The Screening Hypothesis - a new explanation of secondary
product diversity and function
The hypothesis is composed of two parts.
1. The identification of a fundamental constraint that must be a very
significant factor in the evolution of secondary metabolism.
Potent, specific biological activity is a rare property for a molecule
to possess - that is why large screening programmes are needed find useful
biological activity. The low frequency of potent, specific biological activity
is a consequence of the specificity of ligand/binding site interactions.
Some organisms can increased their fitness by making and exploiting biological
active molecules however the low frequency of evolving new molecules places
severe constraints on the evolution of the biochemical pathways leading
to such products.
2. A proposal as to how secondary metabolism might have evolved
with such a constraint - including a prediction as to the metabolic traits
that would expected to minimize the effects of these constraints
How do organisms generate sufficient chemical diversity to enhance their
chances of finding the rare beneficial chemical? How do organisms retain
the capacity to generate so much chemical diversity when individual compounds
or pathways become redundant?. The Screening Hypothesis proposes
that certain metabolic traits (matrix pathways, non-enzymic transformations,
branched pathways, shared pathways and enzymes with a broad substrate tolerance)
would all help increase generation and retention of chemical diversity.
Some consequences of the hypothesis
One should not expect all naturally made chemicals to have a role in the
organisms that make them. Many will have no role and will never have had
any role in the organisms in which they are found. Many chemicals will
simply have been made because the metabolic machinery capable of their
production has a benefit for the producer. If only one product that those
pathways can produce has a beneficial biological activity, the pathways
will be sustained if the costs of possessing that capacity is sustainable.
One should not assume that some biological activity found in a screening
trial conducted by humans has any significance to the role of the chemical
in the organism that produces it. If organisms are producing chemical diversity
they must inevitably produce chemicals with structures that will possess
fortuitous biological activity in non-target organisms.
The metabolic traits predicted by the screening hypothesis will sometimes
make it hard to precisely genetically manipulate secondary product pathways.
For example, it is proposed that in order to enhance the production and
retention of chemical diversity, many enzymes involved in secondary product
biosynthesis will have low substrate specificity. Consequently, if a new
enzyme is introduced into an organism to cause the production of a new
secondary product, there is high probability that existing enzymes in the
transformed organism will further elaborate the new product to produce
more novel diversity.
Some of the metabolic traits predicted (for example low substrate specificity)
might be exploited in biotransformation and bioremediation studies.
The flux of carbon through secondary metabolite pathways must have been
very large throughout the period of life on earth. The metabolic traits
predicted by the Screening Hypothesis may have played a part in encouraging
the catabolism of this huge amount of chemical diversity. The world has
never been a clean place chemically hence organisms must have the capacity
to survive and thrive in the presence of chemical diversity. Most synthetic
chemicals released into the environment in small amounts will find enzymes
that can transform them.
Where can I read more about these ideas?
The following papers provide a more detailed consideration of the Screening
Hypothesis and its implications.
1. Jones CG and Firn RD (1991) Phil Trans Royal Soc 333, 273-280.
A common-sense evolutionary scenario predicts that well-defended plants
should have a moderate diversity of secondary compounds with high biological
activity. We contend that plants actually contain a very high diversity
of mostly inactive compounds. These patterns result because compounds arising
via mutation have an inherently low probability of possessing any biological
activity. Only those plants that make a lot of compounds will be well defended
because only high diversity confers a reasonable probability of producing
active compounds. Inactive compounds are retained, not eliminated, because
they increase the probability of producing new active compounds. Plants
should therefore have predictable metabolic traits maximising secondary
chemical diversity while minimising cost. Our hypothesis has important
implications to the study of the evolution of plant defence.
2. Firn RD and Jones CG (1996) In "Phytochemical Diversity and Redundancy
in Ecological Interactions". ed Romeo et al., Plenum Press, NY. pp. 295-312.
The Screening Hypothesis is a model for the evolution of secondary product
diversity that is based on extensive evidence, accumulated over several
decades, which has shown that specific, high-potency biological activity
is a rare property for a molecule to possess. Evidence to support this
observation comes from studies conducted at the organism, organ, tissue,
cell and molecular levels of organisation.
The proportion of naturally occurring molecules showing any biological
activity will depend on the concentration at which they are assayed. Consequently
it is argued that the meaningful comparative discussion of biological activity
of any collection of endogenous compounds can only be made with reference
to the concentration that will exist at the site of action of the compounds.
When biological activity is only shown by a substance when assayed at concentrations
above those that will occur in natural situations, it may be of pharmacological
interest but such activity should not be assumed to be meaningful in terms
of the discussing the possible role(s) that the substance might play in
the organism that produces it.
The Screening Hypothesis is based on concepts which place very severe
constraints on the evolutionary framework governing the generation, retention
and use of secondary products. It is suggested that these constraints operate
at a high level and that mechanisms to generate and retain chemical diversity
must have evolved at an early stage in the evolution of life. Indeed it
seems likely that secondary products may have been produced as a consequence
of "biochemical inventiveness"which must have been inevitable when primary
metabolism was evolving. "Secondary metabolism" might well have evolved
concurrently with primary metabolism and primitive organisms may have evolved
their "secondary metabolism" to exploit the inevitability of the generation
of some chemical diversity.
Finally we return to the theme of the book, redundancy. It is apparent
that a debate as to whether any secondary product is "redundant" becomes
a semantic argument. If one defines redundant as no longer playing a role
(i.e. the biological activity of a compound, which was once of value to
the producer, is no longer exploited), the Screening Hypothesis would suggest
that the many secondary products never had a role which resulted from their
inherent biological activity hence cannot be considered redundant in this
sense of the word. Likewise if one defines redundant as being superfluous
or over-copious (Geddie W, 1959), the Screening Hypothesis would provide
an arguments against the view that the majority of secondary products evolved
as a result of any superfluous production hence most secondary products
cannot be considered redundant in this sense. The Screening Hypothesis
suggests that the term "redundant" would really only be appropriate in
a very few circumstances. The Hypothesis suggests that the synthesis of
many compounds which bring no short term benefit to the producer is a necessary
part of the overall mechanisms employed by plants and microbes to produce
the occassional chemical which possesses useful biological activity. Most
secondary products are only "redundant" in the way that most antibodies
are "redundant". The production of the majority of these substances results
in no short-term benefit but short-term costs are compensated for by the
longer term benefits that result when the rare biologically active compound
is made. The majority of secondary products and antibodies are not redundant
in any commonly accepted use of the word but are a necessary consequence
of the need to generate chemical diversity.
3. R.D.Firn and C.G. Jones (1998) Avenues of discovery in bioprospecting.
This is a response to a previous Briefing in Nature (392, 535) which considered
the prospects for bioprospecting. We attempted to expain why bioprospecting
has been so unsuccessful - biological activity is a rare property for a
molecule to possess and contrary to common belief, natural products possess
a high potency biological activity are rare. We suggest that combinatorial
biochemistry could be a more productive way of exploiting genetic diversity.
4. R.D.Firn and C.G. Jones (1999) Secondary metabolism and GMOs. Nature
Responds to previous arguments that GMOs can be shown to be non-toxic by
testing or by metabolite screening. We argued that "the rules" of secondary
metabolism will make it hard to predict the outcome of the genetic manipulation
of secondary metabolism. Metabolite screening will be difficult because
of the unknown nature of the possible new products and the fact that a
full analysis of minor compounds in rarely attenpted. There is also a problem
in knowing how secondary metabolism will respond to challenges - which
induced profile do you test?
5. R.D Firn and C.G. Jones (2000) The evolution of secondary metabolism
- a unifying model. Molecular Microbiology 37, 989-994.
Why do microbes make secondary products? That question has been the subject
of intense debate for many decades. There are two extreme opinions. Some
argue that most secondary metabolites play no role in increasing the fitness
of an organism. The opposite view, now widely held, is that every secondary
metabolite is made because it possesses (or did posses at some stage in
evolution) a biological activity that endows the producer with increased
fitness. These opposing views can be reconciled by recognising that, due
to the principles governing molecular interactions, potent biological activity
is a rare property for any molecule to possess. Consequently, in order
for an organism to evolve the rare potent, biologically active molecule,
a great many chemical structures have to be generated, most of which will
possess no useful biological activity. Thus the two sides of the debate
about the role and evolution of secondary metabolism can be accommodated
within the view that the possession of secondary metabolism can enhance
fitness, but that many products of secondary metabolism will not enhance
the fitness of the producer. It is proposed that secondary metabolism will
have evolved such that traits that optimise the production and retention
of chemical diversity at minimum cost will have been selected. Evidence
exists for some these predicted traits. Opportunities now exist to exploit
these unique properties of secondary metabolism to enhance secondary product
diversity and to devise new strategies for biotransformation and bioremediation
6. R.D Firn (2003) Bioprospecting - why is it so unrewarding? Biodiversity
and Conservation 12,: 207-216.
Some economic analyses have placed high values on the chemical diversity
residing in threatened habitats (Principe, 1996; Balick and Mendelsohn,
1992; Rausser and Small, 2000). In particular, bioprospecting (searching
for new biologically active chemicals in organisms) is considered by some
of its advocates to be a route to funding the preservation of biodiversity,
especially in the LDCs. However, the large multinational pharmaceutical
and agrochemical companies devote very little of their research effort
to bioprospecting (Cordell, 2000). Why is this? The answer lies in the
fact that any chemical (whether synthetic or natural product) has a very
low chance of possessing useful biological activity. One model for the
evolution of natural product diversity that is gaining increasing support
suggests that the common belief, that natural products have been selected
by their producers such that only biologically active natural products
are commonly found, is wrong. Given that a random collection of synthetic
or natural products have similar chances of containing a biologically active
chemical and that synthetic chemicals are nearly always easier to synthesise
on an industrial scale, it is to be expected that bioprospecting does not
excite the multinationals. Although Rausser and Small (2000) argued that
scientific advances will make bioprospecting more cost effective in future,
an alternative scenario is presented where current biotechnological developments
will further erode the value of bioprospecting. Hence there should be no
expectation that large income streams will be available from bioprospecting
agreements to help fund the preservation of biodiversity.
7. R.D. Firn and C.G Jones (2003). Natural products - a simple model to
explain chemical diversity. Natural Products Reports, 20, 382-391.
A simple evolutionary model is presented which explains why organisms produce
so many natural products, why so many have low biological activity, why
enzymes involved in natural product synthesis have the properties they
do and why natural product metabolism is shaped as it is.
8. R.D. Firn & C.G.Jones (2004) The evolution of plant biochemistry
and the implications for physiology. In "The Evolution of Plant Physiology".
Some biochemical pathways are common to most plants ("primary metabolism").
Other pathways are widely found but are not universal (e.g. some parts
of the carotenoid or lipid biosynthetic pathways). There are also minor
branches leading from these pathways which are found only in a very small
number of species ("secondary metabolism"). Why is plant metabolism like
this? The Jones-Firn model to explain why plants and microbes produce so
many "secondary metabolites" is based on the fact that the chances of any
molecule possessing potent biological activity is very low. This model
has now been extended by considering what other properties new molecules
could bring to their producer. Two new propery classes are defined and
it is proposed that selection would shape metabolism depending on the type
of property. Furthermore, because particular properties will be associated
with individual molecules, and not with particular pathways, it is predictable
that many pathways will be multifunctional. By considering the evolution
of regulatory systems controlling such pathways it becomes clear that such
regulatory systems will have been shaped by the underlying multifunctionality
of some pathways with the result that "cross talk" is inevitable.
An appreciation of these constraints may help those seeking to understand
any physiological process that involve biologically active molecules (for
example plant hormones or compounds involved in a plant's response to insects,
fungi or bacteria).
The Screening Hypothesis has been criticised and the following work should
be consulted to read these criticisms.
The critics of the Screening Hypothesis
1. Berenbaum MR & Zangerl AR (1996) Phytochemical diversity - adaptation
or random variation. In "Phytochemical Diversity and Redundancy in Ecological
Interactions". ed Romeo et al., Plenum Press, NY. pp. 1-24.
However, it should be made clear to readers that much of the evidence that
is presented by Berenbaum & Zangerl concerns the furanocoumarins which
are compounds which seem to bring about their major biological effects
by covalently interacting with molecules. Making chemically reactive molecules
as a defensive system will give rise to broad spectrum activity and will
result in any molecules sharing the basic reactive parts of the structure
being "active". However, this broad spectrum activity is hard to target,
indeed such chemicals have a high chance of inducing a loss of fitness
in the producer and "resistance" costs to the producer maybe significant.
Evidence from studies of compounds that react covalently with their
targets does not contradict the basic ideas behind the Screening Hypothesis
which is based on the much commoner reversible binding of ligands to proteins.
We would argue that the furanocoumarins are a special case and it will
be interesting to see how common such colvalent interactions are.
Readers might be interested in noting that "site-directed irreversible
inhibitors" were once considered to offer very great potential as drugs
(a book by G Baker in the mid 1960s??) but the approach did not yield the
breakthroughs expected. Did the same happen in organisms?
Please engage us in debate. The hypothesis is there to argued about
and we really do enjoy thinking about its strengths and weaknesses. We
have some more papers in preparation which aim to develop the ideas further.
We are assembling evidence which is consistent with the predictions we
made in the original article consequently we would welcome being made aware
of evidence which supports or contradicts the predictions. We would also
welcome evidence as to the frequency of biological activity in collections
of chemicals. There must be masses of data in the files of screening companies
which would be valuable - we don't need to know the identity of the copounds
or even the exact screen being used!
Richard D Firn,