Differences in Mode of Action between Peloruside A and Paclitaxel, Two Microtubule Stabilizing Agents

Abstract

Peloruside A (peloruside) was isolated from the New Zealand marine sponge Mycale hentscheli. It stabilizes the polymerized form of microtubules and therefore has a similar mechanism of action to paclitaxel, a microtubule stabilizing agent that is currently used in cancer chemotherapy. It has been previously shown that peloruside has a unique binding site on tubulin that is different from the taxoid binding site where most microtubule stabilizing agents bind to. In addition, peloruside is not as susceptible as paclitaxel to the development of multiple drug resistance. The present study has demonstrated differences between the two compounds that are likely to be a result of their different binding sites on tubulin, including synergistic interactions and different activity profiles in resistant cells. Peloruside interacted synergistically with two taxoid site binding drugs, paclitaxel and epothilone A, but not with laulimalide, a microtubule stabilizing agent that shares the peloruside binding site. A peloruside-resistant cell line was developed that showed a 5-fold increase in IC50 and in concentration required for G2/M cell cycle arrest when treated with peloruside. No significant difference from the parental cell line was observed when treated with a number of other microtubule stabilizing and destabilizing agents, including laulimalide. Sequencing of the major tubulin isotypes, alpha- and Beta-tubulin, showed a single point mutation in Beta-tubulin. The mutated amino acid was located within a region that has recently been proposed to be important for peloruside binding, and this resistant cell line could be useful in future modelling studies to identify the unique peloruside binding site. In a proteomics study, it was shown that there are other, non-tubulin-binding related differences in activity between peloruside and paclitaxel. DIGE proteomics and MALDI TOF were used to compare both microtubule stabilizing agents to examine the downstream effects and to identify possible additional targets of peloruside. Peloruside and paclitaxel altered 25 and 42 proteins, respectively, and only 11 proteins identified in this study changed similarly in response to both drugs. The microtubule regulating protein stathmin was altered in response to peloruside but not to paclitaxel. In addition, both compounds gave similar but not identical networks in an Ingenuity pathway analysis. A chemical genetic screen was also used in order to uncover possible secondary targets of peloruside. The nearly complete set of heterozygous deletion mutants was screened for mutants that showed reduced growth after treatment with peloruside. Topoisomerase I was one of the identified hits. Natural and semi-synthetic analogues of peloruside were also screened for biological activity. Peloruside tolerated minor side-chain modifications; however, alterations at the pyranose ring resulted in loss of activity. Based on the above results, it was concluded that peloruside is a good candidate for further development as an anti-cancer agent.