title: Rational design of small molecule probes for investigating the mechanism of action of the chemotherapeutic agents CDDO and artemisinin
Creators: name: Wong, Michael
Publisher: name: University of Liverpool Repository
Year: 2015

description: Adverse drug reactions (ADR) are a major concern for the pharmaceutical industry and health practitioners as they can cause morbidity and in severe cases mortality. ADRs are one of the major reasons why drugs fail during clinical trials so research directed at predicting ADRs to minimise failure is essential. The CDDO (2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) and the synthetic endoperoxide series are two promising classes that have potential for the treatment of cancers and malaria and may revolutionise treatment, within their fields, if approved for clinical use. The two main aims that are presented in this thesis are to; (i) design and synthesise novel analogues and chemical probes to identify potential molecular targets for both the CDDO and endoperoxide series (ii) develop appropriate in vitro test systems to help define the molecular mechanism of each class of drug. CDDO-Me (methyl 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oate) is one of the most potent inducers of Nrf2, a transcription factor that regulates the expression of numerous cell defence genes in mammalian cells. Nrf2 is sequestered in the cytosol by Keap1, which ‘senses’ chemical and oxidative stress via its 27 cysteine residues. Although CDDO-Me is one of the most potent inducers of Nrf2, the molecular target and chemical mechanism is still not defined. Current literature suggests that a reversible 1,4 conjugate addition to specific cysteine residue(s) located on the Keap1 protein results in an increase in Nrf2 levels. In order for SAR work to be performed a synthetic route to CDDO and analogues was developed which involved nine steps using oleanolic acid as starting material. Highlights of the chemistry included addition of the ketone using mCPBA and incorporation of the cyano group in steps 3 and 7 of the synthesis. In addition to preparing the target molecule CDDO a number of additional molecules were prepared to define the importance of functional groups in the A and C rings of CDDO. Genetically modified H4IIE rat hepatoma cells transiently transfected with the an Nrf2-sensitive luciferase reporter gene were used to screen the CDDO-Me analogues, including DDO-Me which lacks a cyano group on the A ring, for their ability to induce Nrf2. NMR studies with model thiols were performed to determine the ability of these compounds to form reversible or non-reversible adducts. Mass spectrometry (MS) was used to confirm the NMR data and interpretations. In total, four probes were identified that reacted in a non-reversible fashion: DDO-Me, DDO-Al and DDO-Az (click probe versions of the parent DDO-Me that can be used to facilitate proteomic studies) and CDDO-Epox (a probe with similar overall structure to CDDO-Me but can react at the β-carbon in a non-reversible fashion; this feature should aid proteomic approaches to reactive cysteine residue identification). To further investigate if these compounds were reactive to cysteine residues within a model protein, recombinant human GSTP1 was used as a model protein for chemically reactive molecules. Cys-47 located on GSTP1 has been shown to react with other electrophones and during our studies LCMS has confirmed that all four of the synthesised active probes were capable of attaching covalently to Cys-47 of GSTP1. The emergence of malaria parasite resistance to most available drugs, including the semi-synthetic artemisinin derivatives artemether and artesunate, has led to efforts to create new synthetic peroxides as potential antimalarial agents. Leading examples of synthetic endoperoxides include OZ277 (arterolane), a molecule in phase III clinical trials in combination with piperaquine, and OZ439, a second generation derivative with improved pharmacokinetics and enhanced in vivo antimalarial activity. 1,2,4,5-Tetraoxanes are another class of endoperoxide with proven excellent antimalarial profiles against both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum and oral activity in murine models of the disease. It is currently widely accepted that endoperoxides have a similar antimalarial mechanism to artemisinin, whereby Fe2+ medicated generation of cytotoxic carbon-centred radicals, results in death of the parasite. It is presumed that C-radicals can react with important key proteins; however, the specific molecular target(s) that leads to eventual parasite death are still unknown. A chemical synthesis of tetraoxane probes that contain a UV chromophore was performed and analogues were subsequently screened for antimalarial activity. The most active tetraoxane identified was exposed to a range of Fe2+ salts and conditions developed to mimic the biological environment. Primary, secondary and novel carbon-centred radical derived products (surrogate markers of bioactivation) were purified using UV-HPLC, characterised and submitted as chemical probes and standards for biological studies. In order for proteomic studies to be initiated, an allyl or azide group was incorporated into a semi-synthetic artemisinin skeleton. The azide (and alkyne) functional group within these probes provides a handle for protein pull down via click chemistry. Azide and acetylenes were chosen over direct linkage to the biotin group to reduce steric hindrance in the semi-synthetic probe. The synthesised click probes were tested for antimalarial activity and were submitted for protein pull down and identification of potential molecular targets. Similarly DDO allyl and azide were synthesised and were tested for Nrf2 induction and further confirmed as viable probes via NMR experiments with simple thiols and GSTP1. In summary, novel CDDO non reversible probes were synthesised and have shown potential as chemical tools to identify the molecular targets/mechanisms by which these compounds activate Nrf2. Tetraoxanes also have been prepared along with artemisinin click probes and the latter have been submitted for click chemistry pull down experiments, within Plasmodium falciparum parasites, to identify potential molecular targets.