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.
Although the search for disease biomarkers continues, the clinical return has thus far been disappointing. The complexity of the body's response to disease makes it difficult to represent this response with only a few biomarkers, particularly when many are present at low levels. An alternative to the typical reductionist biomarker paradigm is an assay we call an "immunosignature." This approach leverages the response of antibodies to diseaserelated changes, as well as the inherent signal amplification associated with antigen-stimulated B-cell proliferation. To perform an immunosignature assay, the antibodies in diluted blood are incubated with a microarray of thousands of random sequence peptides. The pattern of binding to these peptides is the immunosignature. Because the peptide sequences are completely random, the assay is effectively disease-agnostic, potentially providing a comprehensive diagnostic on multiple diseases simultaneously. To explore the ability of an immunosignature to detect and identify multiple diseases simultaneously, 20 samples from each of five cancer cohorts collected from multiple sites and 20 noncancer samples (120 total) were used as a training set to develop a reference immunosignature. A blinded evaluation of 120 blinded samples covering the same diseases gave 95% classification accuracy. To investigate the breadth of the approach and test sensitivity to biological diversity further, immunosignatures of >1,500 historical samples comprising 14 different diseases were examined by training with 75% of the samples and testing the remaining 25%. The average accuracy was >98%. These results demonstrate the potential power of the immunosignature approach in the accurate, simultaneous classification of disease.cancer diagnostic | immunodiagnostic | antibody biomarker | peptide microarray
The Republic of Maldives' black marine sponge Hyrtios erecta has been found to contain three cancer cell-line inhibitory pentacyclic sesterterpenes designated sesterstatins 1-3 (2-4). One of the sesterterpenes, sesterstatin 2, specifically inhibited the Gram-positive opportunist Staphylococcus aureus. All three of the P-388 lymphocytic-leukemia-active (ED50 0.46 to 4.3 micrograms/mL) sesterstatins were obtained in trace quantities (3.0 x 10-7 to 5.4 x 10-7% yields) and represent structural variations on the more usual scalarin-type porifera sesterterpenes. The structures were elucidated by highfield (500 MHz) 2D NMR techniques augmented by HRMS results.
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