Getting the best from plants

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Getting the best from plants Plants have, for a long time, been used for many purposes besides food. A recent Ciba Foundation symposium* considered the applications of plant-derived materials in medicine and agriculture, including the practical and economic aspects of developing these compounds commercially. Plants used in traditional medicine throughout the world are being screened to assess their potential as sources of drugs for western medicine. Michael Balick (Institute of Economic Botany, New York, USA) and Paul Cox (Brigham Young University, Provo, Utah, USA) are working with tribal healers in Belize and Samoa, respectively, to identify those plants occurring frequently in the native pharmacopoeias as likely to possess significant bioactivity. Extracts of these plants (usually a water:ethanol mix mimics most closely the fresh plant material) are then sent to the USA for screening. The tribal healers use plants to treat a variety of diseases but in the USA the emphasis is on screening for anti-cancer or anti-HIV activity. The assumption is that these plants have proven bioactivity in humans but do not cause deleterious side-effects and they therefore provide more promising starting compounds than plants chosen at random. Once a plant extract has been shown to be effective in a screen, the active component has to be identified. At this point the organic chemists take over; their task is to isolate the compound and to develop it for the market by modifications to improve shelf life or to make it more easily administered. This chemical manipulation of natural products provoked a discussion at the symposium on who should benefit financially from a successful drug. Gordon Cragg *Bioactive Compounds From Plants, Ciba Foundation Symposium 154, Bangkok, Thailand, February 20-22 1990. To be published by John Wiley & Sons, October 1990. (~ 1990, Elsevier Science Publishers Ltd (UK)

[National Cancer Institute (NCI), Rockville, Maryland, USA] described the procedure for compensation his institute followed in the development of compounds from plants: similar constraints are imposed by many ethnobotanists. The NCI makes the results of the screening available to the collection contractors for them to pass on to the country where the plant was collected. Local scientists and officials are invited to the NCI for discussions and to work in their laboratories, particularly on the chemical isolation of the compound. Assistance for upgrading facilities is also provided to scientists in the developing country. If a drug is patented by the US government, licensing of that drug to a pharmaceutical company requires that a percentage of the royalties is returned to the country of origin. The issue is complicated when a natural compound has been substantially modified before marketing: the NCI varies the percentage payable according to the amount of development that has been invested in a drug. However, for a commercially successful drug, even a small fraction of the profits can make a major contribution to scientific programmes in developing countries. In the past decade, natural product chemistry has focused on the elucidation of biosynthetic pathways. Ideally, this results in isolation of the enzymes responsible for catalysing each step leading to the formation of a secondary metabolite. Much has been learned about these enzymes and about their regulation within plant cells. Wolfgang Barz (Westf~lische Wilhelms-Universit/it, MOnster, FRG) has determined the pathways leading to the synthesis of the phytoalexins, medicarpin and maackiain, in chickpea (Cicer arietihum). Phytoalexins are part of plant defence mechanisms that are activated in response to stress or injury of various kinds, including microbial or fungal infection. Barz has found that elicitation of the phyto-

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alexin response causes coordinate induction of all enzymes in the pathway from glucose-6-phosphate dehydrogenase to the final pterocarpan synthase (other enzymes involved in primary metabolism are not affected). The commercial importance of phytoalexins is that they are degraded by fungi and are potential starting structures for the synthesis of fungicides. The laborious purification of proteins has been superseded by the application of molecular biological techniques. Marc van Montagu (Rijksuniversiteit Gent, Belgium) explained that if a plant extract can be partially enriched for a specific enzyme, and that enzyme identified on a two-dimensional protein gel, sufficient material can be regained from the gel for peptide analysis of part of the protein. The DNA sequence for this peptide can be deduced and oligonucleotide cDNA probes made. These can be used to screen libraries and to isolate full-length genomic clones, which are then expressed in bacteria to give high yields of the target enzymes. Bacterial expression systems are not ideal because they cannot glycosylate proteins; an alternative is to express the genes in plant cell cultures. Plant cell and tissue cultures have been studied for some years but so far have been used for the commercial production of only a few compounds, including shikonin by Mitsui & Co. in Japan. One problem with the production of secondary metabolites in culture is that these compounds are usually made by specialized tissues within the plant but not by the undifferentiated cells in cultures. The use of these cell cultures to produce the enzymes of the relevant biosynthetic pathway, possibly after introduction of the requisite genes by genetic engineering, may be the logical way forward. The coordinate regulation of the enzymes that make phytoalexins suggests that their activity, and hence phytoalexin production, could be increased quite simply. If synthesis of the enzymes themselves is induced by a common signal, the genes encoding them probably share a common element in their promoter regions, and it should not be too difficult to identify a suitable activating signal. The hard economics of plant

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cultures were presented by Michael Fowler (University of Sheffield, UK). He described the factors that influence production costs and various attempts that have been made to reduce these costs in different systems. His conclusion was that while plant cultures still hold promise for the future, particularly for making enzymes as mentioned above, they are not yet commercially viable sources of natural products. Norman Farnsworth (University of Illinois at Chicago, USA) disagreed, saying that if there is money to be made, people will find ways of reducing production costs. He cited vincristine, which was initially sold by Eli Lilly for $3.1 million per kg and is now available for less than $3000 per kg. On the other hand, Ingo Potrykus (Eidgen6ssische Technische Hochschule, Zfirich, Switzerland) believed that production in culture will never be as cheap as growing the plants in the field, parti-

cularly w h e n global environmental factors are taken into account. Genetic engineering of plants was originally seen as a way of introducing properties that could be conferred by conventional plant breeding more quickly and without the disadvantage of cotransmission of undesirable genes, e.g. improving herbicide resistance without reducing yield. Tim Hall (Texas A & M University, USA) reviewed more diverse applications, such as improving the nutritional properties of seed proteins or the use of RNAs to combat viral infection. Again the optimism was dampened by consideration of practical aspects. Potrykus described the many ways in which genes can be transferred into plant cells, but concluded that routine, efficient transfer of genes into any chosen plant leading to stable expression and transmission to subsequent generations is still a long way off.

Complementarity of peptides specified by 'sense' and "antisense" strands of DNA The rational design of proteins would be greatly facilitated by the ability to design pairs of interacting peptides or proteins and to predict precisely their points of contact. There is at present no simple way of doing this. However, we have proposed a theory that, in many instances, may simplify such design and enable interactive sites to be predicted. The molecular recognition theory arose from the observation that codons for hydrophobic amino acids on one strand of nucleic acid are complemented by codons for hydrophilic amino acids on the other strand, and vice versa a-3. Thus, two peptides derived from complementary nucleic acid sequences in the same reading flame will show a total interchange of their hydrophobic and hydrophilic amino acids when the amino terminus of one is aligned

with the carboxy terminus of the other (Fig. 1). This inverted hydropathic pattern might allow two such peptides to assume complementary conformations conducive to specific interaction 1. While the precise mechanism of interaction remains a mystery, we originally envisioned that the two peptides would assume amphipathic secondary structures (helices or ~ sheets); in a hydrophilic environment, the hydrophobic plane of one would interact with the hydrophobic plane of the other. This, of course, overcomes the frequently cited argument that the theory suggests an interaction of hydrophobic amino acids with hydrophilic amino acids. The prototypical system used for evaluating the interaction of two such peptides involved testing for the binding of corticotropin (ACTH) to a synthetic peptide (HTCA 5'-3')

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The different technologies were put into perspective when Daniel Bellus (CIBA-GEIGY, Basel, Switzerland) posed the participants a specific problem. His company has a compound of plant origin that shows biological activity and has a complex chemical structure; the company requires 100 kg of material for toxicity studies, so in what technology should they invest if they want that amount of material within one year? All the participants agreed that, for rapid results, the money should be invested in a plantation; however, Wolfgang Barz made the proviso that 5% of the money should be assigned to molecular biology. He predicted that within ten years, all the investment would be in molecular biology. JOAN MARSH

The Ciba Foundation, 41 Portland Place, London W I N 4BN, UK.

specified by RNA complementary to that of ACTH (these are termed complementary peptides) (Fig. 1). HTCA 5'-3' bound ACTH in a saturable, high-affinity, specific fashion. Hydropathic inversion of amino acids for complementary codons read in the 5'-3' direction also occurs in the 3'-5' direction. The mRNA for HTCA results in a peptide that binds ACTH regardless of whether the amino acids are assigned in the 5'-3' or 3'-5' direction. We synthesized a molecule, HTCA 3'-5', in which the amino acids are assigned in the nonconventional 3'-5', rather than the 5'-3', direction: HTCA 3'-5' bound ACTH identically to the HTCA 5'-3' (Ref. 3). In spite of the fact that only eight of the 24 amino acids were common to both 5'-3' and 3'-5' HTCA, the two HTCAs were, of course, hydropathically virtually identical, since the genetic code allows for hydropathic interchange in either reading direction (Fig. 2). This data is not meant to imply that RNA is translated or transcribed in the 3'-5' direction, but is merely used to test the prediction that peptides with opposite hydropathic patterns will interact. Additional evidence that peptide interaction is due to hydropathic interchange is the antigenic