Lecture 5 – the Screening Hypothesis – evidence for and against

"All truth passes through three stages. First, it is ridiculed. Second, it is violently opposed. Third, it is regarded as self evident" Arthur Schopenhauer

Recap: Natural Products (NPs) have long been, and still are, the most valued chemicals in society. Much of history is about the control of that wealth. The study of NPs has been hindered by fragmentation of the subject. The evolutionary significance of NPs only began to attract attention a century after Darwin & Wallace (was Gromit involved?) advanced the theory of natural selection. The prevailing view is that NPs are made to benefit the producer – all NPs have a role or had a role. The chemical co-evolution model which has dominated thinking for 30 years came from the top down – ecologists were the driving force. The Screening Hypothesis, an alternative evolutionary model, was built from the bottom up on the basis that specific biological activity is a rare property for any molecule to possess.

Learning outcomes what the predictions of the Screening Hypothesis are what evidence can be found that is consistent with these predictions what evidence has been presented that is inconsistent with the predictions how the model might be tested more fully

Learning outcomes what are the predictions of the Screening Hypothesis what evidence can be found that is consistent with these predictions what evidence has been presented that is inconsistent with the predictions how the model might be tested more fully

what are the predictions of the Screening Hypothesis? 1. some enzymes involved in natural product synthesis will have a broad substrate specificity 2. some enzymes will carry out more than one stage of a biosynthetic pathway (because of 1) 3. matrix pathways will be be quite common (because of 1) 4. some enzymes will produce multiple products (another way of generating diversity and retaining it) 5. many natural products will have no known biological activity

what are the predictions of the Screening Hypothesis? Prediction 1.some enzymes involved in natural product synthesis will have a broad substrate specificity Spearmint Peppermint

Spearmint Peppermint Croteau, R., Karp, F., Wagschal, K.C., Satterwhite, D.M., Hyatt, D.C., and Skotland, C.B. (1991). Biochemical characterisation of a Spearmint mutant that resembles Peppermint in monoterpene content. Plant Phys, 96: Peppermi nt Spearmint

1. some enzymes involved in natural product synthesis will have a broad substrate specificity  “………in conclusion, it is suggested that further analysis of heterologously expressed proteins will confirm that multifunctional enzymes are ubiquitous ”

Phenylpropanoid pathway Prediction 2.some enzymes will carry out more than one stage of a biosynthetic pathway (because of prediction1)

Prediction 3matrix pathways will be be quite common (because of prediction1) Isoprenoid pathways to Gibberellins

Prediction 4some enzymes will produce multiple products (another way of generating diversity and retaining it)  Steele et al. (1998), studying some sesquiterpene synthases, found that one enzyme produced 34 different compounds from a single substrate and another produced 52. Substrate Chemically unstable intermediate Multiple rearrangment products

Learning outcomes what the predictions of the Screening Hypothesis are what evidence can be found that is consistent with these predictions what evidence has been presented that is inconsistent with the predictions how the model might be tested more fully

What evidence has been presented that is inconsistent with the predictions? 1. "Biological activity" is wrongly or poorly defined by Jones & Firn 2. Maybe it is mixtures that count, not single compounds? 3. The furanocoumarins as a paradigm? Most are biologically active. 4. The enzymes involved in furanocoumarins synthesis have a narrow substrate tolerance Berenbaum, M.R. and Zangerl, A.R. (1996) Phytochemical diversity: adaptation or random variation. In Recent Advances in Phytochemistry. Romeo J.T., Saunders J.A., and Barbosa, P. (eds). New York: Plenum Press, pp.1-24.

1. "Biological activity" is wrongly or poorly defined by Jones & Firn Biological ActivityBiomolecular Activity Biomolecular activity is defined as the ability of one particular chemical to reversibly associate with one particular protein such that the functioning of that protein is disturbed Each type of Biological activity will have at least one underlying biomolecular activity

1. "Biological activity" is wrongly or poorly defined by Jones & Firn Indeed the term Biological activity is too vague because it could refer to: ecosystem level population level individual organism level organ or tissue level molecular level

2. Maybe it is mixtures that count, not single compounds? Additive effects: I g A + 1g B = 2g A = 2g B Synergistic effects 1g A + 1g B < 2 g A or 2g B N.B. Any chemical tested on a plant (for fungicidal or insecticidal activity is being tested in a mixture because hundreds of NPs are already in the plant!

2. Maybe it is mixtures that count, not single compounds? Chemical A (high BM activity)Breakdown product (v low BM activity) Enzyme Chemical B inhibits Enzyme When B is absent A gets broken down thus no A effect. When B is given in absence of A no effect seen When A is given with B, then the effect of A and B are seen But A and B both act individually hence each obeys the Law of Mass Action consequently each will have a low probability of potent, specific biomolecular activity

3. The furanocoumarins as a paradigm? Most are biologically active.

These chemicals are highly chemicaly reactive or photoreactive – they form covalent bonds. They are atypical as they do not react reversibly with non-covant interactions. Many related chemicals, sharing the same reactive chemical groups will have similar properties. They are a poor model for Natural Products in general …. But interesting all the same

4. The enzymes involved in furanocoumarins synthesis have a narrow substrate tolerance OK but there is plenty of evidence that this is not universally true. Could it be something to do with their chemical reactivity?

Could the Screening Hypothesis be tested more fully? 1. Is biological activity a rare property for any molecule? HTS could answer this question … indeed it has answered it 2. Are enzymes involved in Natural Product synthesis substrate specific? More substrate testing needed. 3. Genetic manipulation of Natural Product biosynthesis pathways Add genes coding for N P making enzymes into organisms already making them and look for increased chemical diversity

Summary Even without the hypothesis being subjected to specific tests the Screening Hypothesis looks promising: some enzymes involved in natural product synthesis do have a broad substrate specificity some enzymes do carry out more than one stage of a biosynthetic pathway (because of 1) matrix pathways are quite common (because of 1) some enzymes do produce multiple products (another way of generating diversity and retaining it) most Natural Products do not posess specific, potent biomolecular activity …. But that is a circular agrument ….

Essay Titles for Examination 1. Humans have seemingly been obsessed with NPs. Discuss the biological issues that you consider to be relevant to the analysis of that obsession. 2. Natural scents, colours and flavours are NPs of great commercial importance. How easy has it been for breeders to manipulate the content of each of these valuable products? How should an understanding of NP evolution inform future attempts to exploit these NPs?