Chapter I. Isolation of Natural Products As Anticancer Drugs I.1
Total Page：16
File Type：pdf, Size：1020Kb
Chapter I. Isolation of Natural Products as Anticancer Drugs I.1 Introduction Human beings have relied on natural products as a resource of drugs for thousands of years. Plant-based drugs have formed the basis of traditional medicine systems that have been used for centuries in many countries such as Egypt, China and India.1 Today plant-based drugs continue to play an essential role in health care. It has been estimated by the World Health Organization that 80% of the population of the world rely mainly on traditional medicines for their primary health care.2 Natural products also play an important role in the health care of the remaining 20% people of the world, who mainly reside in developed countries. Currently at least 119 chemicals, derived from 90 plant species, can be considered as important drugs in one or more countries.3 Studies in 1993 showed that plant-derived drugs represent about 25% of the American prescription drug market, and over 50% of the most prescribed drugs in the US had a natural product either as the drug or as the starting point in the synthesis or design of the agent.4 There are more than 250,000 species of higher plants in the world, and almost every plant species has a unique collection of secondary constituents distributed throughout its tissues. A proportion of these metabolites are likely to respond positively to an appropriate bioassay, however only a small percentage of them have been investigated for their potential value as drugs. In addition, much of the marine and microbial world is still unexplored, and there are plenty of bioactive compounds awaiting 1 Balandrin, N. F.; Kinghorn, A.D.; Farnsworth, N. R.; Human Medicinal Agents from Plants; Kinghorn, A. D.; Balandrin, N. F., Eds., ACS Symposium Series 534, 1993, 2-12. 2 Farnsworth, N.R.; Akerele, O.; Bingel, A.S.; Soejarto, D.D.; Guo, Z. Bull. WHO, 1985, 63, 965-972. 3 Arvigo, R.; Balick. M; Rainforest Remedies, Lotus Press, Twin Lakes 1993. 4 Grifo, F.; Newman, D. J.; Fairfield, A. S.; Bhattacharya, B.; Grupenhoff, J. T. The Origin of Prescription Drugs, Grifo, F. and Rosenthal, J. Eds., Island Press, Washington D.C 1997: p 131 1 discovery in these two worlds. Besides their direct medicinal application, natural products can also serve as pharmacophores for the design, synthesis or semi-synthesis of novel substances for medical uses. The discovery of natural products is also important as a means to further refine systems of plant classification. I.2 Natural Products as Anticancer Drugs. Cancer continues to be a great threat to human life. It causes the second highest mortality rate in the US, and every year about 1 million new cases of cancer are diagnosed in this country. Nearly one out of every four Americans will develop cancer during his or her life. The number of cancer deaths continued to increase from 1973 to 1990. In 1990 about 510,000 Americans died of cancer.5 It is estimated that from 1970 to 1995, the US government has spent a total of approximately 30 billion dollars through the National Cancer Institute (NCI) on devising improved treatments for cancer.6 In the past twenty years, there has been a lot of progress in the war against cancer. Advances in cellular biology and molecular biology have helped us in understanding different mechanisms of this disease. More and more anticancer drugs and vaccines have been developed. Natural products have contributed significantly to the development of anticancer drugs. According to a recent review,7 among the 79 FDA approved anticancer drugs and vaccines from 1983-2002, 9 of them were directly from the isolation of natural products and 21 of them were natural product derivatives. Also among the 39 synthetic 5 Cooper, G. M. The Cancer Book. Jones and Bartlett Publishers, Boston, MA 1993, 7 6 Pezzuto, J. M. Plant-derived anticancer agents. Biochem. Pharmacol. 1997, 53, 121-133 7 Cragg, G. M.; Newman, D. J. Snader, K. M. Natural Products as Sources of New Drugs over the Period 1981-2002. J. Nat. Prod. 2003, 66, 1022-37 2 anticancer drugs, 13 of them were based on a pharmacophore originated from natural compounds. I.3 The ICBG and NCDDG Programs. Two research programs support our studies of bioactive natural products, the ICBG and the NCDDG program. The International Cooperative Biodiversity Group Program (ICBG program) was funded by the National Institutes of Health (NIH). This program focuses on the plants from two regions: the South American country Suriname (formerly Dutch Guiana) and the African country Madagascar. Both countries have previously been determined to be strategically important for biodiversity. The program has diverse goals in addition to those of natural product isolation or drug discovery. These additional goals include the development of alternative uses for natural resources, education, and economic development for the people of these countries. The research program at Virginia Tech focuses on the isolation and characterization of bioactive compounds, including those with anticancer, anti-malarial and anti-mycobacterial activities. The National Cooperative Drug Development Group (NCDDG) Program was funded by the National Cancer Institute (NCI). This program focuses on the development of novel natural or synthetic compounds as anticancer agents. The work at Virginia Tech is primarily concerned with the isolation of new natural products with novel mechanisms of action. The extracts of these studies are drawn from the NCI repository of natural extracts and include both marine and plant extracts. 3 I.4 The Bioassay Guided Isolation of Natural Products. The discovery of natural drugs is guided by bioassay. Bioassay plays a very important role in every step of the discovery program. First it can be used to detect the bioactivity of the crude extracts and thus guide the selection of extracts for further study. In the isolation steps the bioassay will guide the fraction of a crude sample towards the pure isolated compound. For these purposes, bioassay must be rapid, simple, reliable, reproducible and most important, predictive. It should also model a living organism well. Unfortunately, no bioassay can meet all of the above criteria. In vivo testing (such as on rats) can provide more valid data than in vitro cellular testing; however, animal testing is complicated, slow and expensive, and is normally only used on pure compounds that have demonstrated in vitro ativity. Currently there are a large number of available bioassay systems in the area of anticancer drugs, divided into two groups; cellular assays and cell-free assays. Cellular assays utilize intact cells (yeast cells, mammalian cells, etc.) while cell-free assays utilize isolated systems (enzymes, DNA fragments, etc.) for bioactivity study. These cell-free assays are usually mechanism-based, with a key enzyme or other biomolecule as the target. Cytotoxicity assays are very commonly used in cellular assays. Since cytotoxicity is an activity that is consistent with anticancer activity, the major advantage of cytotoxicity assays is that all potential mechanisms of cellular proliferation can be monitored simultaneously. Thus, the search for new anti-cancer reagents in the past has been primarily focused on extracts showing cytotoxicity to one or two cell lines. The approach has been fruitful and led to the discovery of paclitaxel, among many other 4 compounds. Cytotoxicity-based assays are normally reported as IC50 values (the concentration of a sample that can inhibit 50% growth of a target cell in a single cell line). The cell line employed in the cytotoxicity assay of our group is the A2780 human ovarian cancer cell line. The A2780 assay is a general cytotoxicity assay, which means that in many cases the active compound will simply be toxic, and thus will not be suitable for drug use. The use of cell-free mechanism-based assays is a second approach to drug discovery. These assays utilize isolated assay systems (cellular receptor, enzyme, etc.) to test the bioactivity. Basically these assays are designed to test the unknown extract, fraction, or pure compound in comparison to known antitumor agents in mechanisms that have been clearly delineated. Mechanism-based assays are very selective and sensitive and also reproducible. An important advantage of these assays is that once a lead compound is discovered, its mechanism of action is already known, and lead optimization can thus be carried out more efficiently. Because of these advantages, several mechanism-based assays are currently employed in the NCDDG project, such as assays for inhibitors of Akt-kinase, Myt-1 kinase and DNA polymerase β (pol-β) assay. If a novel compound is found with a similar effect to a known specific compound, it can be classified to the specific mechanistic class. This approach can lead to a more systematic method to discover new anticancer drugs. 5 I.5 Mechanism Based Bioassays Employed in the NCDDG Program. I.5.1. The Akt-kinase Bioassay. Akt (protein kinase B), a serine/threonine kinase, is a critical enzyme in signal transduction pathways involved in cell proliferation, apoptosis and angiogenesis. Akt kinase, together with another kinase (p53), play opposing roles in signaling pathways that determine cell survival.8 In mammalian cells three forms of the Akt enzyme (Akt-a, b, g or Akt-1, 2, 3) are reported that exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites. The principal role of Akt is to facilitate growth factor-mediated cell survival and to block apoptotic cell death, which is achieved by phosphorylating several pro-apoptotic factors. 9 For example, Akt-a is expressed to various degrees in breast cancer cell lines and is important in estrogen- stimulated growth. Treatment of multiple cancer cell lines with the Akt inhibitors could result in reduced survival of both drug resistant and drug sensitive cells.10 Therefore, searching for Akt-inhibitors from natural products could be a useful method for anti- cancer drug development.