No abstract

No abstract

No abstract

The bryostatins are macrocyclic lactones that represent an additional structural class of potent activators of protein kinase C. These marine animal biosynthetic products are of unusual interest because they induce only a subset of the biological responses induced by the phorbol esters. We have now determined the binding affinities of naturally occurring and semisynthetic bryostatins for protein kinase C by competition analysis with [26-3Hlbryostatin 4 as the radioactive ligand. Esterification of the hydroxyl group at C26 caused dramatic loss of activity as did inversion of the asymmetric center at this position. In contrast, neither of the ester groups at C7 and C20 had a major influence on activity. Computer modeling of the phorbol esters, related diterpenes, and indole alkaloids suggested that the C20, C9, and C4 oxygens of phorbol represented critical elements of the phorbol ester pharmacophore. The C26 oxygen of the bryostatins, together with the C1 and C19 oxygens, gave an excellent spatial correlation with this model, with a root-mean-square deviation of 0.16 A (compared to 0.10-0.35 A among phorbol-related diterpenes). The extension of the phorbol ester pharmacophore model to the bryostatins and its agreement with the structureactivity relations for the bryostatin class of compounds provide additional support for the validity of the model. Protein kinase C mediates one arm of the signal-transduction pathway proceeding through inositol phospholipid breakdown (1,2). This pathway is directly involved in the action of a broad range of cellular effectors, including growth factors and oncogenes, and indirectly affects other transduction pathways such as that of the cyclic AMP second-messenger system. Protein kinase C is proposed to be activated by 1,2-diacyl-sn-glycerol compounds and by certain exogenous analogs such as the phorbol ester tumor promoters (3, 4). In addition to the phorbol esters and related diterpenes, a number of structurally distinct natural products have been identified that also bind to protein kinase C with high affinity and that resemble the phorbol esters in their actions at nanomolar concentrations in biological systems. These compounds include the indole alkaloids such as teleocidin and the polyacetates such as aplysiatoxin (5).Recently, considerable attention has focused on an unusual class of potent activators of protein kinase C, the bryostatins. These compounds are macrocyclic lactones isolated from Bugula neritina and other marine bryozoans on the basis of antineoplastic activity against the P388 leukemia cell system (6). The bryostatins at nanomolar concentrations inhibit phorbol ester binding to protein kinase C and stimulate enzymatic activity in vitro to a comparable degree as do the phorbol esters (7-9). Biologically, however, the bryostatins induce only a subset of the typical phorbol ester responses. Moreover, the bryostatins block, in an apparently noncompetitive fashion, the action of the phorbol esters on those responses that they themselves do not induce. Exampl...

Bryostatin 1, like the phorbol esters, activates protein kinase C. However, bryostatin 1 induces only some of the effects in cultured cells which result from phorbol ester treatment, whereas it blocks other responses to the phorbol esters. In mouse keratinocytes in particular, bryostatin 1 induces ornithine decarboxylase, a marker of proliferation, but blocks induction of markers of differentiation. Because of the postulated role of induction of differentiation in tumor promotion, we have now examined bryostatin 1 as a tumor promoter and as an inhibitor of phorbol ester tumor promotion in the initiation-promotion model of skin carcinogenesis. After initiation with 7,12-dimethylbenz[a]anthracene, weekly topical treatments of the backs of mice with 1 microgram (1.1 nmol) bryostatin 1 induced epidermal hyperplasia and inflammation, although not to the extent seen after treatment with the promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment with bryostatin 15 min before each TPA exposure reduced the phorbol ester-induced hyperplasia. Bryostatin 1 was ineffective as a complete tumor promoter and displayed very weak activity as a second stage promoter upon treatment of initiated mice for 30 weeks. Combined exposure of mice to bryostatin 1 and TPA resulted in a substantial inhibition of promotion by TPA. Our in vivo results extend earlier in vitro findings that bryostatin 1 acts as a partial inhibitor of protein kinase C function.

No abstract

The highly cytotoxic, sponge-derived, antimitotic macrolide polyether spongistatin 1 has been previously shown to inhibit microtubule assembly, the binding of vinblastine and GTP to tubulin, and displacement of GDP bound in the exchangeable site of tubulin. We have now examined in detail inhibition by spongistatin 1 of both [3H]vinblastine and [3H]dolastatin 10 binding to tubulin. We found spongistatin 1 to be a noncompetitive inhibitor of the binding of both radiolabeled drugs to tubulin, in contrast to competitive patterns obtained with vincristine versus [3H]vinblastine and with a chiral isomer of dolastatin 10 versus [3H]dolastatin 10. Since dolastatin 10 is itself a noncompetitive inhibitor of vinca alkaloid binding to tubulin, this implies at least three distinct binding sites for the structurally complex and diverse natural products that interfere with each others binding to tubulin and with nucleotide exchange. Spongistatin 1, in contrast to both vinca alkaloids and peptide antimitotic agents like dolastatin 10, does not induce formation of a GTP-independent, morphologically distinctive polymer ("aggregate"). We also examined eight compounds closely related structurally to spongistatin 1 (spongistatins 2-9). The most distinctive in their properties were spongistatins 6 and 8. These two compounds, despite activity comparable to spongistatin 1 as inhibitors of tubulin polymerization and [3H]vinblastine binding, had much reduced activity as inhibitors of nucleotide exchange and [3H]dolastatin 10 binding. Spongistatins 1 and 6 were compared for effects on dolastatin 10-induced aggregate formation in conjunction with effects on [3H]dolastatin 10 binding. Spongistatin 6 was about 4-fold less active than spongistatin 1 as an inhibitor of aggregation and over 20-fold less active as an inhibitor of dolastatin 10 binding.

No abstract