Aplicación del life course en las historias de las personas

6

Alan L. Harvey

Strathclyde Institute for Drug Research, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK. Tel.:

44 141 553 4155, Fax: 44 141 552 8376, E-mail: sidr@strath.

ac.uk

Fig. 1. Origin of the 244 drug prototypes used up to 1995.

Source: Sneader (1996).

minerals

plants

animals microbes

synthesis

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in the 20th century, synthetic chemistry and biotechnology offered alternatives to natural products. For technological convenience, the efforts of drug discovery scientists have tended to be directed at synthetic rather than natural products; however, there is a shortage of successful new chemical entities being introduced to medical practice (Drews, 2000; Baker and Gill, 2005; Cuatrecasas, 2005). This chapter will attempt to show how natural products can still be a useful way to bridge the gap in drug discovery. Studies on natural products can also be a means to link research in academia with that in pharmaceutical companies.

Ethnopharmacology

Broadly speaking, natural products can be used in drug discovery in two main ways: in focused studies directed by information from traditional uses of natural products, and in random screening programmes.

It is often claimed that using ethnopharmacological information will greatly increase the chances of discovering new drugs. However, it is not clear from published information whether this assertion is valid. Two large-scale studies provide some pointers to success rates with natural product screening. The NCI anti-cancer screening programme published that screening of random samples gave a hit rate of 10.4%, while plants collected on the basis of some ethnopharmacological information gave a hit rate of 19.9%. However, plants that were known to be poisonous had a much higher initial hit rate - 50%. The second example comes from the Central Drug Research Institute in Lucknow, India. On a wide range of assays, randomly collected plants had a hit rate of 18.9%, whereas plants with associated ethnopharmacological uses had a hit rate of 18.3%.

More recently, the natural product group at the Strathclyde Institute for Drug Research (SIDR) in Glasgow has worked with collaborators in Latin America to commercialise anti-anxiety leads that were found by studies on plants used locally as sedative teas. A series of flavonoids was found that acted as partial agonists on some subtypes of benzodiazepine sites in the brain. The most interesting compounds could act like benzodiazepines to reduce behaviour associated with anxiety, but without causing sedation or memory impairment, which are common side effects of benzodiazepines (Medina et al., 1990, 1997; Marder and Paladini, 2002).

In another example, a traditionally used topical preparation from the flowers of Calendula officinalis was subjected to bioassay-guided fractionation to try to isolate compounds that might be responsible for the effectiveness of the natural product extract in treating patients with psoriasis. Several related compounds were defined as being cytostatic, rather than cytotoxic, to skin cells in culture (US Patent, 2001).

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HIGH THROUGHPUT SCREENING RANDOM SCREENING AND NATURAL PRODUCTS

Natural products are a superbly diverse chemical collection that can be productively applied to random screening approaches to drug discovery.

This requires natural products researchers to be aware of differences between traditional and modern approaches to drug discovery (Harvey 1998).

Modern drug discovery is dominated by molecular approaches, e.g., with cloned receptors and high throughput screening (HTS). Although molecular strategies also include so-called rational drug design, random screening is probably the most prevalent activity, and also the most relevant for natural products researchers. Random screening is likely to be more productive than other approaches because of the continuing technical challenges faced by the other technologies. For example, with rational drug design, accurate three-dimensional information about the drug’s binding target is needed. Molecular biology gives an abundance of predicted protein sequence data, but potential therapeutic targets are not linear proteins.

Therefore, structural information is required, but currently, X-ray crystallography is too slow to keep pace with the production of sequence information or not feasible because the protein targets are integral membrane proteins, and de novo prediction of structures from linear protein sequences is still an inexact art.

Random screening is the rapid testing of large numbers of compounds on molecular targets—often referred to as ‘high throughput screening’ (or HTS). It is built on molecular biology to provide the assays and robotics and IT to provide the throughput (e.g., up to 500,000 points/week/assay).

However, the success of any HTS programme depends on the chemical diversity that is applied to the assay. This is because success depends on detecting a specific binding interaction between the target and one of the test compounds: the greater the variations in the three-dimensional shapes of the compounds and in their chemical characteristics, the greater the chance of a detectable interaction with the target. Compound supply, therefore, becomes critical for successful HTS programmes, and three-dimensional diversity of compounds is even more important than the number of compounds.

Compounds for HTS can come from two main sources: natural products and synthetic chemistry. Some large collections of synthetic chemicals are available, e.g., from years of in-house synthesis in major pharmaceutical companies, or from collections of the output of many academic laboratories. However, these numbers are still small when the HTS system can assay half a million compounds each week. Due to this,

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many companies have turned to synthetic chemistry based on automated parallel syntheses, using so-called combinatorial chemistry. These methods can provide very high numbers of compounds, but they are generally lacking in three-dimensional diversity. Combinatorial chemistry also suffers from a number of practical issues relating, for example, to the purity of the screening samples, to their stability, and to the reproducibility of the syntheses on scale-up. Few, if any, drugs have reached the market based on initial hits from combinatorial libraries (Newman et al., 2003).

In contrast, natural products offer much greater structural diversity.

In a comparison of published chemical databases, 40% of chemical scaffolds from natural products were found to be absent from synthetic libraries (Henkel et al., 1999). An analysis of the properties of a combinatorial chemistry collection, natural products and drug molecules indicated that the synthetic compounds provided relatively little chemical diversity compared to that of drugs and natural products (Feher and Schmidt, 2003).

Additionally, very few of the world’s natural products have been tested in HTS programmes (Harvey and Waterman, 1998, Harvey, 2000).

What holds back the use of natural products in high throughput screening?

Despite their chemical diversity, natural products are not favoured in HTS because of several real and perceived difficulties. These include their chemical complexity; the difficulty of screening mixtures of compounds in natural product extracts; the time-consuming nature of natural products chemistry; the belief that screening of natural products gives rise to large numbers of artefacts; the supposedly common occurrence of synergistic actions between different components in an extract; the fear of poor reproducibility between different batches of extracts, possibly from seasonal effects on plant secondary metabolism; the uncertainty of being able to obtain resupplies of an interesting extract in large quantities; and the general political problems of access to biodiversity and the implications of the United Nations Convention on Biological Diversity (CBD) (Harvey, 2001).

Given the continued successes deriving from natural product lead compounds (several of the world’s current top 20 most valuable drugs are natural products or derived from a natural product lead; Newman et al., 2003; Butler, 2005), it seems worthwhile for the screening departments of pharmaceutical companies to consider natural products as a source of compounds for screening. The structural diversity is acknowledged to be high, so can the other technical barriers be reduced?

Some of the comparisons of the analysis of databases of structures of

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natural products and synthetic compounds indicate that natural products are not necessarily much more complex chemicals than the synthetic ones (Henkel et al., 1999). Also, there have been many advances in separation chemistry and in techniques for analysis and structural elucidation of natural products (Bindseil, 2001; Wolfender, 2001; Cremin and Zeng, 2002;

Eldridge et al., 2002; Hu et al., 2005). Therefore, following up initial hits made with extracts is not necessarily any longer or more difficult than scaling up and reconfirming hits made from a combinatorial library.

Additionally, the higher resolution of current techniques means that structures can be obtained from much smaller quantities of natural products than before (Hu et al., 2005), opening the way to early production of synthetic material and of analogues for optimisation studies.

Another perceived hindrance to the wider use of natural products in HTS systems is the difficulty of access to biodiversity. Most companies with interests in screening natural products are aware of the CBD, but few companies are very clear on its impact. The CBD has been ratified by most countries in the world in recognition of a need to encourage the preservation of the world’s biodiversity. In addition to its conservation purposes, the CBD provides a framework for the sustainable exploitation of the genetic resources contained in biodiversity. Under the CBD, countries are recognised as having sovereign rights over the biodiversity within their national boundaries. Countries have to develop appropriate regulations to facilitate access to biodiversity for research and development purposes.

Access has to be under principles of prior informed consent and to involve fair and equitable benefit-sharing. Involvement of source countries in research on their biodiversity and technology transfer to the bioresource-rich countries are expected under the CBD.

This raises many practical problems for companies wishing to access samples of natural products for screening. Although many countries have ratified the CBD several years ago, very few have introduced the necessary laws and regulations governing access. Also, it is daunting to companies seeking the broadest range of biodiversity because they would need to establish links with several different countries throughout the world.

Conversely, it is also daunting for research groups working on traditional medicinal leads to know how to gain additional value from natural products that are no longer actively being pursued in the traditional medicines validation programme. Natural product researchers may not find it easy to make appropriate contacts with counterparts in HTS companies. Also, the numbers of samples that they can contribute may be too small to interest a company.

For these reasons, it would be useful for groups of natural products researchers in different countries to operate in networks that pool their

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resources. If such networks also have a single contact for commercialisation of natural products available for random screening, they will facilitate the development of collaborations with industry.

SIDR’s natural product network

An example of such a collaborative research network is that organised by SIDR. It is long-established, being operative for more than 10 years and self-sustaining through commercial interactions. The network was originally based on research contacts between the Phytochemistry Research Group of the University of Strathclyde and several departments and institutes in developing countries. The network is based on a collaborative approach to natural product-based drug research, wherever possible. Each institution in the network signs a legally binding agreement with the University of Strathclyde covering conditions of collecting (sustainable, no endangered species, compatible with local laws and regulations) and a commitment to training, technology transfer and involvement in research projects. Benefit sharing from commercial exploitation coordinated by SIDR is on a 60:40 basis, with 60% of income going to the overseas institution.

Currently, groups in 20 different countries are involved. The natural product library that has been created is largely based on higher plants.

Due to the geographical spread of the participants, samples from over 90%

of the families of higher plants are included in the collection (Figure 2).

Such biodiversity guarantees exceptionally high chemical diversity, making the network’s library particularly attractive for HTS purposes.

The collection has been used in several screening collaborations with the pharmaceutical industry, as well as for academic purposes. Unique

Fig. 2. A comparison of the total plant families (black columns) and families represented in the screening collection of SIDR (light columns).

0

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active molecules have been found against various targets, and these have formed the basis of synthetic programmes aimed at optimising a development candidate. Several routine assays are conducted within SIDR in attempts to find potential lead compounds for future exploitation. The assays are summarised in Table 1.

Table 1. Drug discovery assays in routine use at SIDR

Assay Therapeutic area

Serotonin receptor antagonists Anti-migraine Serotonin uptake inhibitors Anti-depressants Potassium channel blockers Immunosuppressants Tumor necrosis factor blockers Anti-inflammatory

Cell adhesion inhibitors Anti-inflammatory/anti-cancer Tumor cell cytotoxics Anti-cancer

Tumor enzyme inhibitors Anti-cancer Skin cell inhibitors Anti-psoriasis Bacterial replication Antibiotics Bacterial biofilm inhibitors Anti-infectives Glucose uptake promoters Anti-diabetics Parasite growth assays Anti-parasitics Metabolic enzyme inhibitors Anti-diabetics

Proteinase inhibitors Anti-inflammatory and anti-Alzheimer’s Muscarinic receptor agonists Analgesics

Adenosine receptor antagonists Neuroprotectives and cognitive enhancers

Antioxidants Neuroprotectives

Cell growth and nuclear mutations Genotoxicity and mutagenicity Cardiac ion channels Cardiotoxicity

CONCLUSIONS AND FUTURE PERSPECTIVES

From the average numbers of compounds that drop out of industrial drug development during toxicological, pharmacokinetic and clinical testing, it is evident that not all leads from traditionally used natural medicines will succeed as pharmaceutical products. However, natural products can be applied to HTS where their exceptional chemical diversity can provide new lead structures against molecular targets. Despite this, the pharmaceutical industry is not generally enthusiastic about using natural products in HTS systems. The advances in the processing of natural products need to be more widely appreciated, and there is a need for networks of natural

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products researchers to provide convenient and politically appropriate access to the world’s biodiversity and to engage in modern approaches to drug discovery. This is an opportunity for researchers based in academia to contribute to the discovery of leads for new medicines.

References

Baker, A. and J. Gill. 2005. Rethinking innovation in pharmaceutical R&D. J.

Commercial Biotech. 12: 45-49.

Bindseil, K.U. 2001. Pure compound libraries: a new perspective for natural product based drug discovery. Drug Discovery Today 6: 840-847.

Butler, M.S. 2005. Natural products to drugs: natural product derived compounds in clinical trials. Nat. Prod. Report 22: 162-195.

Cremin, P.A. and L. Zeng. 2002. High-throughput analysis of natural product compound libraries by parallel LC-MS evaporative light scattering detection.

Analytical Chem. 74: 5492-5500

Cuatrecasas, P. 2005. Drug discovery in jeopardy. J. Clin. Invest. 116: 2837-2842.

Drews, J. 2000. Drug discovery: a historical perspective. Science 287: 1960-1964.

Eldridge, G.R., H.C. Vervoort, C.M. Lee, P.A. Cremin, C.T. Williams, S.M. Hart, M.G. Goering, M. O’Neil-Johnson and L. Zeng. 2002. High-throughput method for the production and analysis of large natural product libraries for drug discovery. Analytical Chem. 74: 3963-3971.

Feher, M. and J.M. Schmidt. 2003. Property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry. J.

Chem. Inf. Comput. Sci. 43: 218-227.

Harvey, A.L. 1998. Advances in Drug Discovery Techniques, Wiley, Chichester, UK.

Harvey, A.L. 2000. Strategies for discovering drugs from previously unexplored natural products. Drug Discovery Today 5: 294-300.

Harvey, A.L. 2001. Natural Product Pharmaceuticals: A Diverse Approach to Drug Discovery, PJB Publications, Richmond, Surrey, UK.

Harvey, A.L. and P.G. Waterman. 1998. The continuing contribution of biodiversity to drug discovery. Current Opinion in Drug Discovery and Development 1: 71-76.

Henkel, T., R.M. Brunne, H. Muller and F. Reichel. 1999. Statistical investigation into the structural complementarity of natural products and synthetic compounds. Angewandte Chemie International Edition 38: 643-647.

Hu, J.F., E. Garo, H.D. Yoo, P.A. Cremin, L. Zeng, M.G. Goering, M. O’Neil-Johnson and G.R. Eldridge. 2005. Application of capillary-scale NMR for the structure determination of phytochemicals. Phytochem. Anal. 16: 127-133.

Marder, M. and A.C. Paladini. 2002. GABA_A-Receptor ligands of flavonoid structure. Curr. Top. Med. Chem. 2: 853-867.

Medina, J.H., A.C. Paladini, C. Wolfman, M. Levi de Stein, D. Calvo, L. Diaz and C. Peña. 1990. Chrysin (5-7 di OH flavone), a naturally-occurring ligand for benzodiazepine receptors with anticonvulsant properties. Biochem.

Pharmacol. 40: 2227-2232.

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Medina, J.H., H. Viola, C. Wolfman, M. Marder, C. Wasowski, D. Calvo and A.C.

Paladini. 1997. Overview. Flavonoids: A new family of benzodiazepine receptor ligands. Neurochem. Res. 22: 419-425.

Newman, D.J., G.M. Cragg and K.M. Snader. 2003. Natural products as sources of new drugs over the period 1981-2002. J. Nat. Prod. 66: 1022-1037.

Sneader, W. 1996. Drug Prototypes and their Exploitation, Wiley, Chichester, UK.

US Patent 6,225,342; May 1, 2001: Use of Calendula glycosides for the treatment of psoriasis.

Wolfender, J.L. 2001. The potential of LC-NMR in phytochemical analysis.

Phytochem. Analysis 12: 2-22.

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Introduction

For many years the study of natural products from endophytes has been of low priority for research as it was not known that plants serve as a reservoir for untold number of microbes. The whole scenario of natural product drug discovery programme changed with the discovery of Taxomyces andreanea, the producer microorganism of taxol from Taxus brevifolia the world’s first billion dollar anticancer drug. This change not only helped in conserving the world’s diminishing biodiversity but also the price of the drug was reduced as it could be produced via fermentation (Suffness, 1995).

First described by de Bary (1866), endophytes are defined as microorganisms that colonize internal plant tissues. Petrini (1991) defined them as microorganisms that inhabit, at least for one period of their life cycle, plant inner tissues without causing any apparent harm to the host.

Hallmann et al. (1997) described them as those that may be isolated from surface sterilized plant parts or extracted from inner tissues and cause no damage to the host plant. It has now been shown by molecular techniques that microorganisms that are not culturable on usual media and conditions can be found inside plants.

The most frequently isolated endophytes are the fungi. Dreyfuss and Chapela (1994) estimated that there may be at least 1 million species of endophytic fungi alone. Fungal endophytes have been reported from mostly

Fungal Endophytes: A Potential

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