Dibefurin, a novel fungal metabolite inhibiting calcineurin phosphatase activity.

The novel calcineurin inhibitor, dibefurin, has been isolated from the fungal culture AB 1650I-759. The isolation was bioactivity-directed fractionation using an assay which measures the phosphatase activity of calcineurin. The compound was purified by countercurrent, reverse phase and gel filtration chromatographies. Several studies, including crystallographic, NMR and MS, revealed that dibefurin is a novel dimeric compound of a unique structural type.

Simple aromatics identified with a NFAT-lacZ transcription assay for the detection of immunosuppressants.

Determination of the mechanism of action of FK506 and cyclosporin A has yielded new molecular targets involved in signal transduction during T cell activation. A common target of FK506 and cyclosporin A is inhibition of activation of the NFAT transcription factor, for which a specific binding region is present in the promoter of the IL-2 gene. A reporter gene assay has been used to screen for agents that interfere with this early step in T cell activation. Simple aromatic compounds that block NFAT-dependent transcription and show in vitro immunosuppressive activity were isolated from the broth and mycelia of two Streptomyces sp. fermentations. The compounds were active at concentrations that were not directly cytotoxic.

Aselacins, novel compounds that inhibit binding of endothelin to its receptor. I. The producing organism, fermentation and biological activity.

A radioligand test to detect inhibitors of endothelin-1 binding to its receptors in bovine atrial and porcine cerebral membranes was used to screen fungal metabolites from stationary fermentations. Inhibitory activity, observed in culture extracts of two Acremonium species, led to the discovery of aselacins A, B and C. Aselacin A inhibits binding to both membrane fractions with IC50s of approximately 20 micrograms/ml.

MLR-52, (4'-demethylamino-4',5'-dihydroxystaurosporine), a new inhibitor of protein kinase C with immunosuppressive activity.

In the course of screening with the mixed lymphocyte reaction, a new inhibitor of protein kinase C with immunosuppressive activity was isolated from the fermentation broth and mycelia of Streptomyces sp. AB 1869R-359. Although certain similarities exist, this strain is morphologically and physiologically distinct from other reported producers of staurosporine-related compounds. We have found that this strain produces relatively high levels of staurosporine and the new minor compound MLR-52, which possesses the indolo[2,3-a]carbazole chromophore of staurosporine, but differs in the substitution pattern of the sugar moiety. Their structures have been elucidated by mass and NMR spectra. MLR-52 has been shown to inhibit the enzymatic activity of protein kinase C and the murine mixed lymphocyte reaction.

In vivo characterization of a phosphoramidon-sensitive endothelin-converting enzyme in the rat.

Experiments were conducted to characterize the nature of a phosphoramidon-sensitive endothelin-converting enzyme in vivo by evaluating the pressor response to a bolus intravenous (i.v.) injection of endothelin family peptides following administration of phosphoramidon in anesthetized Sprague-Dawley rats. Phosphoramidon given i.v. at 10 mg/kg completely prevented the pressor response to human big endothelin-1-(1-38) (big ET-1). The EC50 for phosphoramidon was determined to be in the range of 1 to 3 mg/kg. The pressor response to big ET-1 60 min after phosphoramidon injection was attenuated by roughly 60% indicating a long inhibitory half-life. Very high doses of big ET-1 (> 20 mg/kg) were capable of over-riding the effect of phosphoramidon and produced characteristic pressor responses suggesting that the inhibition by phosphoramidon can be considered competitive in nature. Human big endothelin-3-(1-41) (big ET-3) produced significant increases in arterial pressure although with less potency and efficacy compared to big ET-1. The pressor response to big ET-3 was also inhibited by phosphoramidon. Phosphoramidon does not act indirectly by interfering with ET-1 receptor-mediated actions since the inhibitor has no effect on the in vivo pressor response to ET-1 and does not antagonize [125I]ET-1 receptor binding or constrictor responses in vitro. These results are consistent with the idea that a phosphoramidon-sensitive endothelin-converting enzyme is capable of cleaving both big ET-1 and big ET-3 to the active peptides in the rat.

A colorimetric microassay for the detection of agents that interact with DNA.

A simple microtiter assay for the detection of compounds that bind DNA is described. Agents that displace methyl green from DNA are detected spectrophotometrically by a decrease in absorbance at 630 nm. The feasibility of using the assay for detecting DNA-active compounds in fermentation extracts was assessed, and the activities of reference compounds in the methyl green assay and an ethidium bromide displacement method were compared.

High-level expression of recombinant human FK-binding protein from a fusion precursor.

The human peptidyl-prolyl isomerase FK-binding protein (FKBP) was cloned as a fusion partner with CMP-KDO synthetase (CKS), and the resultant construct was characterized as an improved high-expression source for FKBP. The CKS-FKBP fusion was expressed as a soluble protein at levels approaching 1 gm/L in Escherichia coli fermentations. The fusion protein was purified to near homogeneity by a one-step ammonium sulfate fractionation of whole cell lysate. After selective cleavage, the fusion precursor produced yields approaching 300 mg of purified FKBP per liter of harvested culture, a approximately 30 to 60-fold increase over that observed for a nonfusion construct. Selective cleavage of the fusion partners was accomplished using either hydroxylamine or specific, limited proteolysis. Once separated from the CKS fusion partner, the FKBP was isolated in a single step by either reversed-phase HPLC or chromatography on Q-Sepharose. For comparison of physical and chemical properties, a nonfusion construct of recombinant human FKBP was expressed in E. coli and isolated. The purified FKBPs exhibited expected SDS-PAGE molecular weights and N-terminal sequences. The proteins had similar proton NMR spectra and binding to [3H]FK-506. The fusion construct, CKS-FKBP, was also found to bind [3H]FK-506. These data indicate that FKBP fused to the C-terminus of CKS folds independently of the fusion partner and suggests the fused FKBP adopts a conformation resembling that of the native protein.

Dunaimycins, a new complex of spiroketal 24-membered macrolides with immunosuppressive activity. III. Immunosuppressive activities of dunaimycins.

The immunosuppressive effects of the dunaimycins, a new complex of spiroketal 24-membered macrolides, were compared to cyclosporin A, ascomycin, and rapamycin. Each dunaimycin was a potent inhibitor of the mitogenic response observed in mixed murine splenocyte or human leukocyte cultures, and like immunosuppressive drugs these compounds were relatively less potent inhibitors of the constitutive proliferation of murine EL4 thymoma cells. Dunaimycin D4S showed no selectivity in inhibiting the mitogenic response of spleen cells to concanavalin A, pokeweed mitogen, lipopolysaccharide, or phytohemagglutinin. Cyclosporin A and ascomycin did not inhibit interleukin 2 dependent proliferation, whereas the dunaimycins and rapamycin blocked the uptake of [3H]thymidine in mixed cultures supplemented with exogenous interleukin 2. In addition, dunaimycin D4S had no apparent affinity for cyclosporin A or FK-506 immunophilins. Although the dunaimycins inhibited the activity of Na+, K(+)-ATPase, inhibition of this enzyme appeared insufficient to explain the biological activity of these new macrolides. Over a narrow concentration range, dunaimycin D4S showed in vivo immunosuppressive activity in the murine popliteal lymph node hyperplasia model.

Antitumor activity and biochemical effects of topsentin.

Topsentin, a bis(indolyl)imidazole marine natural product, inhibited the proliferation of cultured human and murine tumor cells at micromolar concentrations (IC50 values ranged from 4 to 40 microM) and was active against in vivo P388 leukemia (%T/C = 137, 150 mg/kg, QD1-5) and B16 melanoma (%T/C = 144, 37.5 mg/kg, QD1-9) tumors. Effects of 30 microM topsentin (1-hr exposures) on incorporation of radiolabeled precursors by P388 cells indicated inhibition of DNA synthesis (91%) and to a lesser extent RNA synthesis (57%), whereas synthesis of protein was unaffected (0%). Fluorescence spectral changes and competitive binding experiments with ethidium bromide indicated that topsentin interacted with DNA. No evidence for intercalation was observed in DNA unwinding studies, but competitive binding experiments with Hoechst 33342 and CC-1065 indicated that topsentin bound to DNA in the minor groove.

Preliminary characterization of a cloned neutral isoelectric form of the human peptidyl prolyl isomerase cyclophilin.

We report the cloning of a neutral isoelectric form of the human peptidyl prolyl isomerase, cyclophilin, its expression in Escherichia coli, and its purification and comparison to bovine thymus cyclophilin. The cloned protein exhibited a pI of approximately 7.8 and formed a simple 1:1 complex with cyclosporin A. This cloned form had a pI similar to that observed for the neutral isoform (pI approximately 7.4) of human splenocyte cyclophilin. The bovine thymus proteins exhibited anomalous behavior on CM-cellulose chromatography but were resolved into alkaline (pI approximately 9.3) isoforms and a new neutral (pI approximately 7.8) isoform by isoelectric focusing gel electrophoresis and ultimately into at least four discrete isoforms by capillary electrophoresis. For cyclosporin A binding we observe a Kd of approximately 160 nM for an electrophoretically heterogeneous preparation of the natural bovine protein and approximately 360 nM for the more homogeneous preparation of the cloned human neutral isoform. Stopped-flow measurements of the activation energies for peptidyl-prolyl isomerase activity indicate the recombinant human protein has an activation enthalpy of 3.67 kcal/mol and an activation entropy of -47.3 cal/K-mol for cis----trans isomerization.

Antitumor activity and nucleic acid binding properties of dercitin, a new acridine alkaloid isolated from a marine Dercitus species sponge.

A new cytotoxic acridine alkaloid that exhibited antitumor activity in vivo was isolated from a marine Dercitus species sponge collected at a depth of 160 m in the Bahamas. This violet alkaloid, designated dercitin, inhibited the proliferation of cultured murine and human leukemia, lung, and colon tumor cells at nM concentrations (IC50 values of 63-150 nM) and prolonged the life of mice bearing ascitic P388 tumors (%T/C = 170, 5 mg/kg, i.p., QD1-9). Dercitin was also active against i.p. B16 melanoma and modestly inhibited the growth of s.c. Lewis lung carcinoma on the same schedule. DNA blocked the antiproliferative effects of the agent in culture, and incorporation studies indicated that dercitin disrupted DNA and RNA synthesis with less effects on protein synthesis, similar to the effects of known DNA intercalators. After 1-h exposure to 400 nM dercitin, the rates of incorporation of [3H]uridine, [3H]thymidine, and [3H]leucine by cultured P388 cells were inhibited 83, 61, and 23%, respectively. Equilibrium dialysis indicated that dercitin bound calf thymus DNA with an affinity of 3.1 microM and maximal binding of 0.20 mol dercitin/mol base pair. Binding involved intercalation as evidenced by ability to relax supercoiled phi X174 DNA (half maximal concentration for dercitin relaxation was 36 nM). The effects of dercitin on DNA mobility were reversible, and complete relaxation of DNA with topoisomerase I in the presence of dercitin followed by phenol extraction resulted in the appearance of supercoiled DNA. Dercitin, at microM concentrations, had a small effect in the K+-sodium dodecyl sulfate assay using cultured P388 cells, suggesting minimal inhibition of topoisomerase activity. But, dercitin completely inhibited DNA polymerase I/DNase nick translation of DNA at 1 microM. Relaxation of DNA at a given concentration was greater than inhibition of nick translation suggesting that the effects of dercitin on enzyme activity were secondary to changes in DNA conformation. Results indicate that dercitin is a new marine natural product that probably exerts its biological effects through intercalation into nucleic acids.

Antitumor activity and mechanism of action of the novel marine natural products mycalamide-A and -B and onnamide.

Three novel heterocyclic compounds, mycalamide-A and -B and onnamide, were isolated from Mycale sp. and Theonella sp. sponges collected in New Zealand and Okinawan waters. Each exhibited potent in vitro toxicity and in vivo efficacy against murine and human tumor cells. Concentrations of each that inhibited replication of cultured murine lymphoma P388 cells by 50% were 5 nM or less. Mycalamide-A and -B were also potent inhibitors of HL-60, HT-29, and A549 human tumor cell replication (50% inhibitory concentration less than 5 nM), while values for onnamide were greater (50% inhibitory concentrations between 25 and 200 nM). Mycalamide-A (10 micrograms/kg) and -B (2.5 micrograms/kg) were moderately active against P388 leukemia (increase in life span, approximately 50%), while onnamide was inactive (40 micrograms/kg; increase in life span, 15%). Mycalamide-A was also active against B16 melanoma, Lewis lung carcinoma, M5076 ovarian sarcoma, colon 26 carcinoma, and the human MX-1, CX-1, and Burkitt's lymphoma tumor xenografts. Mechanism of action studies indicate that the three agents inhibited protein synthesis. For example, after 1-h exposures to 20 nM mycalamide-A and -B, the rates of [3H]leucine incorporation into acid-precipitable material of cultured P388 cells were inhibited 54 and 99%, while the effects on incorporation of [3H]uridine and [3H]thymidine were less. The relative effects of 20 to 2000 nM mycalamide-A on protein, RNA, and DNA synthesis were consistent with those observed during exposure of P388 cells to 1 microM emetine, a known inhibitor of protein synthesis. Also, the three agents inhibited translation of RNA into protein in a cell-free lysate of rabbit reticulocytes. Although mycalamide-A disrupted DNA metabolism, the agent apparently did not intercalate into DNA, and a mixture of four deoxynucleosides (250 microM each) did not decrease the antiproliferative effects of the agent. Collectively, these data indicate that this class of compounds represents novel antitumor agents which should be further evaluated to define their potential.

A mammalian cell agar-diffusion assay for the detection of toxic compounds.

A method using murine P-388 leukemia or human HT-29 colon carcinoma cells was developed for the bioautography of potential antitumor agents. Of 18 cancer chemotherapeutic drugs and natural products tested, all were detected by toxicity at 0.01 or 1.0 micrograms with P-388 cells, and 11 of the 18 were detected at 10 micrograms or less with HT-29 cells. Bioautography of a crude extract of Pseudoplexaura wagenaari and subsequent purification yielded the known compound crassin acetate. With modification, the assay detected specifically toxic DNA-binding agents.

Comparison of Coulter volumes with radiometrically determined intracellular water volumes for cultured cells.

During methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells, proliferation is inhibited, morphologic and biochemical changes occur, and giant, often multinucleated, cells form. We have used the increase in cell volume as a marker of the mature syncytiotrophoblastlike phenotype. Uninduced and differentiated BeWo cells are not spherical, and theoretical considerations suggested that deviations in shape could result in significant errors in Coulter volume. To determine if the values obtained by electrical pulse sizing reflected the actual mass of BeWo cells, we have evaluated the relationship between Coulter volumes and intracellular water volumes obtained using a shape-independent estimate for eight cell types. A close correlation (r2 = 0.97) was found, indicating that cell volume changes in populations of irregularly shaped cells can be accurately measured using a Coulter instrument.

Evidence of multifactorial mechanisms in an adriamycin-resistant HL-60 promyelocytic leukemia cell line.

HL-60/AR leukemia cells, which were 60-fold resistant to the growth inhibitory activity of adriamycin, remained sensitive to the antiproliferative and differentiation-inducing activities of aclacinomycin A. The replication of HL-60/AR and of adriamycin sensitive parental HL-60 cells was inhibited by greater than 80% by 30 nM aclacinomycin A and the majority of cells (about 60 to 70%) of each line underwent granulocytic differentiation when treated with this agent, as assessed by the reduction of nitroblue tetrazolium. Measurement of the initial rates of uptake of daunorubicin and steady-state levels of adriamycin in sensitive and resistant lines indicated that transport differences do not fully account for the insensitivity of HL-60/AR cells to these anthracyclines. Furthermore, 30-fold greater levels of cell-associated adriamycin were required in HL-60/AR cells for toxic effects equivalent to those occurring in parental HL-60 cells. Analysis of DNA histograms of adriamycin treated HL-60 cells indicated that cell-cycle progression was blocked in G2-M, while this antibiotic blocked progression of resistant HL-60/AR cells in the S phase. These results suggest that, in addition to alterations in membrane permeability, differential sensitivity of multiple biochemical targets may be important in the toxicity and the development of resistance to anthracyclines. Furthermore, the finding that HL-60/AR cells do not exhibit cross-resistance to aclacinomycin A indicates that this oligosaccharide-containing anthracycline may have utility in the treatment of adriamycin resistant neoplasms.

Inhibition of methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells by thymidine.

During exposure to methotrexate, cultured human choriocarcinoma (BeWo) cells stop proliferating, enlarge, and undergo a complex differentiative response that resembles in utero development of quiescent syncytiotrophoblasts. In the present work, complete inhibition of proliferation and maximal cell enlargement required exposure to 1 microM methotrexate, whereas colony-forming ability, determined after transfer of cells to drug-free medium, was unaffected over a wide range of concentrations (10(-12)-10(-5) M). BeWo cells were sensitive to the antifolate effects of methotrexate since thymidylate synthase activity and incorporation of [14C]formate into DNA, RNA, and protein were reduced by greater than 90% after short drug exposures, and progression of cells through S phase of the cell cycle was blocked by prolonged drug exposures. When methotrexate was coadministered with hypoxanthine and thymidine or leucovorin, its antiproliferative and differentiative effects were blocked. When methotrexate was coadministered with either hypoxanthine or thymidine, its antiproliferative activity was unaffected, whereas expression of syncytiotrophoblastic markers was blocked in the presence of thymidine but not in the presence of hypoxanthine. Exposure of BeWo cells to fluorodeoxyuridine also stimulated cell enlargement and expression of syncytiotrophoblastic markers, and these effects were blocked by coadministration of thymidine. Thus BeWo cells, which were sensitive to the antifolate effects of methotrexate, were not killed during cytostasis but instead entered a reversible differentiated state, apparently resulting from thymidylate starvation and consequent inhibition of DNA synthesis.

Density-dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells.

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug-induced conversion of BeWo cells from the cytotrophoblastlike to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of "giant" cells, a process shown to require exogenous purines, and expression of placental (heat-stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate-induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two-threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate-induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate-induced differentiation of BeWo cells was inhibited in a density-dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.

The effects of hypoxanthine on methotrexate-induced differentiation of cultured human choriocarcinoma (BeWo) cells.

When cultured human choriocarcinoma (BeWo) cells are exposed to methotrexate, proliferation ceases and cells undergo a complex differentiative response that resembles development of normal trophoblast. Although thymidylate starvation has been shown to be causative in methotrexate-induced expression of syncytiotrophoblastic markers by BeWo cells, the role of purine deprivation is uncertain since previous studies utilized growth media containing exogenous purines. This work investigated the effects of hypoxanthine on methotrexate-induced cell enlargement, expression of placental alkaline phosphatase, and morphological differentiation to the syncytiotrophoblast-like phenotype. When methotrexate exposures (1 microM, 48 h) were conducted in a purine-free basal medium supplemented with dialyzed fetal bovine serum, RNA synthesis was greatly reduced and cell enlargement did not occur. Specific methods for removing purines (charcoal extraction and xanthine oxidase treatment) decreased the ability of serum to support cell enlargement during methotrexate exposures, whereas addition of hypoxanthine to culture fluids restored its ability to support maximal increases in cell mass, confirming that purines were the factors lost during dialysis. In contrast, morphologically differentiation to the syncytiotrophoblast-like phenotype and increased expression of placental alkaline phosphatase were unaffected by the availability of purines during exposure to methotrexate.