Investigation of the function of delta-cadinene synthase with aza-analogues and site directedmutagenesis

Terpenes are one of the most structurally varied families of natural products with extraordinary chemical properties that have been exploited for numerous applications. Sesquiterpene synthases are a family of metal-dependent enzymes that catalyse the cyclisation of farnesyl diphosphate (FDP) into a myriad of complex C15-isoprenoid hydrocarbons, the sesquiterpenes. δ-Cadinene synthase (DCS) from Gossypium arboreum (cotton tree) catalyses the formation of δ-cadinene (DCN), a bicyclic intermediate in the biosynthesis of important phytoalexins such us gossypol. Two mechanistic proposals have been made for the formation of δ-cadinene: a 1,10-ring closure mechanism leading to the key intermediate germacradienyl cation, or a 1,6-ring closure leading to thealpha-bisabolyl carbocation. Previous investigation with fluorinated FDP analogues were in partial agreement with both scenarios and hence it was not possible to distinguish unambiguously between the two possible cyclisation reactions. To investigate the catalytic mechanism of DCS, enantiopure samples of the azaanalogues of alpha-bisabolyl cation and germacradienyl cation were needed. These compounds are designed as stable structural and electrostatic mimics of the putative short-lived carbocationic intermediates generated by terpene synthases, and hence often act as potent reversible competitive inhibitors (low Ki) of these enzymes. Here, the enantioselective total synthesis of R- and S- aza-analogues of the alpha-bisabolyl cation are described as well as the partial racemic synthesis of azagermacradienyl cation. Both enantiomers of aza-bisabolyl cation were goodmimics of α-bisabolene. They were competitive inhibitors of DCS, providing evidence for a 1,6-cyclisation closure. The second part of the project involved the investigation of the role of tryptophan 279 for the desolvation of the active site of DCS and therefore for the formation of DCN. Seven mutants of W279 were created. The data obtained showed that W279 is essential to prevent water from entering the active site and form the hydroxylate terpenoid germacradien-4-ol (GD4ol). Mutagenesis studies yielded a mutant, W279A, capable of making GD4ol as the sole product

Silent catalytic promiscuity in the high-fidelity terpene cyclase δ-cadinene synthase

δ-Cadinene synthase (DCS) is a high-fidelity sesquiterpene synthase that generates δ-cadinene as the sole detectable organic product from its natural substrate (E,E)-FDP. Previous work with this enzyme using substrate analogues revealed the ability of DCS to catalyse both 1,10- and 1,6-cyclisations of substrate analogues. To test whether this apparent promiscuity was an artefact of alternate substrate use or an inherent property of the enzyme, aza analogues of the proposed α-bisabolyl cation intermediate were prepared since this cation would be formed after an initial 1,6-cyclisation of FDP. In the presence of 250 μM inorganic disphosphate both (R)- and (S)-aza-bisaboyl cations were potent competitive inhibitors of DCS (Ki = 2.5 ± 0.5 mM and 3.44 ± 1.43 μM, respectively). These compounds were also shown to be potent inhibitors of the 1,6-cyclase amorpha-4,11-diene synthase but not of the 1,10-cyclase aristolochene synthase from Penicillium roquefortii, demonstrating that the 1,6-cyclase activity of DCS is most likely an inherent property of the enzyme even when the natural substrate is used and not an artefact of the use of substrate analogues

Nucleophilic water capture or proton loss: single amino acid switch converts δ-Cadinene synthase into germacradien-4-ol synthase

δ-Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ-cadinene and about 2 % germacradien-4-ol, which is also generated from FDP by the cyclase germacradien-4-ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site-directed mutagenesis of δ-cadinene synthase. If W279 in δ-cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS-W279A is a catalytically highly efficient germacradien-4-ol synthase (kcat/KM=1.4×10−3 μm s−1) that produces predominantly germacradien-4-ol in addition to 11 % δ-cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release

Cervical cancer benefits from trabectedin combination with the β-blocker propranolol: in vitro and ex vivo evaluations in patient-derived organoids

Background: Cervical cancer (CC) is characterized by genomic alterations in DNA repair genes, which could favor treatment with agents causing DNA double-strand breaks (DSBs), such as trabectedin. Hence, we evaluated the capability of trabectedin to inhibit CC viability and used ovarian cancer (OC) models as a reference. Since chronic stress may promote gynecological cancer and may hinder the efficacy of therapy, we investigated the potential of targeting β-adrenergic receptors with propranolol to enhance trabectedin efficacy and change tumor immunogenicity.Methods: OC cell lines, Caov-3 and SK-OV-3, CC cell lines, HeLa and OV2008, and patient-derived organoids were used as study models. MTT and 3D cell viability assays were used for drug(s) IC50 determination. The analysis of apoptosis, JC-1 mitochondrial membrane depolarization, cell cycle, and protein expression was performed by flow cytometry. Cell target modulation analyses were carried out by gene expression, Western blotting, immunofluorescence, and immunocytochemistry.Results: Trabectedin reduced the proliferation of both CC and OC cell lines and notably of CC patient-derived organoids. Mechanistically, trabectedin caused DNA DSBs and S-phase cell cycle arrest. Despite DNA DSBs, cells failed the formation of nuclear RAD51 foci and underwent apoptosis. Under norepinephrine stimulation, propranolol enhanced trabectedin efficacy, further inducing apoptosis through the involvement of mitochondria, Erk1/2 activation, and the increase of inducible COX-2. Notably, trabectedin and propranolol affected the expression of PD1 in both CC and OC cell lines.Conclusion: Overall, our results show that CC is responsive to trabectedin and provide translational evidence that could benefit CC treatment options. Our study pointed out that combined treatment offset trabectedin resistance caused by β-adrenergic receptor activation in both ovarian and cervical cancer models