Bioactivity of marine organism-derived natural products

Abstract

Background: HIV/AIDS is one of the most devastating diseases in the world with approximately 36 million people living with the virus in 2012 and approximately 2.7 million new infections in that same year. Antiretroviral therapy (ART) successfully reduce infection and decrease symptoms; but, the emergence of viral drug resistance due to drug induced mutations in viral genes can render treatment ineffective. Infection with HIV not only weakens the immune system leading to AIDS and increasing the risk of opportunistic infections, but also increases the risk of several types of cancer. These facts underscore an urgent need to develop new anti-HIV and cancer drugs with fewer or no side-effects. Research into drug discovery and development using natural products is increasingly becoming better established. Marine organisms as a source of natural products delivered numerous novel compounds with sensational multiple pharmacological properties. Thousands of novel compounds and their metabolites with diverse biological activities ranging from antiviral to anticancer have been isolated from various marine sources. With natural products, there are endless opportunities for discovering novel compounds that can be used as drugs or backbones of drug leads. Methods: In this thesis, thirteen marine organisms were investigated for inhibitory properties against HIV-1 enzymes as well as for potential cytotoxicity. Cytotoxicity (The ability to kill cancer cells) of the extracts was determined using tetrazolium dyes. Direct enzyme assays were used to determine the inhibitory properties of the extracts against HIV-1 protease (PR) and reverse transcriptase (RT). The most active extract Litophyton arboreum was then subjected to silica gel chromatography in order to isolate, purify and identify the active compounds. These active compounds were then tested for cytotoxic and HIV-1 enzyme inhibitory activities. The cytotoxicity results were subsequently confirmed by real-time cell electronic sensing and the enzyme studies were supported by in silico analysis docking using MOE software. The most active compound (7β-acetoxy-24-methylcholesta-5-24(28)-diene-3, 19-diol) also underwent an ecological study that demonstrated the role of this compound in the activity of the extract as well as the organism‘s seasonal distribution in its habitat. The HPLC qualitative profiles of the seasonal total extracts were performed and the concentration of the active compound in each extract was determined and related to the activities and seasonal distribution of the organism in the community. Results and discussion: Cytotoxicity results of the 13 marine organism‘s extracts in (Hela, U937 and Vero cells) showed strong activity of L. arboreum against U-937 (IC50; 6.5μg/ml ±2.3) with a selectivity index (SI) of 6.45, while Sarcophyton trochliophorum showed strong activity against HeLa cells (IC50; 5.2 μg/ml ±1.2) with an SI of 2.09. Other species showed moderate to weak cytotoxicity against both cell lines. Two extracts showed potent inhibitory activity against HIV-1 protease; these were Cassiopeia andromeda (IC50; 0.84 μg/ml ±0.1) and Galaxura filamentosa (2.6 μg/ml ±1.3). It was interesting to note that the most active extracts against HIV-1 PR, C. andromeda and G. filamentosa showed no cytotoxicity in the three cell lines at the highest concentration tested (100 μg/ml). L. arboreum extract was the only extract that showed activities in all the bioassays tested in this study. The bioassay-guided fractionation using different chromatographic and spectroscopic techniques in the analysis of the Red Sea soft coral L. arboreum led to the isolation of nine compounds; Further fractionation and purification of the active fractions resulted in the isolation and identification of nine known compounds (for which the structures are provided in Figure 4.2) sarcophytol M (1), alismol (2), 10-O-methyl alismoxide (4), alismoxide (5), (S)-chimyl alcohol (6), 7β-acetoxy-24-methylcholesta-5- 24(28)-diene-3,19-diol (7), erythro-N-dodecanoyl-docosasphinga-(4E,8E)-dienine (8), and 24-methylcholesta-5,24(28)-diene-3β,7β,19-triol (9). Compound 7 demonstrated strong cytotoxicity against HeLa cells (CC50 4.3±0.8 μM), with a selectivity index of SI 8.1 which was confirmed by real time cell electronic sensing (RT-CES). Compounds 2, 7 and 8 showed strong inhibitory activity against HIV-1 PR at IC50‘s of 7.2 ±0.7, 4.85±0.2 and 4.80 ±0.9 μM respectively. Docking studies gave comparable scores when comparing the binding mode of the active compounds to that of acetyl pepstatin, a known HIV-1 PR inhibitor. Interestingly, compound 8 showed potent HIV-1 PR inhibitory activity and did not demonstrate cytotoxicity against the cell lines used in the investigation. Also, compounds 2 and 5 showed cytostatic action in HeLa cells where the compounds inhibited the cells proliferation but did not kill the cancer cells; both compounds can be considered as potential leads in the development of virostatic cocktails that medicine - A drug which inhibits viral replication. Changes in the biological activities of the seasonal extracts of the soft coral L. arboreum showed the highest activity in the autumn extract. The study also evaluated the concentration of the active triterpene erythro-N-dodecanoyl-docosasphinga-(4E,8E)- dienine) and its influence on the organism‘s maturation and distribution which showed a positive correlation between the compound‘s concentration and oocyte maturation as well as the dominance and distribution of the organism. Conclusion: The findings presented in this thesis demonstrated that marine organisms remain one of the most interesting sources for the discovery of bioactive compounds against cancer and HIV. This study is the first to report the anticancer (cytotoxic behavior specific for cancer cell lines) and anti HIV activities of most of the screened organisms. It is also the first report for the metabolites isolation, identification and biological evaluation of L. arboreum collected from Sharm El sheikh, Red Sea. Some of the isolated metabolites demonstrated potent anti-HIV-1 protease activity, with high safety margins. The detailed IC50 study showed potent inhibitory activity of HIV-1 PR by compound 7β- acetoxy-24-methylcholesta-5-24(28)-diene-3,19-diol [7] (IC50 4.8 μM), in addition to compound 8 (4.86 μM), which also showed no toxicity against any of the cell lines under investigation. This work and many other published reports support the high cytotoxicity of polyhydroxylated sterols from marine organisms, which highlights the importance of this chemical skeleton in the discovery of potential lead compounds from marine sources. This study demonstrated a seasonal change in the biological activities of the soft coral L. arboreum demonstrated the effect of the triterpene on the organism‘s maturation and distribution. The autumn season appear to be the best time for L. arboreum collection for biological purposes, where the highest distribution and highest intensity of the compounds were found. The findings presented here suggest that the screened Red Sea marine organisms investigated could be an interesting source of bioactive compounds and deserve further bioassay-guided isolation procedures to determine the identity and structure of the active compounds. The biological activities of nine compounds isolated from L. arboreum delivered promising leads for further biological evaluation.