The biosynthesis of the alpha,beta-epoxyketone pharmacophore in natural product proteasome inhibitors

• Applicant: Professor Dr. Leonard Kaysser
• Subject Area: Pharmacy
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 259754399

The proteasome has advanced as a validated therapeutic target over the last decade. In July 2012 Carfilzomib (Kyprolis®) a tetrapeptide epoxyketone was approved for the use in patients with relapsed and refractory multiple myeloma in the USA. It was developed from the natural product epoxomicin comprising a unique pharmacophore of two strongly electrophilic groups in the immediate proximity to each other: the alpha,beta-epoxyketone warhead. This structural group allows a highly specific and irreversible binding to the chymotrypsin-like moiety of the eukaryotic proteasome. Until recently nothing was known about the biosynthesis of the alpha,beta-epoxyketone. In our preliminary work, we identified and heterologously expressed the epoxomicin and the eponemycin gene clusters. Notably, the formation of the alpha,beta-epoxyketone warhead could not be deduced from the gene clusters. We therefore predict that unusual biotransformations take place in the conversion of the terminal polyketide. In this project we intend elucidate the biosynthetic pathway which leads to the alpha,beta-epoxyketone. To this end we will primarily focus on the analysis of heterologous gene deletion mutants. We also plan to perform isotope-enriched precursor feeding experiments. Moreover, the catalytic mechanisms of respective enzymes will be investigated using in vitro biochemistry and protein crystallography. In addition to that, genome mining revealed a homologous pathway which we assume to direct the formation of matlystatin-like protesome inhibitors. To verify this finding, we will isolate and heterologously express the corresponding gene cluster. This experiment should serve as a proof of concept for our future genome-guided drug discovery campaigns. This research proposal will give us deep insides in the biology and biogenesis of the therapeutically important class of epoxyketones and will facilitate the generation of proteasome inhibitors with improved properties.

• DFG Programme: Research Grants
• International Connection: Brazil
• Participating Person: Dr. Daniela Trivella