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Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

ABSTRACT: Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC4504003 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

Zhou Yongjun Y Murphy Annabel C AC Samborskyy Markiyan M Prediger Patricia P Dias Luiz Carlos LC Leadlay Peter F PF

Chemistry & biology 20150601 6

Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assemb ...[more]

PMID: 26091168

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Project description:BackgroundEpidermal growth factor receptor (EGFR) and epidermal growth factor receptor pathway substrate 8 (Eps8) have been widely reported to be expressed in various tumors. Eps8 is an important active kinase substrate of EGFR that directly binds to the juxtamembrane (JXM) domain of EGFR to form an EGFR/Eps8 complex. The EGFR/Eps8 complex is involved in regulating cancer progression and might be an ideal target for antitumor therapy. This study focused on the screening of small-molecule inhibitors that target the EGFR/Eps8 complex in breast cancer and non-small cell lung cancer (NSCLC).MethodsIn silico virtual screening was used to identify small-molecule EGFR/Eps8 complex inhibitors. These compounds were screened for the inhibition of A549 and BT549 cell viability. The direct interaction between EGFR and Eps8 was measured using coimmunoprecipitation (CoIP) and JXM domain replacement assays. The antitumor effects of the inhibitors were analyzed in cancer cells and xenograft models. An acute toxicity study of EE02 was performed in a mouse model. In addition, the effect of the EE02 inhibitor on the protein expression of elements downstream of the EGFR/Eps8 complex was determined by western blotting and protein chip assays.ResultsIn this study of nearly 390,000 compounds screened by virtual database screening, the top 29 compounds were identified as candidate small-molecule EGFR/Eps8 complex inhibitors and evaluated by using cell-based assays. The compound EE02 was identified as the best match to our selection criteria. Further investigation demonstrated that EE02 directly bound to the JXM domain of EGFR and disrupted EGFR/Eps8 complex formation. EE02 selectively suppressed growth and induced apoptosis in EGFR-positive and Eps8-positive breast cancer and NSCLC cells. More importantly, the PI3K/Akt/mTOR and MAPK/Erk pathways downstream of the EGFR/Eps8 complex were suppressed by EE02. In addition, the suppressive effect of EE02 on tumor growth in vivo was comparable to that of erlotinib at the same dose.ConclusionsWe identified EE02 as an EGFR/Eps8 complex inhibitor that demonstrated promising antitumor effects in breast cancer and NSCLC. Our data suggest that the EGFR/Eps8 complex offers a novel cancer drug target.

Dataset Information

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

Publications

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

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