Studies of Bioactive Natural Products and Mechanism-Based Bioassays

Abstract

An extract of the sponge <i>Rhabdastrella globostellifera</i> was active in an assay measuring stabilization of the binding of DNA with DNA polymerase &beta;. From this extract, four isomalabaricane triterpenoids were isolated and characterized, three of which were active in the binding assay. All compounds were active in the A2780 ovarian cancer cell line assay.&#13; Bioassay-guided fractionation of an extract of a sponge of species <i>Dysidea</i> using the A2780 bioassay yielded the known scalarane sesterterpenoid heteronemin in good yield. Four derivatives of heteronemin were prepared semisynthetically from the natural product, tested for their bioactivity, and their structure-activity dependence was observed.&#13; Bioassay guided-fractionation of an extract of a <i>Tuemoya</i> sp. green alga, using an assay for inhibitors of the enzyme Tie2 kinase, afforded a two sulfated cycloartanol triterpenoids. Both the major and minor compounds were identified by spectroscopic methods.&#13; Bioassay-guided fractionation of an extract of <i>Petalonyx parryi</i> yielded three known oleanane triterpenoids which inhibited the lyase domain of DNA polymerase &beta;. The structures were confirmed by NMR spectroscopic techniques. This is the first reported study of the chemical components of <i>Petalonyx parryi</i>.&#13; As part of our antitumor natural product drug discovery efforts, several extracts were selected for bioassay-guided fractionation based on their activity in initial in vitro screens. A new dereplication method using aminopropyl SPE cartridges was applied to six of these extracts, and four of the extracts were dropped due to the presence of long-chain fatty acids (LCFAs). We present results for the testing and application of this SPE-based method for LCFA dereplication.&#13; The cell cycle kinase Chk1 is an interesting target for the development of agents which might potentiate DNA damaging agents. Typical assays for Chk1 involve the use of expensive or radioactive reagents. To facilitate the development of new assays using shorter peptide substrates, small libraries of peptides have been synthesized and tested for their activity as Chk1 substrates. Several of the substrates synthesized displayed activity in the Chk1 assay.&#13;