027 showed remarkable activity and was selected for further development. TZT-1027 was found to inhibit the assembly of porcine brain microtubule proteins and to depolymerize the polymerized microtubule proteins. Therefore, its mechanism of antitumor activity seems to be at least partially ascribed to the inhibition of microtubule assembly. We further studied the binding site of TZT-1027 on tubulin. Scatchard analysis of 3H-TZT-1027 binding data suggested two binding sites including a high affinity site and a low affinity site. TZT-1027 affected the binding of vinblastine (VBL) on tubulin but its binding site isn't identical to the VBL binding site. TZT-1027 induced apoptosis within 24 h, not only in human leukemia cells such as HL-60, but also in solid tumors such as human prostate carcinoma cells DU145 and human mammary carcinoma cells MCF-7. TZT-1027 showed good antitumor activity against human xenografts (MX-1 and LX-1) without causing serious body weight reduction, which resulted in tumor regression. We examined the effect of TZT-1027 on the established tumor vasculature using the dye perfusion into tumor. TZT-1027 exhibited considerable antivascular activity in tumor sections in addition to excellent cytotoxic effect.     

A possible reason for the phalloidin tolerance of hepatoma cells

Summary In contrast to normal liver cells, AS-30D hepatoma cells are insensitive to phalloidin. The lack of the typical phalloidin response in the latter cells is not due to a deficiency of contractile proteins. Actin prepared from hepatoma cells is able to form filamentous structures and is stabilized in a manner similar to muscle actin.Isolated liver cells were exposed to a medium containing phalloidin and removed after 20 min by centrifugation. The supernatant was inculated again with fresh cells. The procedure was repeated four times. The phalloidin response decreased to about 19% of the control because of the uptake of phalloidin during each incubation. When the same procedure was carried out with AS-30D hepatoma cells, and aliquots of the supernatants were tested with hepatocytes no marked decrease of the phalloidin response was seen. This indicates that hepatoma cells do not consume the toxin as do normal liver cells.This work was supported by the Deutsche Forschungsgemeinschaft     

Synthesis, in vitro pharmacology, and molecular modeling of very potent tacrine-huperzine A hybrids as acetylcholinesterase inhibitors of potential interest for the treatment of Alzheimer's disease.

Eleven new 12-amino-6,7,10,11-tetrahydro-7, 11-methanocycloocta[b]quinoline derivatives [tacrine (THA)-huperzine A hybrids, rac-21-31] have been synthesized as racemic mixtures and tested as acetylcholinesterase (AChE) inhibitors. For derivatives unsubstituted at the benzene ring, the highest activity was obtained for the 9-ethyl derivative rac-20, previously prepared by our group. More bulky substituents at position 9 led to less active compounds, although some of them [9-isopropyl (rac-22), 9-allyl (rac-23), and 9-phenyl (rac-26)] show activities similar to that of THA. Substitution at position 1 or 3 with methyl or fluorine atoms always led to more active compounds. Among them, the highest activity was observed for the 3-fluoro-9-methyl derivative rac-28 [about 15-fold more active than THA and about 9-fold more active than (-)-huperzine A]. The activity of some THA-huperzine A hybrids (rac-19, rac-20, rac-28, and rac-30), which were separated into their enantiomers by chiral medium-pressure liquid chromatography (chiral MPLC), using microcrystalline cellulose triacetate as the chiral stationary phase, showed the eutomer to be always the levorotatory enantiomer, their activity being roughly double that of the corresponding racemic mixture, the distomer being much less active. Also, the activity of some of these compounds inhibiting butyrylcholinesterase (BChE) was tested. Most of them [rac-27-31, (-)-28, and (-)-30], which are more active than (-)-huperzine A as AChE inhibitors, turned out to be quite selective for AChE, although not so selective as (-)-huperzine A. Most of the tested compounds 19-31 proved to be much more active than THA in reversing the neuromuscular blockade induced by d-tubocurarine. Molecular modeling of the interaction of these compounds with AChE from Torpedo californica showed them to interact as truly THA-huperzine A hybrids: the 4-aminoquinoline subunit of (-)-19 occupies the same position of the corresponding subunit in THA, while its bicyclo[3.3.1]nonadiene substructure roughly occupies the same position of the corresponding substructure in (-)-huperzine A, in agreement with the absolute configurations of (-)-19 and (-)-huperzine A.     

Discovery and Optimization of 1,3,4-Trisubstituted-pyrazolone Derivatives as Novel, Potent, and Nonsteroidal Farnesoid X Receptor (FXR) Selective Antagonists

LBVS of 12480 in-house compounds, followed by HTRF assay, resulted in one nonsteroidal compound (11) with antagonistic activity against FXR (69.01 ± 11.75 μM). On the basis of 11, 26 new derivatives (12a-z) were designed and synthesized accordingly. Five derivatives (12f-g, 12p, 12u, and 12y) showed better antagonistic activities against FXR than compound 11. Remarkably, the most potent derivative, 12u (8.96 ± 3.62 μM), showed antagonistic capability approximately 10 times and 8-fold higher than that of the control (GS) and the starting compound 11, respectively. 12u was further confirmed to have high binding affinity with FXRαLBD, FXR specificity over six other nuclear receptors, and potent antagonistic activity against FXR in two cell testing platforms. 12u strongly suppressed the regulating effects of CDCA on FXR target genes. The therapeutic potential of 12u was identified by lowering the contents of triglyceride and cholesterol in human hepatoma HepG2 cells and in the cholesterol-fed C57BL/6 mices.     

Tailored Cyclodextrin Pore Blocker Protects Mammalian Cells from Clostridium difficile Binary Toxin CDT

Some Clostridium difficile strains produce, in addition to toxins A and B, the binary toxin Clostridium difficile transferase (CDT), which ADP-ribosylates actin and may contribute to the hypervirulence of these strains. The separate binding and translocation component CDTb mediates transport of the enzyme component CDTa into mammalian target cells. CDTb binds to its receptor on the cell surface, CDTa assembles and CDTb/CDTa complexes are internalised. In acidic endosomes, CDTb mediates the delivery of CDTa into the cytosol, most likely by forming a translocation pore in endosomal membranes. We demonstrate that a seven-fold symmetrical positively charged β-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin, which was developed earlier as a potent inhibitor of the translocation pores of related binary toxins of Bacillus anthracis, Clostridium botulinum and Clostridium perfringens, protects cells from intoxication with CDT. The pore blocker did not interfere with the CDTa-catalyzed ADP-ribosylation of actin or toxin binding to Vero cells but inhibited the pH-dependent membrane translocation of CDTa into the cytosol. In conclusion, the cationic β-cyclodextrin could serve as the lead compound in a development of novel pharmacological strategies against the CDT-producing strains of C. difficile.