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Time flies like an arrow: Fruit flies like a banana
Perspectives in Science (2015) 6, 113—120
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/pisc
Time flies like an arrow: Fruit flies like a banana夽 Athel Cornish-Bowden CNRS-BIP, Aix-Marseille Université, 31 Chemin Joseph-Aiguier, B.P. 71, 13402 Marseille, France Received 7 July 2014; accepted 27 May 2015 Available online 17 October 2015
KEYWORDS Circadian rhythms; Logic of metabolism; Drug development; Evolution; Nanomaterials; Fuels
Summary The 2014 Beilstein Bozen Symposium on Chemistry and Time was as diverse in topics, and hence as difficult to summarise in a few words, as all of its predecessors. We can all agree, however, that time is fundamental in all of science, affecting everything we do, and everything that happens in a chemical system, and so the diversity of topics is just a reflection of reality, and the Symposium in Prien left all of its participants with the feeling that they had learned something interesting and novel. © 2015 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Contents Introduction ................................................................................................................ 114 Biological time ............................................................................................................. 114 Clocks and life.........................................................................................................114 Circadian rhythms ..................................................................................................... 114 Sleep research.........................................................................................................114 Ageing ................................................................................................................. 115 Metabolism ................................................................................................................. 115 Metabolism and time .................................................................................................. 115 The logic of metabolism ............................................................................................... 115 Failures of metabolic and signalling pathways ......................................................................... 115 Modelling metabolism ................................................................................................. 115 Drug discovery ............................................................................................................. 116 Why must metabolic and signalling pathways be so complicated? ...................................................... 116
夽 This article is part of a special issue entitled ‘‘Proceedings of the Beilstein Bozen Symposium 2014 — Chemistry and Time’’. Copyright by Beilstein-Institut www.beilstein-institut.de E-mail address: [email protected]
http://dx.doi.org/10.1016/j.pisc.2015.05.004 2213-0209/© 2015 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Getting drugs to their targets..........................................................................................116 Nanomaterials ......................................................................................................... 116 Evolutionary time .......................................................................................................... 117 Directed evolution.....................................................................................................117 Evolution of defence mechanisms ..................................................................................... 117 Chemistry as creation science ......................................................................................... 117 Electron transport and the energy crisis .................................................................................... 117 Hydrogen as a clean fuel .............................................................................................. 117 Understanding electron transport......................................................................................117 Nanocrystallography........................................................................................................118 Concluding remarks ........................................................................................................ 118 Conflict of interest ......................................................................................................... 118 References ................................................................................................................. 118
Introduction The title of the article is, of course, a play on the different meanings of the word flies, but it is more relevant to note that the opening word, time, has a variety of meanings as well, and has many diverse effects on physical, chemical and biological processes. It spans 25 orders of magnitude, from the 14 billion years of the astrophysicists to the femtoseconds (Chapman et al., 2011) of the crystallographers, and at every stage on the scale it is relevant to chemistry and biology. Much of this range was discussed during the 2014 Beilstein Bozen Symposium on Chemistry and Time, and it is not easy to summarise it all succinctly. One thing is clear, however: everyone who was lucky enough to participate in this symposium went away having learned some interesting information that they did not know before.
Biological time Clocks and life In his lecture Tim Clark described biology as ‘‘stuff happening in dirty water’’, but those of us who are biologists like to think of biology as more interesting than that implies, and time is no less important in the timescales that we notice in living systems as it is for the millions of years considered by geologists, astrophysicists and evolutionary biologists, or the femtoseconds that allow structures to be determined before they disintegrate. I shall start by thinking about the most familiar time scales, from seconds to days to years that define our everyday lives. All animals are profoundly influenced by time, and humans are no exception. We live by the clock, but we sometimes forget the underlying physiology, thinking that we can ignore the problems created by variations in day length during the year, as well as those — more obvious — caused by moving through several time zones in a few hours, made easy by modern air travel. Our physiology did not evolve to experience artificial light during the periods we wish to be awake, but research on the nature of sleep and the effects on it of an artificial clock that ignores changes in day length has been surprisingly sparse. Despite the alarming evidence of widespread pathologies due to sleep-related problems, with costs believed to
approach 1% of gross domestic product in some countries, sleep research is barely in the top 100 of the funding categories of the US National Institutes of Health (Roenneberg, 2013).
Circadian rhythms Research into the nature of the circadian clock has, of course, been applied for many years, at least since the classic review of Hess and Boiteux (1971), and pursued subsequently by others (Goldbeter, 1996). However, these studies have been designed primarily to understand the biochemical mechanisms of circadian rhythms, for example, the role of cycling of the monosaccharide -Nacetylglucosamine in them (Ma and Hart, 2014), as described by Gerald Hart, or the structure and function of cryptochromes, proteins that regulate clock genes (Schmalen et al., 2014). Akhilesh Reddy described how some other proteins, the peroxiredoxins, are closely related to nontranscriptional mechanisms that contribute to timekeeping, and their circadian oscillation is conserved across the whole range of life: bacteria, archaea and eukaryotes (Edgar et al., 2012). He explained how this is related to the different effects of the same nutritional intake at different times of day on human body weight: better to consume calories at breakfast than the same amount at dinner.
Sleep research By contrast, the effects on health of interfering with rhythms has been given much less attention, despite the observation that shift work, for example, can greatly increase the likelihood of cancer (Knutsson et al., 2013), or that sleep disturbances are associated with early Parkinson’s disease (Breen et al., 2014). Till Roenneberg described the human sleep project (Roenneberg, 2013), which is designed to fill in some of the gaps: it uses a questionnaire to collect information from more than 150,000 respondents around the world about their sleeping habits, in the hope of obtaining a better understanding of the relationship between sleep and health. A particularly interesting part concerns the Quilombos, an Afro-Brazilian population that is genetically fairly homogeneous, but very diverse in terms of living habits: some live
Time flies like an arrow in rural communities with no electricity, whereas others live in modern cities.
Ageing A different aspect of the physiology of time comes from studies of ageing, which have a major and increasing importance for human health, because, as Adam Antebi described, Alzheimer’s disease is not only increasing but is expected to increase more steeply than ever in the future as the world population ages, reaching about three times present levels in the next 40 years (Hebert et al., 2013), and the incidence of other forms of senility is increasing similarly. So we need to understand how the different stages of life are specified, what brings about the transitions between them, and how they are related to longevity. The nematode Caenorhabditis elegans has proved a useful model for studying these questions (Antebi, 2013). As an animal with a precisely defined set of cells, and a normal lifetime short enough for convenient study that can be modified by various techniques, it allows many of them to be addressed. For example, the steroid hormone receptor-microRNA switch is found to affect timing circuits and longevity.
Metabolism Metabolism and time Metabolism is intimately linked with time, which plays a crucial role in life. On the one hand organisms need to maintain themselves in homeostasis despite frequent and unpredictable changes in their environment; in other words they must achieve self-organisation (Letelier et al., 2011), but all of their efforts eventually fail, as they inevitably age and finally die. All of the metabolic reactions of organisms must have kinetic and dynamic properties consistent with their need to stay alive.
The logic of metabolism The vast array of these reactions can appear hopelessly arbitrary and unmemorable to the student encountering them for the first time in elementary biochemistry courses. However, Antoine Danchin pointed out that closer study reveals a great deal of logical organisation, albeit it with some ‘‘historical’’ constraints inherited from the origin of life, and others from the impossibility of achieving total specificity in enzyme-catalysed reactions: errors in following the informational rules can result in the appearance of a ‘‘shadow metabolism’’ (Danchin and Sekowska, 2013). Selenium toxicity, for example, results from the ability of selenium atoms to substitute for sulphur atoms in some conditions, but not in all, but some bacteria have learned to use selenocysteine as a ‘‘paralogous’’ metabolite, making it the 21st amino acid that is genetically coded (in some organisms). A similar example might appear to be the capacity of arsenic to replace phosphorus in some reactions, and some authors have claimed that arsenicbased life can be observed in some extreme environments (Wolfe-Simon et al., 2011). However, this claim has been
115 rejected almost universally by authors who understand the inescapable chemical fact that arsenate esters are far too labile to exist for a significant time in cells, for example some of my colleagues in Marseilles (Schoepp-Cothenet et al., 2011). The notion of arsenic-based life derives from failure to take account of time as a determinant of which kinds of biological structures are possible. Hiroki Ueda and his colleagues are applying the idea of metabolic logic to circadian clocks, asking, for example, what conditions will allow a temperature-insensitive clock (Jolley et al., 2012). A very simple system, consisting of just one enzyme and one peptide substrate, can produce a temperature-insensitive reaction, but it cannot generate oscillations. However, these become possible with a slightly more complex system with two enzymes (such as a kinase and a phosphatase) with one peptide substrate, and it appears that protein phosphorylation is a limiting step in the mammalian circadian clock.
Failures of metabolic and signalling pathways No matter how logical and well-designed a metabolic system may appear to be, however, it can never operate perfectly, in part on account of the impossibility of perfect specificity mentioned already, and Dario Alessi pointed out that most human diseases can be attributed to failures in signalling pathways. Cancer, for example, was suggested as long as 90 years ago to be a metabolic disorder (Warburg et al., 1924), but this has only become widely recognised in the past decade, in part as a result of the discovery of the upstream protein kinase that acts on the AMP-activated protein kinase as a complex containing LKB1, a known tumour suppressor (Hawley et al., 2003). This led to a great deal of further research, as recently reviewed (Hardie and Alessio, 2013). One may add to this the realisation that there is an unexpected link between cancer and metabolic control analysis. It has been known for more than a century than cancer cells are typically aneuploid (Hansemann, 1890), with an incorrect number of chromosomes, but it remained far from obvious why this might be related to the clinical manifestations of cancer. Metabolic control analysis, in contrast, is concerned with the relationship between enzyme activity and metabolic fluxes, with an important generalisation that changing the activity of an enzyme typically has no significant effect on metabolic flux (Kacser and Burns, 1973). However, that is for changes in one or at least a small number of enzyme activities; it is very different in an aneuploidal cell in which many enzymes simultaneously have abnormal concentrations, and hence abnormal activities. In this case serious metabolic disturbances can arise that can, when they become sufficiently severe, lead to cancer (Duesberg et al., 2001).
Modelling metabolism Because of the large number of metabolic reactions, most of them only poorly characterised, it remains a huge task to construct a model of the whole of metabolism, but heroic efforts to do this are in progress. On the one hand there are a few pathways, such as glycolysis in the human erythrocyte (du Preez et al., 2008), the parasite Trypanosoma brucei
116 (Bakker et al., 1999), or yeast (Van Eunen et al., 2012), for which an adequate supply of usable kinetic data is available, but these remain exceptional — a situation that the Beilstein STRENDA initiative (Kettner and Cornish-Bowden, 2014) is striving to overcome — and for most metabolic systems kinetic modelling is a dream for the future. Large-scale models, therefore, have to be based on stoichiometric information: knowing which reactions are present, but without knowing their kinetics. The scale of such an effort can be judged from Recon 2 (Swainston et al., 2013), a reconstruction of human metabolism consisting of 6852 biochemical and transport reactions distributed over eight intracellular compartments, 2469 unique metabolites, and enzymes coded by 1766 genes. It will be obvious that a project of such magnitude requires input from many research groups around the world, and for that reason Neil Swainston and his colleagues call Recon 2 a ‘‘community-driven’’ reconstruction. As an example of the type of problem that can be addressed with a reconstruction of this kind one can mention the effect of deleting the gene for the enzyme fumarase in renal cancer (Frezza et al., 2011). However, without wishing to devalue this type of study one may regret, in a symposium on Chemistry and Time, the absence of any kinetic information.
Drug discovery Why must metabolic and signalling pathways be so complicated? One of the ways in which organisms can overcome the lack of perfect specificity in individual processes is to evolve more complicated systems that involve several components, and this is a major reason for the apparently unnecessary complexity of living organisms. Tom Blundell pointed out that traditional ideas of signalling pathways would give very noisy — and hence inadequate — signals, but multi-component protein assemblies can greatly improve the signal-to-noise ratio in cell regulation, molecular interactions being stabilised cooperatively as the components are assembled into a signalling complex. This allows signalling systems to be both selective and reversible, but from the researcher’s point of view it leads to two challenges (Winter et al., 2012): how to design drugs that target protein—protein interfaces, and how to understand the mechanisms of the assembly. Francesco Gervasio described a different approach to understanding ligand—protein interactions, pointing out that a major problem for modelling binding kinetics is the lack of information in most cases of the detailed Gibbsenergy profiles along association pathways. An effective computational strategy can, however, allow binding kinetics to be calculated (Juraszek et al., 2013), as well as the conformational changes that occur on binding (Sutto and Gervasio, 2013). Akhilesh Reddy’s report of the effects of circadian rhythms, mentioned already, is clearly relevant here: the effectiveness of a drug must depend not only on the dose but also on when in the circadian cycle it is administered. What is optimistically called rational drug design, a term that was already treated with scepticism as long ago as 1966 (Belleau, 1966), is often pursued with no consideration of metabolism
A. Cornish-Bowden at all. For example, a supplement to Nature entitled ‘‘intelligent drug design’’ (Various authors, 1996) nowhere included the words ‘‘metabolism’’ or ‘‘metabolic’’, and a similar special issue of Science on drug discovery (Various authors, 2000) was barely any better (Cárdenas and CornishBowden, 2000). The point that is overlooked in these efforts is that what (most) drugs do is to modify metabolism, and the circadian point is that to be effective they have to act on the metabolic state that exists at the time of administration.
Getting drugs to their targets I first learned of Lipinski’s rules in a lecture by Christopher Lipinski himself at the first Beilstein Bozen Symposium that I attended in 2002 (Lipinski, 2002). The rules had been first proposed some years earlier (Lipinski et al., 1997), and have been enormously influential in the pharmaceutical industry: the original paper has now been cited more than 3000 times, with more than 300 in each of the years 2011—2013. Nonetheless, Douglas Kell has strongly argued (Kell, 2013) that this approach is largely misconceived, and that that is why adherence to the rules has not been very effective in drug discovery. The problem is that they allow prediction of the properties that allow a molecule to enter a cell by passive diffusion, but that is not at all what one needs to know, because most effective drugs are transported into cells by specific carriers. Trevor Howe argued in addition that kinetic considerations also needs to be taken into account, as equilibrium assays are not likely to be enough. He described the Innovative Medicines Initiative Kinetics for Drug Discovery Programme, a European project costing around 21 million euros over five years, and involving seven major drug companies and ten universities in four countries (Müller-Fahrnow, 2010). This idea was developed by Emad Tajkhorshid, who discussed how membrane transport can be simulated computationally with high resolution in both time and space, for example in ATP-binding cassette transporters (Moradi and Tajkhorshid, 2013).
Nanomaterials Even if membrane transporters are the principal mediators of active exchange of materials across cellular membranes, they are not the only ones. Drug delivery systems do not have to rely on natural transporters, and nanomaterials are attracting attention as an alternative, as presented by Nathan Gianneschi. He and his group showed that active accumulation of nanoparticles in tumours can be achieved in a specific assembly event that responds to biochemical signals associated with tumour tissue (Chien et al., 2013). It is impossible to prepare and test every conceivable kind of nanoparticle, because, as Dave Winkler said in his presentation, there are potentially more drug-like molecules that obey the laws of chemical valence than there are atoms of matter in the universe. Instead it is essential to have powerful computational methods that allow the biological effects of potential nanomaterials to be modelled and predicted in silico. For this we need to know not only the possible useful biological effects of such nanomaterials, but also any potential dangers that they may represent. Novel
Time flies like an arrow sparse machine-learning methods based on Bayesian neural networks are thus being developed for this purpose (Winkler et al., 2014), and they prove to have useful predictive value.
Evolutionary time
117 different genera of protozoans, the major pathogens Trypanosoma and Leishmania, only 80 million years ago. Not only that, but as Robert Field described, it has a very large number of protein-coding genes, similar to that of humans, and transcriptomic studies are allowing understanding of how it makes, uses and controls molecules and processes.
Directed evolution Chemistry as creation science As everyone knows, evolution by natural selection is excruciatingly slow. The last universal common ancestor (‘‘LUCA’’) of all extant organisms is thought to have existed more than 3 × 109 years ago, but evolution has been slow enough to allow many of its characteristics to be deduced from those of modern organisms. Moreover, natural selection is a poor algorithm for optimisation, akin to what computer scientists call greedy algorithms, which work by making the best local choice at every step, in the hope of finding a global optimum, but with no vision of where this optimum is likely to be. However, as argued by Philippe Marlière, not only are biotechnologists unwilling to wait millions of years to arrive at useful results; they also believe that they can proceed faster by means of directed evolution under human selection and synthetic biology. For example artificial nucleic acids (Herdewijn and Marlière, 2012) can propagate genetic information in vivo deliberately insulated from the normal processes of replication and transcription. Quite apart from its technological promise, directed evolution can lead us to a better understanding of how the genetic code developed: it can hardly have coded for as many as 20 amino acids at the outset, but which ones did it have and how did others arise? Jeffrey Wong’s coevolution theory (Wong, 2005), which postulates that ‘‘new’’ codons were given to new amino acids by their metabolic precursors, probably provides part of the answer, but one can also study the question by asking how an enzyme that used only a few amino acids could be improved by expanding its resources: for example, how could a chorismate mutase with only nine amino acids be improved by allowing it access to 20 (Muller et al., 2013)? This type of study has led to some surprising results: for example, one might expect that mutating leucine into such a similar amino acid as valine might offer little advantages, but such apparently innocuous substitutions can be observed in parallel in independent evolutionary trajectories; this suggests that even building blocks with very similar structures can be highly beneficial for fine-tuning enzyme structure and function.
Evolution of defence mechanisms Given the time available, many organisms, including agriculturally important pests such as the leaf beetle Chrysomela populi, have arrived at defence mechanisms against predators, in which, as Wilhelm Boland described, the larvae have ‘‘bioreactors’’ on their backs that discharge droplets of toxins when they are attacked (Discher et al., 2009). The green alga Euglena gracilis, familiar to most people who have taken biology practical classes, is not only very versatile in its metabolism, allowing it respond efficiently to redox stress, but also has a complicated evolutionary history. One might expect it to be closely related to other algae and plants, but in fact it separated from quite
Creationism is normally regarded as being in complete opposition to evolutionary science, and that is certainly true if we restrict attention to natural biology (Cornish-Bowden et al., 2014). However, Leroy Cronin pointed out in his presentation that synthetic chemistry is a creationist science: chemists envisage the molecules they want to create and then design them on the basis of chemical principles, an approach completely different from natural selection. He is exploring the possibility of inorganic systems that can evolve and provide insight into the prebiotic evolution that predated the world of nucleic acids (Cronin, 2013).
Electron transport and the energy crisis Hydrogen as a clean fuel The approaching energy crisis is one of the major problems that humanity will need to solve in the coming decades if disaster is to be avoided. It is easy to say, and I have said it myself, that large savings in energy can be made by adopting less wasteful habits, but, as Stenbjörn Styring pointed out in his lecture, that cannot solve the problem: it can only save energy in the developed countries, whereas many populations that live today in a highly undeveloped state can be expected to increase their energy requirements enormously by 2050. Moreover, electricity accounts for only 17% of energy consumption today, and more efficient generation of electrical power can only improve a small part of the problem. Many groups today are accordingly working on ways of using solar energy to produce more efficient fuels that can be oxidised without producing carbon dioxide, most notably hydrogen gas (Magnuson et al., 2009). Attempts to produce fuels by natural photosynthesis in living organisms, such as bacteria or algae (Matsumura et al., 2014), or active enzymes obtained from them (de Poulpiquet et al., 2014), are attracting considerable attention, but an alternative is to use biomimetic methods in which purely chemical catalysts are designed on the principles learned by studying such organisms (Simmons et al., 2014).
Understanding electron transport Applying any of these approaches successfully will require increased understanding of electron transfer in proteins, the subject of Israel Pecht’s presentation. How are the rates of electron transfer between redox centres controlled, tuned for efficiency, and made capable of avoiding harmful products? These aspects can be probed by studying model systems such as the bacterial copper proteins azurin and laccase, and examining the effects of mutations on rates and
118 structure (Farver and Pecht, 2011). Such proteins are also attractive for potential uses in bioelectronic systems, which can be studied by examining their conductance properties in the solid state (Ron et al., 2010). As described by Tim Clark, a problem that arises in simulating electron transport is that the processes to be simulated take place in time domains that differ by many orders of magnitude. For example, electronic devices constructed with self-assembled monolayers of flexible organic molecules need to be coupled with very much faster electron transport through the system. A practical approach — not strictly valid but giving useful results in practice — involves simulation of electron movement on static snapshots from simulations on the long time scale of the conformational changes (Leitherer et al., 2014). This type of calculation requires the power of modern parallel processing, but this is feasible because ‘‘a smart phone today has the power of a top-100 computer of 1990’’.
Nanocrystallography At present serial femtosecond nanocrystallography allows X-ray diffraction snapshots to be obtained of fully hydrated nanocrystals at room temperature (Chapman et al., 2011). The advantage of this is that most damage processes occur on much longer time scales, and so large membrane protein complexes can be examined before they have had time to disintegrate. This already represents a major advance on what was possible comparatively recently, but it has the disadvantage that the Linac Coherent Light Source is not only an extremely expensive piece of equipment, but it also occupies a large amount of space: the SLAC National Accelerator Laboratory in California is about 3.2 km in length. In her concluding presentation in the symposium Petra Fromme foresaw the construction in the next few years of an accelerator that could operate in the attosecond range of time, would be of the order of a metre in length, and would be relatively inexpensive (by the standards of linear accelerators). If this is achieved it will represent a huge technological advance.
Concluding remarks In conclusion, the 2014 Beilstein Bozen Symposium on Chemistry and Time assembled a very range of expert lecturers who were very successful in illustrating the importance of time at many different levels of chemistry and biology.
Conflict of interest The author declares that there is no conflict of interest.
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Time flies like an arrow: Fruit flies like a banana夽 Athel Cornish-Bowden CNRS-BIP, Aix-Marseille Université, 31 Chemin Joseph-Aiguier, B.P. 71, 13402 Marseille, France Received 7 July 2014; accepted 27 May 2015 Available online 17 October 2015
KEYWORDS Circadian rhythms; Logic of metabolism; Drug development; Evolution; Nanomaterials; Fuels
Summary The 2014 Beilstein Bozen Symposium on Chemistry and Time was as diverse in topics, and hence as difficult to summarise in a few words, as all of its predecessors. We can all agree, however, that time is fundamental in all of science, affecting everything we do, and everything that happens in a chemical system, and so the diversity of topics is just a reflection of reality, and the Symposium in Prien left all of its participants with the feeling that they had learned something interesting and novel. © 2015 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Contents Introduction ................................................................................................................ 114 Biological time ............................................................................................................. 114 Clocks and life.........................................................................................................114 Circadian rhythms ..................................................................................................... 114 Sleep research.........................................................................................................114 Ageing ................................................................................................................. 115 Metabolism ................................................................................................................. 115 Metabolism and time .................................................................................................. 115 The logic of metabolism ............................................................................................... 115 Failures of metabolic and signalling pathways ......................................................................... 115 Modelling metabolism ................................................................................................. 115 Drug discovery ............................................................................................................. 116 Why must metabolic and signalling pathways be so complicated? ...................................................... 116
夽 This article is part of a special issue entitled ‘‘Proceedings of the Beilstein Bozen Symposium 2014 — Chemistry and Time’’. Copyright by Beilstein-Institut www.beilstein-institut.de E-mail address: [email protected]
http://dx.doi.org/10.1016/j.pisc.2015.05.004 2213-0209/© 2015 The Author. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Getting drugs to their targets..........................................................................................116 Nanomaterials ......................................................................................................... 116 Evolutionary time .......................................................................................................... 117 Directed evolution.....................................................................................................117 Evolution of defence mechanisms ..................................................................................... 117 Chemistry as creation science ......................................................................................... 117 Electron transport and the energy crisis .................................................................................... 117 Hydrogen as a clean fuel .............................................................................................. 117 Understanding electron transport......................................................................................117 Nanocrystallography........................................................................................................118 Concluding remarks ........................................................................................................ 118 Conflict of interest ......................................................................................................... 118 References ................................................................................................................. 118
Introduction The title of the article is, of course, a play on the different meanings of the word flies, but it is more relevant to note that the opening word, time, has a variety of meanings as well, and has many diverse effects on physical, chemical and biological processes. It spans 25 orders of magnitude, from the 14 billion years of the astrophysicists to the femtoseconds (Chapman et al., 2011) of the crystallographers, and at every stage on the scale it is relevant to chemistry and biology. Much of this range was discussed during the 2014 Beilstein Bozen Symposium on Chemistry and Time, and it is not easy to summarise it all succinctly. One thing is clear, however: everyone who was lucky enough to participate in this symposium went away having learned some interesting information that they did not know before.
Biological time Clocks and life In his lecture Tim Clark described biology as ‘‘stuff happening in dirty water’’, but those of us who are biologists like to think of biology as more interesting than that implies, and time is no less important in the timescales that we notice in living systems as it is for the millions of years considered by geologists, astrophysicists and evolutionary biologists, or the femtoseconds that allow structures to be determined before they disintegrate. I shall start by thinking about the most familiar time scales, from seconds to days to years that define our everyday lives. All animals are profoundly influenced by time, and humans are no exception. We live by the clock, but we sometimes forget the underlying physiology, thinking that we can ignore the problems created by variations in day length during the year, as well as those — more obvious — caused by moving through several time zones in a few hours, made easy by modern air travel. Our physiology did not evolve to experience artificial light during the periods we wish to be awake, but research on the nature of sleep and the effects on it of an artificial clock that ignores changes in day length has been surprisingly sparse. Despite the alarming evidence of widespread pathologies due to sleep-related problems, with costs believed to
approach 1% of gross domestic product in some countries, sleep research is barely in the top 100 of the funding categories of the US National Institutes of Health (Roenneberg, 2013).
Circadian rhythms Research into the nature of the circadian clock has, of course, been applied for many years, at least since the classic review of Hess and Boiteux (1971), and pursued subsequently by others (Goldbeter, 1996). However, these studies have been designed primarily to understand the biochemical mechanisms of circadian rhythms, for example, the role of cycling of the monosaccharide -Nacetylglucosamine in them (Ma and Hart, 2014), as described by Gerald Hart, or the structure and function of cryptochromes, proteins that regulate clock genes (Schmalen et al., 2014). Akhilesh Reddy described how some other proteins, the peroxiredoxins, are closely related to nontranscriptional mechanisms that contribute to timekeeping, and their circadian oscillation is conserved across the whole range of life: bacteria, archaea and eukaryotes (Edgar et al., 2012). He explained how this is related to the different effects of the same nutritional intake at different times of day on human body weight: better to consume calories at breakfast than the same amount at dinner.
Sleep research By contrast, the effects on health of interfering with rhythms has been given much less attention, despite the observation that shift work, for example, can greatly increase the likelihood of cancer (Knutsson et al., 2013), or that sleep disturbances are associated with early Parkinson’s disease (Breen et al., 2014). Till Roenneberg described the human sleep project (Roenneberg, 2013), which is designed to fill in some of the gaps: it uses a questionnaire to collect information from more than 150,000 respondents around the world about their sleeping habits, in the hope of obtaining a better understanding of the relationship between sleep and health. A particularly interesting part concerns the Quilombos, an Afro-Brazilian population that is genetically fairly homogeneous, but very diverse in terms of living habits: some live
Time flies like an arrow in rural communities with no electricity, whereas others live in modern cities.
Ageing A different aspect of the physiology of time comes from studies of ageing, which have a major and increasing importance for human health, because, as Adam Antebi described, Alzheimer’s disease is not only increasing but is expected to increase more steeply than ever in the future as the world population ages, reaching about three times present levels in the next 40 years (Hebert et al., 2013), and the incidence of other forms of senility is increasing similarly. So we need to understand how the different stages of life are specified, what brings about the transitions between them, and how they are related to longevity. The nematode Caenorhabditis elegans has proved a useful model for studying these questions (Antebi, 2013). As an animal with a precisely defined set of cells, and a normal lifetime short enough for convenient study that can be modified by various techniques, it allows many of them to be addressed. For example, the steroid hormone receptor-microRNA switch is found to affect timing circuits and longevity.
Metabolism Metabolism and time Metabolism is intimately linked with time, which plays a crucial role in life. On the one hand organisms need to maintain themselves in homeostasis despite frequent and unpredictable changes in their environment; in other words they must achieve self-organisation (Letelier et al., 2011), but all of their efforts eventually fail, as they inevitably age and finally die. All of the metabolic reactions of organisms must have kinetic and dynamic properties consistent with their need to stay alive.
The logic of metabolism The vast array of these reactions can appear hopelessly arbitrary and unmemorable to the student encountering them for the first time in elementary biochemistry courses. However, Antoine Danchin pointed out that closer study reveals a great deal of logical organisation, albeit it with some ‘‘historical’’ constraints inherited from the origin of life, and others from the impossibility of achieving total specificity in enzyme-catalysed reactions: errors in following the informational rules can result in the appearance of a ‘‘shadow metabolism’’ (Danchin and Sekowska, 2013). Selenium toxicity, for example, results from the ability of selenium atoms to substitute for sulphur atoms in some conditions, but not in all, but some bacteria have learned to use selenocysteine as a ‘‘paralogous’’ metabolite, making it the 21st amino acid that is genetically coded (in some organisms). A similar example might appear to be the capacity of arsenic to replace phosphorus in some reactions, and some authors have claimed that arsenicbased life can be observed in some extreme environments (Wolfe-Simon et al., 2011). However, this claim has been
115 rejected almost universally by authors who understand the inescapable chemical fact that arsenate esters are far too labile to exist for a significant time in cells, for example some of my colleagues in Marseilles (Schoepp-Cothenet et al., 2011). The notion of arsenic-based life derives from failure to take account of time as a determinant of which kinds of biological structures are possible. Hiroki Ueda and his colleagues are applying the idea of metabolic logic to circadian clocks, asking, for example, what conditions will allow a temperature-insensitive clock (Jolley et al., 2012). A very simple system, consisting of just one enzyme and one peptide substrate, can produce a temperature-insensitive reaction, but it cannot generate oscillations. However, these become possible with a slightly more complex system with two enzymes (such as a kinase and a phosphatase) with one peptide substrate, and it appears that protein phosphorylation is a limiting step in the mammalian circadian clock.
Failures of metabolic and signalling pathways No matter how logical and well-designed a metabolic system may appear to be, however, it can never operate perfectly, in part on account of the impossibility of perfect specificity mentioned already, and Dario Alessi pointed out that most human diseases can be attributed to failures in signalling pathways. Cancer, for example, was suggested as long as 90 years ago to be a metabolic disorder (Warburg et al., 1924), but this has only become widely recognised in the past decade, in part as a result of the discovery of the upstream protein kinase that acts on the AMP-activated protein kinase as a complex containing LKB1, a known tumour suppressor (Hawley et al., 2003). This led to a great deal of further research, as recently reviewed (Hardie and Alessio, 2013). One may add to this the realisation that there is an unexpected link between cancer and metabolic control analysis. It has been known for more than a century than cancer cells are typically aneuploid (Hansemann, 1890), with an incorrect number of chromosomes, but it remained far from obvious why this might be related to the clinical manifestations of cancer. Metabolic control analysis, in contrast, is concerned with the relationship between enzyme activity and metabolic fluxes, with an important generalisation that changing the activity of an enzyme typically has no significant effect on metabolic flux (Kacser and Burns, 1973). However, that is for changes in one or at least a small number of enzyme activities; it is very different in an aneuploidal cell in which many enzymes simultaneously have abnormal concentrations, and hence abnormal activities. In this case serious metabolic disturbances can arise that can, when they become sufficiently severe, lead to cancer (Duesberg et al., 2001).
Modelling metabolism Because of the large number of metabolic reactions, most of them only poorly characterised, it remains a huge task to construct a model of the whole of metabolism, but heroic efforts to do this are in progress. On the one hand there are a few pathways, such as glycolysis in the human erythrocyte (du Preez et al., 2008), the parasite Trypanosoma brucei
116 (Bakker et al., 1999), or yeast (Van Eunen et al., 2012), for which an adequate supply of usable kinetic data is available, but these remain exceptional — a situation that the Beilstein STRENDA initiative (Kettner and Cornish-Bowden, 2014) is striving to overcome — and for most metabolic systems kinetic modelling is a dream for the future. Large-scale models, therefore, have to be based on stoichiometric information: knowing which reactions are present, but without knowing their kinetics. The scale of such an effort can be judged from Recon 2 (Swainston et al., 2013), a reconstruction of human metabolism consisting of 6852 biochemical and transport reactions distributed over eight intracellular compartments, 2469 unique metabolites, and enzymes coded by 1766 genes. It will be obvious that a project of such magnitude requires input from many research groups around the world, and for that reason Neil Swainston and his colleagues call Recon 2 a ‘‘community-driven’’ reconstruction. As an example of the type of problem that can be addressed with a reconstruction of this kind one can mention the effect of deleting the gene for the enzyme fumarase in renal cancer (Frezza et al., 2011). However, without wishing to devalue this type of study one may regret, in a symposium on Chemistry and Time, the absence of any kinetic information.
Drug discovery Why must metabolic and signalling pathways be so complicated? One of the ways in which organisms can overcome the lack of perfect specificity in individual processes is to evolve more complicated systems that involve several components, and this is a major reason for the apparently unnecessary complexity of living organisms. Tom Blundell pointed out that traditional ideas of signalling pathways would give very noisy — and hence inadequate — signals, but multi-component protein assemblies can greatly improve the signal-to-noise ratio in cell regulation, molecular interactions being stabilised cooperatively as the components are assembled into a signalling complex. This allows signalling systems to be both selective and reversible, but from the researcher’s point of view it leads to two challenges (Winter et al., 2012): how to design drugs that target protein—protein interfaces, and how to understand the mechanisms of the assembly. Francesco Gervasio described a different approach to understanding ligand—protein interactions, pointing out that a major problem for modelling binding kinetics is the lack of information in most cases of the detailed Gibbsenergy profiles along association pathways. An effective computational strategy can, however, allow binding kinetics to be calculated (Juraszek et al., 2013), as well as the conformational changes that occur on binding (Sutto and Gervasio, 2013). Akhilesh Reddy’s report of the effects of circadian rhythms, mentioned already, is clearly relevant here: the effectiveness of a drug must depend not only on the dose but also on when in the circadian cycle it is administered. What is optimistically called rational drug design, a term that was already treated with scepticism as long ago as 1966 (Belleau, 1966), is often pursued with no consideration of metabolism
A. Cornish-Bowden at all. For example, a supplement to Nature entitled ‘‘intelligent drug design’’ (Various authors, 1996) nowhere included the words ‘‘metabolism’’ or ‘‘metabolic’’, and a similar special issue of Science on drug discovery (Various authors, 2000) was barely any better (Cárdenas and CornishBowden, 2000). The point that is overlooked in these efforts is that what (most) drugs do is to modify metabolism, and the circadian point is that to be effective they have to act on the metabolic state that exists at the time of administration.
Getting drugs to their targets I first learned of Lipinski’s rules in a lecture by Christopher Lipinski himself at the first Beilstein Bozen Symposium that I attended in 2002 (Lipinski, 2002). The rules had been first proposed some years earlier (Lipinski et al., 1997), and have been enormously influential in the pharmaceutical industry: the original paper has now been cited more than 3000 times, with more than 300 in each of the years 2011—2013. Nonetheless, Douglas Kell has strongly argued (Kell, 2013) that this approach is largely misconceived, and that that is why adherence to the rules has not been very effective in drug discovery. The problem is that they allow prediction of the properties that allow a molecule to enter a cell by passive diffusion, but that is not at all what one needs to know, because most effective drugs are transported into cells by specific carriers. Trevor Howe argued in addition that kinetic considerations also needs to be taken into account, as equilibrium assays are not likely to be enough. He described the Innovative Medicines Initiative Kinetics for Drug Discovery Programme, a European project costing around 21 million euros over five years, and involving seven major drug companies and ten universities in four countries (Müller-Fahrnow, 2010). This idea was developed by Emad Tajkhorshid, who discussed how membrane transport can be simulated computationally with high resolution in both time and space, for example in ATP-binding cassette transporters (Moradi and Tajkhorshid, 2013).
Nanomaterials Even if membrane transporters are the principal mediators of active exchange of materials across cellular membranes, they are not the only ones. Drug delivery systems do not have to rely on natural transporters, and nanomaterials are attracting attention as an alternative, as presented by Nathan Gianneschi. He and his group showed that active accumulation of nanoparticles in tumours can be achieved in a specific assembly event that responds to biochemical signals associated with tumour tissue (Chien et al., 2013). It is impossible to prepare and test every conceivable kind of nanoparticle, because, as Dave Winkler said in his presentation, there are potentially more drug-like molecules that obey the laws of chemical valence than there are atoms of matter in the universe. Instead it is essential to have powerful computational methods that allow the biological effects of potential nanomaterials to be modelled and predicted in silico. For this we need to know not only the possible useful biological effects of such nanomaterials, but also any potential dangers that they may represent. Novel
Time flies like an arrow sparse machine-learning methods based on Bayesian neural networks are thus being developed for this purpose (Winkler et al., 2014), and they prove to have useful predictive value.
Evolutionary time
117 different genera of protozoans, the major pathogens Trypanosoma and Leishmania, only 80 million years ago. Not only that, but as Robert Field described, it has a very large number of protein-coding genes, similar to that of humans, and transcriptomic studies are allowing understanding of how it makes, uses and controls molecules and processes.
Directed evolution Chemistry as creation science As everyone knows, evolution by natural selection is excruciatingly slow. The last universal common ancestor (‘‘LUCA’’) of all extant organisms is thought to have existed more than 3 × 109 years ago, but evolution has been slow enough to allow many of its characteristics to be deduced from those of modern organisms. Moreover, natural selection is a poor algorithm for optimisation, akin to what computer scientists call greedy algorithms, which work by making the best local choice at every step, in the hope of finding a global optimum, but with no vision of where this optimum is likely to be. However, as argued by Philippe Marlière, not only are biotechnologists unwilling to wait millions of years to arrive at useful results; they also believe that they can proceed faster by means of directed evolution under human selection and synthetic biology. For example artificial nucleic acids (Herdewijn and Marlière, 2012) can propagate genetic information in vivo deliberately insulated from the normal processes of replication and transcription. Quite apart from its technological promise, directed evolution can lead us to a better understanding of how the genetic code developed: it can hardly have coded for as many as 20 amino acids at the outset, but which ones did it have and how did others arise? Jeffrey Wong’s coevolution theory (Wong, 2005), which postulates that ‘‘new’’ codons were given to new amino acids by their metabolic precursors, probably provides part of the answer, but one can also study the question by asking how an enzyme that used only a few amino acids could be improved by expanding its resources: for example, how could a chorismate mutase with only nine amino acids be improved by allowing it access to 20 (Muller et al., 2013)? This type of study has led to some surprising results: for example, one might expect that mutating leucine into such a similar amino acid as valine might offer little advantages, but such apparently innocuous substitutions can be observed in parallel in independent evolutionary trajectories; this suggests that even building blocks with very similar structures can be highly beneficial for fine-tuning enzyme structure and function.
Evolution of defence mechanisms Given the time available, many organisms, including agriculturally important pests such as the leaf beetle Chrysomela populi, have arrived at defence mechanisms against predators, in which, as Wilhelm Boland described, the larvae have ‘‘bioreactors’’ on their backs that discharge droplets of toxins when they are attacked (Discher et al., 2009). The green alga Euglena gracilis, familiar to most people who have taken biology practical classes, is not only very versatile in its metabolism, allowing it respond efficiently to redox stress, but also has a complicated evolutionary history. One might expect it to be closely related to other algae and plants, but in fact it separated from quite
Creationism is normally regarded as being in complete opposition to evolutionary science, and that is certainly true if we restrict attention to natural biology (Cornish-Bowden et al., 2014). However, Leroy Cronin pointed out in his presentation that synthetic chemistry is a creationist science: chemists envisage the molecules they want to create and then design them on the basis of chemical principles, an approach completely different from natural selection. He is exploring the possibility of inorganic systems that can evolve and provide insight into the prebiotic evolution that predated the world of nucleic acids (Cronin, 2013).
Electron transport and the energy crisis Hydrogen as a clean fuel The approaching energy crisis is one of the major problems that humanity will need to solve in the coming decades if disaster is to be avoided. It is easy to say, and I have said it myself, that large savings in energy can be made by adopting less wasteful habits, but, as Stenbjörn Styring pointed out in his lecture, that cannot solve the problem: it can only save energy in the developed countries, whereas many populations that live today in a highly undeveloped state can be expected to increase their energy requirements enormously by 2050. Moreover, electricity accounts for only 17% of energy consumption today, and more efficient generation of electrical power can only improve a small part of the problem. Many groups today are accordingly working on ways of using solar energy to produce more efficient fuels that can be oxidised without producing carbon dioxide, most notably hydrogen gas (Magnuson et al., 2009). Attempts to produce fuels by natural photosynthesis in living organisms, such as bacteria or algae (Matsumura et al., 2014), or active enzymes obtained from them (de Poulpiquet et al., 2014), are attracting considerable attention, but an alternative is to use biomimetic methods in which purely chemical catalysts are designed on the principles learned by studying such organisms (Simmons et al., 2014).
Understanding electron transport Applying any of these approaches successfully will require increased understanding of electron transfer in proteins, the subject of Israel Pecht’s presentation. How are the rates of electron transfer between redox centres controlled, tuned for efficiency, and made capable of avoiding harmful products? These aspects can be probed by studying model systems such as the bacterial copper proteins azurin and laccase, and examining the effects of mutations on rates and
118 structure (Farver and Pecht, 2011). Such proteins are also attractive for potential uses in bioelectronic systems, which can be studied by examining their conductance properties in the solid state (Ron et al., 2010). As described by Tim Clark, a problem that arises in simulating electron transport is that the processes to be simulated take place in time domains that differ by many orders of magnitude. For example, electronic devices constructed with self-assembled monolayers of flexible organic molecules need to be coupled with very much faster electron transport through the system. A practical approach — not strictly valid but giving useful results in practice — involves simulation of electron movement on static snapshots from simulations on the long time scale of the conformational changes (Leitherer et al., 2014). This type of calculation requires the power of modern parallel processing, but this is feasible because ‘‘a smart phone today has the power of a top-100 computer of 1990’’.
Nanocrystallography At present serial femtosecond nanocrystallography allows X-ray diffraction snapshots to be obtained of fully hydrated nanocrystals at room temperature (Chapman et al., 2011). The advantage of this is that most damage processes occur on much longer time scales, and so large membrane protein complexes can be examined before they have had time to disintegrate. This already represents a major advance on what was possible comparatively recently, but it has the disadvantage that the Linac Coherent Light Source is not only an extremely expensive piece of equipment, but it also occupies a large amount of space: the SLAC National Accelerator Laboratory in California is about 3.2 km in length. In her concluding presentation in the symposium Petra Fromme foresaw the construction in the next few years of an accelerator that could operate in the attosecond range of time, would be of the order of a metre in length, and would be relatively inexpensive (by the standards of linear accelerators). If this is achieved it will represent a huge technological advance.
Concluding remarks In conclusion, the 2014 Beilstein Bozen Symposium on Chemistry and Time assembled a very range of expert lecturers who were very successful in illustrating the importance of time at many different levels of chemistry and biology.
Conflict of interest The author declares that there is no conflict of interest.
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