Discovery of potential anticancer and neuroprotective agents from North Queensland plants

Plants represent a largely untapped resource for drug discovery. There are approximately 25,000 plant species in Australia alone, and 9,000 of these are found in Queensland. This study aimed to discover drug leads from North Queensland plants by screening extracts for pharmacological activity. Two pharmacological targets were selected. The first screen aimed to find novel cytotoxic compounds with potential as anticancer agents. The second screen aimed to find compounds with therapeutic potential in schizophrenia or neurological disorders that involve neuroinflammation and neurodegeneration, such as Huntington’s Disease and AIDS-related dementia. Various imbalances in the relative levels of kynurenine pathway metabolites, particularly kynurenic acid, quinolinic acid and 3-hydroxykynurenine, have been implicated in these conditions. In order to discover potential drug leads that might be applied to rectify these imbalances, the extracts were screened for inhibition of three key enzymes of this pathway, namely kynurenine-3-hydroxylase, kynureninase and kynurenine aminotransferase. Samples (365) from 125 species of plants from North Queensland were collected and extracted. After removal of tannins to prevent interference with the assays, the plant extracts were screened for cytotoxicity and for enzyme inhibition. Cytotoxicity was assessed in vitro using the P388D1 mouse lymphoma cell line. The enzyme inhibition assays involved the use of crude enzyme preparations from rat liver or kidney, and products were quantified by HPLC with electrochemical or fluorescence detection. Extracts with the greatest kynurenine-3-hydroxylase inhibition and selected cytotoxic extracts were subjected to bioassay-guided fractionation in order to isolate and identify the active compounds. No extracts possessed sufficient kynureninase or kynurenine aminotransferase inhibition to warrant investigation. Three cytotoxic quinonemethide triterpenes were isolated from Maytenus cunninghamii (Celastraceae). Netzahualcoyoic acid (25, IC50 value = 0.12 μM, 0.11–0.13 μM 95% CI range) and Δ15-celastrol (32, IC50 value not determined) are new structures, while celastrol (22, IC50 value = 0.37 μM, 0.30–0.45 μM 95% CI range) is a known cytotoxic agent. Δ15-celastrol represents a possible biosynthetic intermediate between celastrol and netzahualcoyoic acid. Further work is needed to assess the cytotoxic profile of netzahualcoyoic acid and its acid-rearranged products in the National Cancer Institute’s 60 cell line panel. The known cytostatic compounds podophyllotoxin (36), deoxypodophyllotoxin (37) and picropodophyllotoxin (38) were isolated from Callitris intratropica (Cupressaceae). This is the first reported isolation of podophyllotoxins from this species. Bioassay-guided fractionation of kynurenine-3-hydroxylase inhibitory extracts led to the isolation of two new triterpenes, 11α,28-dihydroxylupenone (43) and 2α,3β-dihydroxyfriedelan-29-oic acid (53), from Maytenus disperma, as well as two new 24-oxomaytenonic acids (58 and 60) and four known triterpenes from M. cunninghamii. A biosynthetic pathway was proposed for compounds isolated from M. cunninghamii and related compounds. Five other species afforded a total of five additional known triterpenoids. Triterpenes were identified as a new class of potent and selective competitive inhibitors of kynurenine-3-hydroxylase. The most active was uncaric acid (78, Kic = 0.023 ± 0.002 μM), isolated from Dolichandrone heterophyllum (Bignoniaceae), and it is one of the most potent kynurenine-3-hydroxylase inhibitors that has been reported. The next most active inhibitors of this study were the 24-oxofriedelan-29-oic acids (60, Kic = 0.061 ± 0.005 μM; 62, Kic = 0.077 ± 0.011 μM; 58, Kic = 0.12 ± 0.02 μM) and celastrol (22, Kic = 0.14 ± 0.03 μM). Important structure-activity relationships relating to triterpene skeleton, functional groups, and ring conformations were observed. It is proposed that the triterpene inhibitors would make ideal drugs for the treatment of many neuroinflammatory and neurodegenerative disorders, and that uncaric acid, 2α-hydroxy 24-oxomaytenonic acid (60) and celastrol should be investigated in vivo