Molecule of the month August 2015: NAD(P)(H)

The molecule I have chosen for this month comes on the back of several recent discussions regarding metabolism, energy and in particular the Krebs cycle. All of which involve a molecule that is ubiquitous in living organisms, but doesn't quite have the "glamour" of ATP. Nicotinamide adenine dinucleotide (as seen left, A) comes in two forms: oxidised: NAD+ and reduced: NADH (sometimes written as NADH2 or NADH + H+, and originally written as DPNH): see B, left. I shall use NAD and NADH here for simplicity.  The key to the function of this coenzyme lies in its ability to act as a hydrogen donor (eg the reductive amination of 2-oxoglutarate by glutamate dehydrogenase, GDH) or as an acceptor (in the oxidative deamination of glutamate dehydrogenase). In other words, NAD acts as an oxidising agent, as it accepts electrons, or a reducing reagent in the NADH form. However, NAD(H) has some "moonlighting" functions in cells, that I shall come to later. The addition of a parenthetic "P" in my title refers to the phosphorylated form of NAD (see top RHS). This version fulfills the same function as NAD, from a redox perspective, but the presence of the additional 2' phosphate on the adensine moiety will clearly mean that some enzymes utilise NAD and some NADP. However, some can use both! And surprise surprise, GDH from humans is one such enzyme.   

Biosynthesis of NAD. Two British Biochemists(Harden and Young), working at the newly formed Lister Institute in the first part of the last century, discovered a heat stable molecule that stimulated a number of yeast fermentation reactions. They called this extract a coferment. We now know that this coenzyme is synthesized in mammals, plants and bacteria, but in slightly different ways. Indeed these often complementary aspects of coenzyme biosynthesis provide one of the many reasons why our own microbiota prove beneficial. In all organisms, amino acids generally provide the starting materials for the biosynthesis of molecules like NAD. In man, the starting point is the two ringed amino acid, tryptophan (Trp or W): in bacteria the acidic amino acid aspartate (Asp or D) may be the starting point. However the first key step is the production of the dicarboxylate, quinolinic acid: a pyridine ring, with two carboxylic acid substituents at 2 o'clock and 4 o'clock (technically pyridine 2,3 dicarboxylic acid). The structure of quinolinic acid is shown top left, and you should note the Nitrogen, which is a key atom in this molecule (and subsequently in NAD). Following the addition of a ribose phosphate and an adenylate, the final step on the way to NAD is the amidation of the pyridine ring. To make NADP, the enzyme NAD kinase obliges. Finally, I should mention the concept of salvage pathways, which has become something of a biochemical backwater, since I was a student. However this is a simple concept: if you eat food that contains fragments of NAD (or indeed molecules like ATP or CTP etc), you can top up your NAD levels through a group of so called salvage pathways. A nice evolutionary bonus!

Having manufactured NAD either de novo, or via a salvage process, the coenzyme participates in numerous enzyme catalysed reactions. I have already mentioned the GDH reaction, but in my recent post about metabolism, it was clear to the metabolic pioneers at the turn of the last century, that a 2 H transfer reaction was likely to be a common feature in the conversion of the food we eat into energy. NAD binds tightly (with dissociation constants in the sub-millimolar range) to dehygrogenase enzymes that recognise alcohols, glyceraldehyd-3-phosphate, sugars and fatty acids to name a few molecules that have the functional term "dehydrogenase" appended. (I should note that NADH generally binds more tightly than NAD). The binding site for NAD is often called the Rossman fold (see the image above on the RHS: the red molecule is NAD), after the eminent crystallographer, Michael Rossman, who studied these classes of enzymes in the 1970s. The mechanism of dhydrogenases differs subtly between the different "classes", but all share a common feature: the positively charged Nitrogen accepts a hydride from the substrate. In some enzymes the hydride is transferred above (A) and in others below (B)  the plane of the pyridine ring. The other proton is usually bound to an amino acid side chain. The reverse is true for the NADH to NAD direction. If you are interested in the details of dehydrogenase enzymes, there are many excellent sections in text books, or reviews: I like the descriptions in Alan Fersht's classic text and this web site will give you more detailed  information.

NAD, NADP or NAD(P)? The presence of the additional phosphate in NADP presents a significant challenge for an NAD-specific enzyme: such a lot of additional negative charge! In the opposite sense, an NADP-specific enzyme might require a strong set of interactions with the phosphate moiety in order for the enzyme to grasp the substrate (or indeed the transition state), during hydride transfer. In general, enzymes are specific for one or the other cofactor, whilst some like GDH in some species, can handle both with similar effieciency. There is a general rule of thumb however, and that is that anabolic dehydrogenases tend to be NADP-specific, whilst catabolic dehydrogenases prefer NAD. As always, rules of thumb in Biology can be your undoing!

Moonlighting functions for NAD. As I said earlier, NAD sometimes acts as a substrate in reactions that are not of the "redox" class. Some proteins are regulated by differential ADP ribosylation: such enzymes, called ADP ribosyltransferases, utilise NAD as the source of this chemical group. In the nucleus of some (if not all?) eukaryotes sequential ADP ribosylation of nucleic acids is common. This so called poly-ADP-ribose polymeration (catalysed by PARPs) is an NAD-dependent reaction, but again not of the redox type. This enzyme is the target for several anticancer drugs, known as PARP inhibitors. You can read about the Sheffield's  role in the PARP story here, from Thomas Helleday's lab page. 

Final points. I have focused here on NAD as the electron donor-acceptor in dehydrogenase reactions, but there is an alternative coenzyme that is found (often) tightly bound to the enzyme (with Kds in the nanomolar range). This is FAD (and its partner FADH). I shall come back to FAD in a separate post. However it does allow me to finish with a practical point. Earlier I said that many studies have been published on NAD-dependent dehydrogenases and one of the main reasons for this is that whilst NAD in solution is colourless, and only absorbs light at uv wavelengths of less, than 280nm. NADH on the other hand has a very convenient absorbance maximum at 340nm (see figure top RHS). This fact alone makes NAD-dependent dehydrogenases very easy to assay. Therefore, armed with such a sensitive and robust absorbance characteristic, enzymologists have used the dehydrogenases as test beds for understanding many generic properties of enzyme catalysis. I also will leave you with the important point. It is the NADH (and FADH) that is generated via the Krebs Cycle, that indirectly gives rise to ATP during respiration, via the electron transfer chain. Again this will be discussed in more detail separately, but the generation of ATP via the mitochondrial ATP synthase is only possible as electrons cascade down the electron transfer chain from NADH and (FADH). 

I hope you agree with me that NAD as a suitable candidate as a molecule of the month!

Life Sciences UTC 2013-2015 : A new adventure in Science Education

By way of a perspective on two years of intensive outreach at Liverpool Life Sciences University Technical College...

Introduction Having left my own school, St. Edwards College, in Liverpool in 1977; apart from occasional visits, I hadn't been back to the city until I began a sabbatical year at the Liverpool School of Tropical Medicine in the summer of 2012. During that year, I met up with a longstanding friend, Dr. Geoff Wainwright, co-founder of the Business Development organisation 2Bio, specialising in translation of early stage innovation. Geoff in turn introduced me to Nigel Ward, founder of the North Liverpool Academy (which would subsequently become the Northern Schools Trust, of which more later). This was all made possible by the agreement of my own Department (Molecular Biology and Biotechnology) at the University of Sheffield, who agreed to the secondment. By the end of 2012, we had agreed to join forces to create a new educational vehicle for young scientists at the Liverpool Life Sciences Technical College down in the rapidly growing Baltic Triangle, near the east end of the old docks. Since then I have been Blogging, tweeting, emailing and talking to everyone about how working with young students from age 14-19 has completely changed my views on education. Over the coming summer vacation, the students will take a break and the new intake will get ready for the start of Autumn term. I thought it would be useful to reflect on the last two years and try to capture the essence of why I believe the UTC is developing an important, new way forward for Science education, that I believe addresses most, if not all of the concerns that we hear in the press about: "...the problem with school leavers".

Recruitment. I shall begin with the students, without whom there would be no UTC! Our youngest students are just shy of 14 years of age, when they bustle into the Innovation Labs for the first time. The sixth form students are mostly 16, with one or two a year older. Prior to entry into the UTC, the students are interviewed over a period of between a month and a year before they arrive; and a premium is placed on their enthusiasm, their attendance and their behaviour records. Oh and of course, a declared interest in Science! Some students will follow courses at GCSE and A level, while others will follow the BTEC route; and yet others will follow a mixed pathway. The two major routes for UTC students can be described as Natural Sciences and Health Care. You may feel that 14 or even 19 is too soon to take career decisions, I happen to think it gives many of the students a strong sense of purpose.

Year 1. All students (Y10/11/12/13) follow the National Curriculum: the delivery, management and achievements of which are scrutinised just like in any other "school" by Ofsted. The remainder of their time in school, which takes the form of a 9-5 working day, is filled with our "REAL" programme of Research Enhanced Active Learning, which you can read about in an earlier post. The aim of this is simple: following a 6 week induction course in laboratory skills, where new students who typically enter at Y10 or Y12 are trained and assessed in a set of methods that we have called our "skills passport". In short the students become proficient in first year PhD level Molecular Biology methods: this is formally assessed and students can repeat these tests until they move up the scale from Silver, through Gold to Platinum. A detailed description of our skills test can be found here and in documents in the sidebar of this blog. The first objective of our skills training is to develop an appreciation and understanding of contemporary laboratory skills and to simultaneously support the development of their experimental dexterity. However much more importantly, it is a device for developing independence in the laboratory alongside an aptitude for teamwork. There is a strong focus on experimental planning and organisational skills and I cannot stress enough: the ability to communicate their science. The impact of this approach is palpable when students return in November after their first half-term break. Gone is the fear of a largely alien laboratory environment, and what emerges are confident, curious and questioning young scientists. As estate agents brochures often say: "an internal inspection is strongly recommended": so too is a visit to the UTC Innovation Labs! Just look at our @lifesciencesutc twitter feed!

As students move through the various phases of assessment, they begin their first serious laboratory experiment: for the last two years we have carried out a preparation of His-tagged GFP. You can read about this here. This allows me also to give you a flavour of some of the many valuable contributions of our partners. This particular project, incorporating microbiology and biochemistry, with a hint of molecular biology, maps closely onto the technology base at Actavis and was facilitated by the tireless support of Pro-Lab Diagnostics, who helped enormously with equipment and media during our early months. Working in small teams the students worked through an industry inspired approach to obtaining a sample of bright green fluorescent protein using affinity chromatography. This incorporated most of their skills, but importantly taught them the importance of sample labelling, keeping good notes, sample storage and tracking (over 6 weeks). Finally the students presented their results, good, bad and indifferent to a small group of research leader from Actavis (then Eden Biodesign). I was bowled over, and importantly so were the Actavis team! Since then we have developed a range of different presentation formats, from reports, posters, talks and infographics, all of which are key to developing a cohort of confident, young scientists. 

The final phase of the student experience in the Innovation Labs is the development of student-led (well actually student-devised), projects. Again, I have written about Jack's and Kellie's projects in detail in earlier posts (a plate from Jenny's project on the microbial diversity in domestic drains is shown left). At University, undergraduate students, typically in year 3, select a project from a long list and work (the details will differ from Institution to Institution) alongside an academic supervisor, often with a couple of other students and sometimes a PhD student, to tackle an experimental problem. In my own group at Sheffield, students might be asked to purify an enzyme or clone a gene, or maybe crystallise a protein. The project is generally supervisor driven. I decided to turn the tables on the students at the UTC; since their interests at this stage in their education are much more diverse, and get them to come up with the idea themselves. Through literature searching and a little steering away from major nuclear physics experiments, or those involving human sacrifice, the students eventually develop an embryonic practical idea. This approach has been an unexpected success. Why? It is simple, and teachers know this intuitively. If a student takes ownership of a piece of work, they are driven to pursue it. Hence the "activation energy" barrier is removed and the student engages fully in the project. I am not saying that all science projects should be student led, some students find this very challenging. However, I do believe the option should be made available. Again this is a formative experience for the students: sometime working alone and others in small groups. Once again this is an integral part of shaping our future scientific minds.

I want to finally try and capture why I think the Life Sciences UTC experience works well. It isn't just the Innovation Lab experience that marks out the "phenotype" of UTC students. There is alchemy alongside the chemistry! I shouldn't of course use such a word, but I do because I haven't yet got to the bottom of the mechanism that underpins the ability for students to gain so much from their education at the UTC. For students to benefit from the challenges that I and now Dr. John Dyer will place before them, they have to be prepared and they have to take it all seriously. The behavioural code at the UTC, captured by the strap line: "every day's an interview" makes it very easy to manage large groups of students and sustain their interest for long periods of concentration, often in potentially hazardous laboratories. In addition we have several additional components to the school calendar.

Master Classes. The last two years has seen the UTC play host to some of the  country's most impressive industrialists, scientists, medics and educationalists. In the comfortable seats of our cinema, or in the labs/classrooms, students have sat, listened and engaged in discussion with an impressive collection of individuals. Sometimes, as we all know from any typical work experience, these master classes can cut into curriculum classes, or disrupt some other planned event. However, as a big supporter of seminars myself, they have significantly raised aspirations of our students. From a discussion of the use of zebra fish as model organisms, to contemporary surgical methods, and synthetic biology in the Life Science industries, to how I won my Nobel Prize! The master class series has been a key part of the last two years. In the picture chemistry students are being given a hands on introduction to NMR thanks to the University of Liverpool and Dr. Kate Copper who is taking students through the method using their RSC funded portable NMR spectrometer.

Placements. Our partners, who are all listed on the UTC Website, are not only supporters, visitors and presenters of Master Classes, but they also provide all of our students with the opportunity to experience the work place first hand. I cannot tell you how rewarding it is to see their lab books after a few weeks on placement. Or the warm emails from senior staff at the Royal Hospital, Pro-Lab, RedX Pharma, Croda etc etc. (not wishing to leave anyone out at all!). The placement scheme builds student confidence in a way that Schools and Universities cannot. They will remain a major part of the UTC programme and I will return to the partners in a subsequent post.

Visitors. The UTC has attracted a huge number of visitors since the minute the doors opened (and actually beforehand!). I don't think a day has passed without me giving a precis of what the Innovation labs are up to on a given day, or preparing to impress a VIP (On the RHS is Sir Mark Walport who shared his enthusiasm for model organism biology with Y10 and Y12 students). Of course we want to broadcast the message to politicians, industrialists, scientists, teachers and the public in general. However, I have come to realise that the regular throughput of visitors who generally focus on talking to the students and asking them what they are doing as they pipette a few microlitres of DNA into a microfuge tube. As the students gain confidence, they become our best ambassadors and in doing so once again we develop their communication skills and there is nothing more important than scientists of the future being comfortable with communicating with the general public of all ages as well as intimidating experts from industry and academia. The roll call over the last year can be viewed most easily on our twitter feed @lifesciencesutc.

UTC Transmits. The brainchild of Lyndsay Macauley at the UTC, this is a mini-symposium hosted by the University of Liverpool, in which a range of eminent speakers from scientists to politicians, including our own students, deliver short talks on a specific theme (see left). During an afternoon, students from the sixth form attend a symposium and engage in a discussion about the topics presented as if at a professional conference. By combining science, health issues, social issues, personal experiences and politics, students are able to contextualise their classroom and innovation lab knowledge in a way that I have so enjoyed, that I will be introducing to my undergraduates when I get back to Sheffield. This, combined with the Master Classes really inspires the students.

The teachers, the support staff and the governors. Clearly the programme

at the UTC that I have been describing represents only around 25% of their school week. The remainder of the time is taken with curriculum classes and enrichment. As I said earlier the teaching team, let by the UTC's senior management team provide students with a typical portfolio of GCSE, A Level and BTEC pathways all of which match the students' aptitudes, abilities and aspirations. Without the support of the teachers, some in the lab sessions, but all in providing our students with the support to help them achieve their academic ambitions, the UTC's REAL programme would not be possible. I cannot thank them enough for letting me into their world! The school staff are in turn supported by a set of governors, of whom I have already mentioned Nigel Ward and Geoff Wainwright, but there are representatives from parents, partners and the University. Their commitment to supporting me, again both directly and indirectly has been vital to what I believe to be an incredible organisation. Again, I express my thanks to them.

Finally, I have to return to the students, with whom I have shared so much and learned so much. I firmly believe that the frustrations expressed by employers University academics and politicians about the abilities and aptitudes of school leavers can be overcome by challenging students in a supportive environment. The UTC model at Liverpool is one way. Of course I understand the need for teachers and schools to be assessed by Ofsted and given performance labels, but much more importantly we need to look closely at the school experience in a more rounded way and I hope that you will come and visit the UTC to experience first hand, how we getting on with the challenge. A little anecdote from my first few weeks, perhaps explains why I knew this whole involvement with schools was going to work. 

The Nobel Foundation announces the annual recipients of the various prizes in early October. The prizes are announce via live streaming from Stockholm and I decided to leave the projector screen on to capture the announcements in the lab. Eventually, after the usual Press Conference delays, the announcement came. First in Swedish! Then in English. At the end, the students, all glued to the screen, burst into a spontaneous round of applause. I knew then this is where I wanted to be! The Nobel Prize week is now a tradition at the UTC and I shall be back there in October 2015!

I have a lot of people to thank in addition to those mentioned, however,  I shall defer that to a later Blog over the summer, when the site will be revised a little to reflect the new order for September 2015!

The Hans Krebs Legacy lives on

I became aware of the connection between Sir Hans Krebs and Sheffield when I first walked underneath the glass panel that framed the iconic Krebs Cycle in Firth Court, the home of the then Department of Biochemistry (now the Department of Molecular Biology and Biotechnology). When I was asked yesterday to explain in around 60 seconds, the significance of his work and his connection with the University of Sheffield, I started asking myself some searching questions. In the end, on a mobile 'phone in my car, I tried to capture at least some of the the essence of what I believe to be an intellectual tour de force: the synthesis of a coherent explanation for the transformation of the food we eat (sugars fats etc), the air we breath and the water we drink, into the energy that propels life. Of course, like any piece of Science, the Krebs Cycle was preceded by great work (that of Emil Fischer, Otto Warburg, among others) and it was succeeded by the work of those who mapped the pathway of ATP production, culminating in Peter Mitchell's incredibly innovative chemiosmotic theory, with John Walker and Paul Boyer providing the molecular icing on the cake nearly fifty years later.

In 2012, during a splendid symposium in honour of one of the greatest elder statesmen of British Biochemistry, Sir Frederick Gowland Hopkins, founding Professor of Biochemistry at the University of Cambridge (1914), I had the privilege to relate the story of Hans Krebs' journey from Berlin to Sheffield. From his rural experiences in Hildesheim as a young boy, through the trenches of World War I, Hans Krebs forged a path through medicine, before finding himself in the laboratory of Otto Heinrich Warburg, in Dahlem Berlin. Originally entitled the Kaiser Wilhelm Institute for Biology (today: the Max Planck Institute, where incidentally as a young academic at Sheffield I spent many enjoyable visits to talk DNA modification with Professor Thomas Trautner). It is hard to imagine what Dahlem-Berlin must have been like in the 1920s. Around the corner from Warburg and Krebs, Albert Einstein, Otto Hahn and Lise Meitner where shaking the foundations of Physics (and the Electron Microscope would be unveiled in the early 1930s by Ernst Ruska). The rarefied atmosphere of Dahlem where both Science and the Arts were undergoing a truly revolutionary upheaval. While Kurt Weill and Lotte Lenya (top right) created music together, bruised, post-Versailles German pride fuelled the emergence of National Socialism which soon led to the collapse of this short lived intellectual and cultural "mecca". 

In July 1933, Hans Krebs was dismissed from his employment in Freiburg University, fresh from his elucidation of the first molecular "cycle" in Biology: the urea cycle. With the support of  Sir Frederick Gowland Hopkins, Krebs was soon in Cambridge, but only a year later, he found himself in Sheffield, where  there began a remarkable period of achievements: both personal and scientific. In his Science: using small conical flask, with a side arm, the Warburg manometer combined a mercury displacement tube, coupled to the above flask, calibrated with great precision using mls of mercury! Tissue slices would be mixed with substrates by tipping the side arm and the consequent evolution of gases were measured as the whole apparatus, mounted in a cylindrical shaking water bath, rattled away in the corner of the lab. Careful measurements, forensic observation, meticulous recording of inventories of compounds transformed, gases evolved, pH changes at carefully controlled temperatures, produced the data from which the Krebs Cycle was forged.

The recognition of patterns in data has the potential to transform a simple, phenomenon into something of universal significance. When Krebs (and his colleagues) began trying to reconcile their manometry data with that of others; he recognised something of great significance and the proposal of a cyclical pathway that we now call the Krebs Cycle. Earlier today, the Krebs family through his marriage married to Margaret Fieldhouse, and all of whom grew up in Sheffield, decided to auction the Nobel Medal, awarded to their father. With the £275 000, they intend to establish the Krebs Trust Fund, to support refugee scientists. This splendid legacy seems to me to be completely consistent with everything I have read or heard about the man. It will be extremely interesting to see how this initiative impacts on Science.

In the mood for indigo?

"Mood Indigo" is the name given to one of my all time favourite Duke Ellington compositions. It is also the colour that we associate with Blue Jeans, and remains one of the most important textile dyes in use today. Once it was a major export item of India, derived from the plant Indigofera tinctora, more recently it has become a major natural product of Guatemala and El Salvador. However, the majority of indigo produced today is manufactured by chemical synthesis. It has however been so familiar in the UK as a colour, that Isaac Newton identified indigo as one of the 7 colours of the visible spectrum: red, orange, yellow, green, blue, indigo and violet. Indigo as a molecule offers not only considerable insight into the mechanism of absorbance spectroscopy, but is an important model compound for helping us to understand the subtlety of intramolecular hydrogen bonding.   

One of the most enjoyable aspects of the last two years at the UTC, has been my re-acquaintance with textile dyes: my PhD involved the application of triazine dyes in the purification of proteins; but this was over 30 years ago! Indigo is not a triazine dye, but it does help us understand the concept of of electron(ic) transitions in chemistry, which are so important in Biology. Indigo also gives us a beautiful illustration of the importance of Hydrogen bonds in molecular recognition. Indigo is a colour that most people would call blue. Isaac Newton however placed indigo in between blue and violet in the visible spectrum, which he famously published in his book "Opticks" in 1704. However, it wasn't until the middle of the 20th century that the field of quantum mechanics began to offer an explanation for the origins of colour at the molecular level (let's forget transition metal salts for now).   

The electromagnetic spectrum spans over 15 orders of magnitude: from the weak, but tunable wavelengths of radio waves to the powerful energy of X rays and gamma rays, that can tear through covalent bonds. Indigo absorbs light that is in the orange part of the visible spectrum, thereby appearing blue (oops, indigo!). The light absorbed, promotes the reorganization of an electron in the indigo molecule: the relatively low level of energy required to induce an electronic transition in the central double bond, is in part a consequence of the planar structure adopted by indigo. Which is in turn a result of the hydrogen bonds at "12 o'clock" and "6 o'clock" (dotted lines in the structure). Without this hydrogen bond stabilization, it is believed that the molecule would rotate more freely around the central double bond, making the electronic transition much less probable and requiring a higher input of energy. At this point, I should point out that whilst there is undoubtedly a relationship between the intramolecular hydrogen bonding in indigo and its absorbance characteristics, there are experiments in which the oxygen is replaced by sulphur that suggest other factors are at work. Sulphur lacks the electronegativity required to form H-bonds and so suggests perhaps that intermolecular stacking plays an important role in constraining the molecule into planer stacks. This is also a feature found in DNA bases, where stacking interactions also play an important role in stabilising the double helix. There can never be an end to experiments until we obtain the truth! The Book entitled "Organic Photochromes" by A.V. El'tsov provides a detailed discussion of these issues (for aficionados).

I think this is a lovely example of how quantum theory helps us to put the phenomenon of absorbance, and therefore colour on a mathematical footing. However, the hydrogen bonding component, makes me think of the elegant
interactions found in Biological polymers. Recall Watson and Crick base pairs in DNA, the base pairing of the anticodon loop and the triplet codons during translation of mRNA on the ribosome (RHS), or the elegant hydrogen bonding between substrates and protein amino acid side chains in the active sites of enzymes. For me, the last two years of using dyes to demonstrate ion exchange chromatography and spectroscopy has reminded me of the  sheer beauty of their visual appeal and has made me completely re-evaluate the way I will teach the importance of Chemistry in explaining Biology. 

Last week, John and I welcomed a group of primary school children to the Innovation labs and we chose to engage them with some demonstrations of colour in Biology, Chemistry and Physics. The excitement and enthusiasm as they watched a dye change back and forth from yellow to blue, by the sequential addition of an acid and a base, was truly inspirational. I hope I have persuaded at least some of you to share these views and to make more use of colour chemistry in science education.

Drug Hunting and Cancer: The future of Medical Sciences Part 2

In the second part of my triptych, I am following the logic of the New Scientist in exploring what the future holds for Drug Discovery, in particular in the area of cancer research. In many ways, the promises of successful drug hunting are a little like Elmer J Fudd's success in hunting down Bugs Bunny. He gets close, it looks a cert.... but somehow Bugs always gets away! (I also intend to cover new antibiotics, a particularly hot potato in the world of medicine and its associated politics, but the Blog was getting too long, so this will be in a supplementary section, to follow). Let me first look at a brief history of drugs, to provide some perspective on how Science has impacted on drug discovery over the last 20 years and then why microbes and tumour cells remain a major challenge.

Drugs are not new. Moreover, the spectacular advances in Molecular Life Sciences that many would say began after the first field trials of Penicillin, throughout World War II, and included Watson and Crick's structure for DNA, Sanger's sequencing of the first protein (the hormone insulin), not forgetting Kendrew and Perutz's ground-breaking work in protein crystallography. It was in fact a slightly distracted doctor who during the Victorian era picked the wrong"potion" from his shelf and inadvertently, but successfully, administered the first dose of paracetamol (well a closely related compound), to treat a patient with fever. In fact apart from the medicinal herbs mixed and formulated by village "pharmacists" that have their roots (sorry!) in traditional cures, it was the Victorians and their 20th Century proteges that in my view laid a formidable foundation for 20th and 21st century medicine. 

The antimalarial compound, artemesinin

In fact it could be argued that desperation is the mother of drug discovery! If the regulatory barriers to drug release that exist today had been in place over the last 120 years, many of the compounds we can buy over the counter may not be available. And it could be argued that the synthetic organic chemists that have been the mainstay of the pharmaceutical industry, in particular since the 1960s, would have been severely handicapped without the clues from aspirin, paracetamol, not to mention simple anaesthetics! In fact I have recently been to a number of drug discovery talks where chemists, armed with the sophistication of combinatorial instruments, molecular modelling software and the latest separation devices are revisiting Natural Products for their new ideas. It seems to me that it is the interplay between Natural Product Chemistry, Synthetic Chemistry, Synthetic Biology and Molecular Biology and its new polyomics approaches, that will provide the biggest breakthroughs.

What is cancer? Most of you will be familiar with someone who has suffered from cancer, a disease whose name strikes fear into the heart of most. We know from experience that while many recover from cancer, the threat of its return haunts the victim (and their family and friends) and that many die from the disease. I am sure you have heard doctors say that "cancer is not one disease but many". And that the prognosis for one person can be quite different for the next. I think this tells you straight away that cancer results from some degree of disruption to the complex cellular regulatory processes that keep us healthy. Cancer can result from the inappropriate activity of one or more biological control pathways: this may take the form of  bad timing or the  bad location of a process, or alternatively it can arise from too much or too little of a key control molecule. Cancer is rarely caused by the loss of a single, isolated function, like the loss of an enzyme activity in a metabolic pathway. In fact if that appears to be the case, then that molecule usually turns out to play roles in other cellular processes, that were initially overlooked. In short the more treatable diseases are like the failure of a light bulb: fixed by replacement, but cancer is more like the failure of your electrical fuse box, which can be catastrophic.

Cancer has been around in the literature since the early days of medicine and has been written about by medical pioneers like Galen and Hippocrates, and until relatively recently (around 50 years ago), it was assumed to be "incurable", with surgery (early treatment), radiotherapy (from 1900 onward) and chemotherapy (from 1950s: the most successful early drug being the DNA replication inhibitor methotrexate) providing patients with limited hope. During the 1980s, the remarkable advances in microbial molecular cloning paved the way for a new understanding of the causes of cancer. The genes responsible for eliciting cancer if they are mutated, lost or rearranged, were isolated for the first time. The initial group of cancer genes were said to be dominant (take a look here) and were given the name "oncogenes". You can read about them here, but suffice to say they encode proteins that normally regulate cellular processes, and these processes are often called signalling pathways. Some of these molecules regulate the expression of families of genes, or the transduction of signals between and within cells. Again, I hope you can see where this is going, from a drug discovery perspective. If you are trying to use a drug to interrupt a simple metabolic step, catalysed by a single enzyme, you are more likely to succeed than if your drug is targeted at an oncoprotein, that modulates the activity of many cellular processes. This work, shortly followed by the discovery of recessive cancer genes, called tumour suppressors, and exemplified by p53 has provided the pharmaceutical industry on the one hand with valuable insight, but on the other has highlighted the challenge of finding a "cure for cancer".

Today, anticancer drugs are among the most sophisticated (and consequently expensive) medicines in use. Treatments with the latest drugs can be £100 000 per year, compared with methotrexate, which would cost around £100 per year. Clearly there are major economic and political challenges here, which partly led to the formation of the NICE organisation to help provide an evidence base for regulating  drug prescription. The drugs in use are the products of research that can cost nearly £1bn (usually cited as $1bn), and therefore drug companies need a means of recovering their costs. (These issues are highly controversial, but are not the topic for discussion here).  Take Herceptin (otherwise known as Trastuzumab: the -mab suffix indicates this is drug is based on a monoclonal antibody). The drug is marketed by Genentech for certain classes of breast cancer (note the web address! This shows you how long the company has been in business). Briefly, the activity of a class of epidermal growth factor (EGF) receptors (these membrane bound molecules are the triggers for cell growth when EGF binds to them), specifically HERs 1-4, are modulated by Trastuzumab. It is thought that the drug acts mainly by promoting the degradation of HER-2, leading to a reduction in cell growth, but other mechanisms are also involved that are less well known. HER-2 is hyperactive in many breast tumours and by administration of Trastuzumab, the tumour is inhibited. The drug also promotes the "intervention" of the body's immune defences, giving rise to a powerful anti cancer effect. This drug has been a major success: but at a price. Nevertheless, our understanding of the immune system is beginning to provide inroads into the treatment of an increasing number of cancers. 

In the New Scientist special edition, the emergence of targeted drugs that not only treat the diseased cells and tissues, but also send a digital report on the status of the disease, are suggested to be around the corner! I shall exemplify this with the recent work published in the journal Cell on Photostatins (PST), also covered in a recent article in the Economist. If a molecule could be synthesised that interferes selectively with a Biological process and whose activity can be tuned by a non-invasive pulse of light (hv), then drugs become not only therapeutic but also diagnostics. The term "theranostics" would encompass this type of compound. In the above image, cells are shown as a blue nucleus with green radiating fibres: these are microtubules (take a look here). The addition of the PST molecule, followed by a specific pulse of light, leads to the breakdown in the microtubule network. Since microtubules are central to the cell's many growth and cell division functions, you can see how this approach has enormous potential for precision/personalised medicines. The introduction of a simple test for the over-expression of HER-2 protein: the target for Herceptin, has been approved and marketed by Dako. The company's Hercep Test is one of the earliest examples of a theranostic agent. The combination of these two functions in the form of drugs administered to treat and report on the targeted cells, is a quantum leap in the treatment of complex diseases like cancer and one that offers exciting prospects. It reminds me of the excitement I felt when at the age of around 12, I saw the film "Fantastic Voyage"; a Jules Verne type fantasy, in which a submarine and crew are miniaturised and injected into a scientist's veins to fix a life threatening blood clot. The prospect of controlled nano drones is probably more likely to be the future reality, as we begin to combine targeted intervention, reporting and surveillance. I agree with the New Scientist view that progress across these fronts will begin to transform modern medicine. However, I also believe that the biggest challenge is not whether we can innovate, but how on earth do we pay for it all!

"A change is gonna come"

The lyric of the late, great Sam Cooke, which hit the charts 50 years ago, in 1965, seems a long way from the classic short story published 100 years ago, in 1915 by one of my top 5 authors, Franz Kafka: Die Verwandlung, or The Metamorphosis. It opens in as compelling a manner as George Orwell's 1984 or Charles Dickens' A Tale of Two Cities (if you haven't read them, now's as good a time as any, with summer holidays coming on). The first part begins with a frightening awakening, quite removed from the mood of the opening of Swann's Way by Marcel Proust, the first in his 7 part work collectively entitled "À la recherche du temps perdu". Kafka's story draws on a mythological tale (Ovid) based on a real biological phenomenon: metamorphosis.

"One morning, when Gregor Samsa woke from troubled dreams, he found himself transformed in his bed into a horrible vermin. He lay on his armour-like back, and if he lifted his head a little he could see his brown belly, slightly domed and divided by arches into stiff sections. The bedding was hardly able to cover it and it seemed ready to slide off at any moment. His many legs, pitifully thin compared with the size of the rest of him, waved about helplessly as he looked."

"What on earth am I rambling on about?" (I can hear you gasp!). I am thinking about a recent documentary on BBC4 in which the film maker David Malone  beautifully contrasts the developmental biology of metamorphosis in caterpillars, tadpoles and sea urchins, with the "psychological metamorphosis" which he exemplifies through the plight of a soldier returning from war. If you want another recommendation it is the final part of Sebastian Faulks' novel Birdsong, which addresses this traumatic social phenomenon. 

We have chosen to make the darkling beetle, (RHS) Tenebrio molitor one of our model organisms at the UTC. You can drop into the cell growth room any time and see these stages of metamorphosis in this beetle. Think about the larvae, the pupae and the adult beetle. Then consider the fact that each "form"  is encoded by the same set of genes or genome. Two totally different creatures derived from the same genome. I challenge you all to not be fascinated by the task of explaining how such a complex outcome: two essentially distinct  creatures from one "blueprint".  This presents opportunities for Biology, Chemistry, Physics and Maths.

The summer for me will involve getting to grips with new software to decode the data obtained by our outgoing Grenland Biodesign team, in particular Jack , Rigsby, Sarah, Matthew and Will, who worked with Ashraf (one of my PhD students) who provided the genomic DNA samples for Dr. Chritiane Hertz-Fowler's team at the Genome Centre (University of Liverpool) to produce the raw genome data. What a great legacy for the UTC: our own model organism and an emerging genome that we can call our own. September 2015 will see a new cohort of students at Y10 and Y12, a real change is gonna come, but I know that those leaving this year will leave a long lasting legacy!