Atoms and Empty Space: the Structural Biology of Specificity

The eminent structural biologist Laurence Pearl, Professor of Structural Biology in the Genome Damage and Stability Centre at the University of Sussex, has strong links with Birkbeck. He studied for his PhD under Professor (now Sir) Tom Blundell at Birkbeck in what was then the Department of Crystallography (and the only full university department so named in the UK). The latest of the many honours and awards that he has received is the Biochemical Society's prestigious Novartis Medal for 2018. This is an annual award, made "in recognition of contributions to the development of any branch of biochemistry" and the winner is invited to present a lecture either at the Society's London headquarters or at one of its many conferences. Pearl gave his Novartis Medal Lecture, entitled Atoms and Empty Space: the Structural Biology of Specificity at the London office of the Biochemical Society on 13 November 2018.

This lecture was live-streamed on Facebook. It is linked from the Biochemical Society Facebook page, but you don't need to have a Facebook account to view it; just click on the video link below. (It is about an hour long.)

Video © Biochemical Society 2018

To summarise the lecture briefly, Pearl began with a well-known but still controversial quote from the Greek philosopher Democritus: "Nothing exists except atoms and empty space. All else is opinion." He then summarised the beginning of his scientific career as a postgraduate student at Birkbeck. During his PhD he learned computer programming and wrote - or contributed to - some of the first structural bioinformatics programs, as well as studying the structure of the aspartic protease, endothiapepsin (PDB 4APE). This was in the early 1980s, when the world was becoming aware of the scourge of AIDS and the necessity of targeting its viral cause, HIV. Sequencing the tiny HIV genome revealed a protease with similar sequence motifs to aspartic proteases, but less than half as long. It was Pearl, by then a postdoc at the Institute of Cancer Research in London, and Willie Taylor from Birkbeck who predicted, years before the structure was solved, that HIV's protease would be active as a dimer. (There is more about this in section 7 of PPS, on Quaternary Structure.) The HIV protease structure - exactly as Pearl and Taylor had predicted it - became an important tool for the discovery of protease inhibitors as drugs against AIDS. The first protease inhibitor to enter the clinic was named saquinavir after the amino acid motif SQNI that led them to predict the enzyme's dimeric structure.

Not surprisingly, this discovery also proved to be a gateway to Pearl's career as an independent researcher. He set up his lab in 1989 in the biochemistry department at UCL, with research projects in three areas: DNA repair, signal transduction, and a group of unrelated proteins that help other proteins to fold and that have collectively been named chaperones. He spent the rest of his lecture describing just a few of the many proteins that his group has studied during the last 30 years: first at UCL, then back at the Institute of Cancer Research and since 2009 at Sussex. These included the enzyme uracil DNA glycosylase, which is involved in the specific repair of cytosine residues in DNA that have been deaminated to form uracil. His co-worker on this project was Renos Savva, who is now a senior lecturer at Birkbeck and director of our MSc course in Biobusiness. The structure of this enzyme (PDB 1UDI) explains the exquisite specificity of this enzyme. Another, much smaller DNA repair enzyme, mismatch uracil glycosyase, was found to have the same fold. His more recent work, which uses electron microscopy as well as X-ray crystallography, includes the structure and mechanism of glycogen synthase kinase, and the structure and dynamics of further DNA damage and repair systems. Listen to the lecture if you want to find out more!

The Many Uses of Bioinformatics

Every year, Birkbeck hosts a lecture by a distinguished scientist to honour the memory of the founder of its Crystallography Department, J.D. Bernal. “Sage” as he was called by all who worked with him had an enormous range of research interests spanning both science and society; he is widely considered one of the most brilliant scientists never to have won a Nobel Prize. The 2014 Bernal Lecture, held on March 27, was given by Professor Janet Thornton, the director of the European Bioinformatics Institute (EBI) at Hinxton near Cambridge.

Professor Dame Janet Thornton, © BBSRC 2014

Introducing the lecture Professor David Latchman, Master of Birkbeck, described it as a unique occasion: the only time he has introduced as a guest lecturer someone who he had interviewed for a job. Thornton includes both Birkbeck and UCL on her CV: appropriately, her last post in London was that of Bernal Professor, held jointly at both colleges. She moved on to “even greater heights” as director of one of Europe’s top bioinformatics institutions in 2003.

Thornton began her lecture with a quote from Bernal: “We [academics] can go on being useless up to a point, with confidence that sooner or later some use will be found for our studies”. That quote is of particular relevance to the subject that she has made her own: bioinformatics. She had already begun her research career in 1977, when Fred Sanger invented the process that was used to obtain the DNA sequence of the human genome. That endeavour, which was completed in 2003, took over ten years and cost billions of dollars. Sequencing a human-sized genome, which has about 3 billion base pairs of DNA, now takes maybe 10 minutes and costs about a thousand dollars. While a decade ago we had one “Human Genome”, we now have lots. Mega-sequencing projects already planned or in progress include projects to sequence about 8,000 Finns, and the entire 50,000 population of the Faeroe Islands; one to sequence paired tumour and normal genomes from 20,000 cancer patients; and the UK10K project, which is investigating the genetic causes of rare diseases.

It is now almost extraordinarily simple and cheap to obtain genomic data, but real challenges remain in interpreting and understanding it so that it can be used in medicine. This is the province of bioinformatics, and Thornton devoted much of her presentation to explaining five ways in which gene (and protein) sequence information is being applied to both basic and clinical medical research:

1. Understanding the molecular basis of disease
2. Investigating differences in disease risk caused by human genetic variation
3. Understanding the genomics of cancer
4. Developing drugs for infectious diseases, including neglected diseases
5. Investigating susceptibility to infectious disease

There are rather more than 20,000 genes in the human genome, far fewer than were originally predicted. Tiny differences between individuals in many of these either directly cause a genetic disorder or confer an increased – or in some cases decreased – risk of developing a disease. The genetic causes of some diseases, such as the bleeding disorder haemophilia, were known many years before the “genome era”: others have been discovered more recently. Mapping known mutations onto the structure of the enzyme copper, zinc superoxide dismutase has revealed the cause of the inherited disorder amyotrophic lateral sclerosis, a form of motor neurone disease. And knowing the genome sequence has already made an enormous contribution to our understanding of the mechanisms of disease development, contributing to improvements in diagnosis and the design of novel drugs.

We now understand that cancer is a genetic disease: it arises when mutations in a group of cells cause them to grow and divide excessively. A cancer is no longer classified just by its location (for example, a breast or lung cancer) but by the particular spectrum of genetic variations in its cells. About 500 different genes are known to be mutated in cancer, some much more often than others. For example, about 60% of cases of melanoma, a type of skin cancer, contain one specific mutation in the gene BRAF. This codes for a protein that can direct cells to grow and divide, and the cancer-causing mutation sticks this protein into the ON position, so this signal is always sent. Scientists in a company called Plexxicon used their knowledge of this mutation and the structure of the protein to design a drug, vemurafenib, which prevents the BRAF protein from signalling. This can cause a dramatic, if short-term improvement in melanoma patients, but, crucially, it only works in patients whose cancers carry this mutation. It is one of the first developed examples of a “personalised medicine” that is only used alongside a diagnostic test for a genetic variation. There will soon be many more.

Genomics is also proving very useful in the fight against infectious disease. Antibiotic resistance is one of the greatest emerging threats to human health, and scientists have to use all the tools at their disposal, including genomics and bioinformatics, as they try to stay one step ahead of rapidly mutating pathogens. Sequencing is widely used to track the sources of outbreaks of infection and of resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) in hospitals, and it is the only way of determining the exact nature of an infection. One of the most dramatic examples of the use of genomics in infectious disease control occurred in 2011, when a novel strain of E. coli O104 caused about 4,000 cases of serious food-borne illness and 50 deaths in Germany. This was originally linked to cucumbers imported from Spain but a global effort to trace its specific sequence variants proved that the source of the infection was beansprouts grown on a farm near Hamburg.

There was much more to Thornton’s wide-ranging lecture than simply bioinformatics and medicine: more, indeed, than it is possible to do justice to in a single blog post. She went on to describe some of the benefits of genomics for agriculture and food security. These included designing new strategies for controlling pests and diseases, maximising the efficiency of biomass processing, and even managing biodiversity. It is necessary to measure biodiversity in order to manage it properly; it is now possible to define a short stretch of DNA sequence that fully identifies a species or sub-species (a so-called “DNA barcode”) and these are beginning to be used to track some very diverse organisms, including the 400,000 known species of beetle.

The lecture ended with a short discussion of some of the challenges facing bioinformatics and genomics in the second decade of this century, largely relating to difficulties with storing, manipulating and understanding the enormous quantity of data that is being generated. Mining this data mountain for the benefit of mankind is a task that is beyond either the academic community or the biotech industry alone. It will require novel ways of doing science that involve governments and charities as well as academia and industry. The new Centre for Therapeutic Target Validation, launched at Hinxton on the same day as Thornton’s Bernal Lecture, is a pioneering example of such a partnership. It has been set up by the EBI, the Sanger Institute where a third of the original human genome sequence was obtained, and pharmaceutical giant GSK, and its scientists aim to use the whole range of available genomic data to select and evaluate new targets for novel drugs.

Bioinformatics is covered in section 6 of the PPS course. Students who take the second-year option Techniques in Structural Molecular Biology will return to it then, where the material focuses on selecting protein targets for structural genomics initiatives: a task that is linked to that of selecting drug discovery targets.

This post will be cross-posted on the Birkbeck Events blog.

New protein structures presented at the 2013 BCA Winter Meeting

The work of crystallographers in the UK is supported through the British Cryatallographic Association, which has about 700 members based in academia and industry. It is organised into four groups representing different disciplines within crystallography, including one for structural biologists called, not surprisingly, the Biological Structures Group. Every December, this group organises a one day conference to present some of the most recent developments in structural biology. I have blogged these meetings before, and searching this blog for "winter meeting" will find a few of those posts.

The 2013 meeting was billed as both a "final" event in the centenary year of the Braggs' landmark discoveries and part of the build-up to the International Year of Crystallography, but these were not the only anniversaries highlighted there. 2013 also marked the sixtieth anniversary of the publication of the structure of DNA. The 2013 Winter Meeting was held in King's College London, which played a very important part in that discovery: Maurice Wilkins and Rosalind Franklin, who obtained the X-ray diffraction patterns that led to the discovery of the double helix, were based there. (Wilkins shared the Nobel Prize for this discovery with Watson and Crick; Franklin died in 1958, four years before that prize was awarded.) And the first precise physical model of the double helix is still on display in the college.

Maurice Wilkins' original DNA model

The first researcher to speak at the meeting was Birkbeck's own Professor Bonnie Wallace. Her work on the structures of voltage gated sodium channels has been described on this blog before, most recently in April 2013. These proteins are responsible for the transport of ions in and out of cells, an essential signalling mechanism in all multi-cellular organisms. Their structures, however, are among the most intractable of all membrane proteins (PPS section 11, to be released in May, covers this fast moving field). Wallace has used a combination of X-ray crystallography, spectroscopy and molecular dynamics to explore the structure and mechanism of sodium channels in bacteria. The bacterial sodium channel is simpler than the mammalian equivalent, consisting of a tetramer in which helices from each monomer line the pore. The Wallace group's most recent strucure (PDB 3ZJZ) shows the position of the C-terminal domain of these channels for the first time. This domain consists of a coiled coil formed by one helix from each monomer that is linked to the rest of the protein by a flexible region. Moving the coiled coil up and down causes a conformational change that allows the channel to open and close.

The technique of rational or structure-based drug design, which involve modelling the interactions between a library of potential ligands and a protein binding site, has proved particularly successful in the design of anti-viral drugs. Several inhibitors of HIV protease and of influenza virus neuraminidase that were designed in this way have become very successful drugs. David Stuart from the University of Oxford and the Diamond synchrotron gave a talk illustrating how structure-based in silico techniques are now being applied to design drugs against another virus family: the Picornaviruses. Members of this large family are responsible for a diverse range of diseases, ranging in humans from polio to the common cold. The foot-and-mouth virus, which affects livestock and which devastated parts of the UK countryside in 2001, is also a member of this family.

One of the viruses studied in Stuart's goup is a human picornavirus that causes similar symptoms to the foot-and-mouth virus and that represents a serious threat to public health in East Asia. The disease is known as hand foot and mouth virus, and the virus as CAV16: like all picornaviruses, it consiss of a single strand of RNA enclosed within an icosahedral (20-sided) protein capsid. The intact virus particles are very fragile and diffraction patterns must be captured before the particles disintegrate in the X-ray beam. Stuart and his Chinese collaborators have used one of the microfocus beamlines at Diamond to take snapshots of the virus structure at several points during its life cycle. One of these is of an "uncoating intermediate" that shows one of the viral proteins (VP1) emerging from the capsid so that it can be embedded in the membrane of a host cell Ren et al., 2013). Stuart and his co-workers are now designing compounds to bind to these intermediate structures and prevent the virus from entering its human host cells.

All cells, whether prokaryotic or eukaryotic, contain long molecules of DNA that must be packaged in order to fit into the confined space available. Fortunately for developers of anti-bacterial drugs (and users of antibiotics) bacterial cells package DNA using a different mechanism from mammalian ones. In bacteria, enzymes called topoisomerases bind to, cut and re-join double-stranded DNA so that it can be unwound or untangled ahead of replication. Ivan Laponogov, a postdoctoral research assistant at King's College, described recent work in his group on the structure of one of these enzymes. Bacterial topoisomerase II ia a target for an important class of antibiotics, the fluoroquinolones, but resistance to these drugs is increasing.

These enzymes are powered by ATP and act as "clamps", capturing one double-helical strand of DNA and passing it through a break in another to remove supercoils and knots in the nucleic acid structure. The structure presented at this meeting was the first of a complete topoisomerase dimer bound to DNA in the "open clamp" position. This structure was solved with and without a fluoroquinolone drug (levofloxacin) bound. The structure with drug bound showed that molecule intercalating between DNA bases at the point where the nucleic acid would be cleaved, preventing that cleavage. The structure without the drug showed the DNA in a different position; the position of a functionally important magnesium ion also changed between the structures.

Many essential cellular processes involve a post-translational modification in which poly-(ADP ribose) or PAR is added to amino acid side chains, and the processing of this molecule involves a wide variety of enzymes. Inhibitors of one of these, poly-(ATP ribose) polymerase or PARP, have recently been developed as drugs against cancer. David Leys from the University of Manchester described his work on the structure of another enzyme in the PAR life cycle: poly-ADP-ribose glycohydrolase (PARG), which catalyses the removal of PAR from proteins.

Mammalian PARG enzymes have three domains, a N-terminal regulatory region and two C-terminal domains forming the catalytic region; the equivalent bacterial enzymes lack the N-terminus. Leys and his groups first solved structures of a bacterial PARG bound to ADP-ribose (PDB 3SIG) and to a known inhibitor with a similar structure. They found that a C-terminal helix in the protein was clamped around the terminal ribose of PAR, enabling the release of a single ADP-ribose from the polymer. This basic mechanism is similar in the mammalian enzyme. More recently, the Leys group has solved the structure of PARG bound to an intact PAR substrate (PDB 4L2H); modelling studies based on this structure suggest that the enzyme acts predominantly as an exo-glycohydrolase, that is, it catalyses the removal of one residue at the end of the polymer chain. Understanding the structure and mechanism of these enzymes should enable us to develop small-molecule inhibitors of PARG, and these may one day rival the PARP inhibitors as anti-cancer drugs.

A hundred years on from the "invention" of crystallography and sixty years on from the structure of DNA, these elegant, fascinating and complex structures presented at one meeting give a snapshot of recent progress in structural biology. Furthermore, each of these structures has already provided insights into human disease that may yet lead to the development of useful drugs.

Twenty Years of Structural Biology in Drug Discovery

Approximately once a term, scientists working in structural biology and related areas in and near London, meet under the auspices of the London Structural Biology Club to discuss their recent research. These Club meetings generally involve four or five lectures followed by an informal discussion over beer and pizza, generously provided by a sponsoring company.

Most speakers at LSBC meetings are academic researchers and a typical presentation will highlight a novel structure or two, emphasising, for a specialist audience, some of the trickier aspects of how they were solved. The most recent meeting, however – held on 3 July 2012 in Birkbeck’s Clore Management Centre, and sponsored by specialist light scattering company Avid Nano – included one rather unusual talk. Dave Brown recently left the pharmaceutical giant Pfizer after sixteen years in its structural biology group. He now combines an academic post at the University of Kent at Canterbury with work in a new biotech company, Cangenix Molecular Solutions. During his time at Pfizer, Brown contributed his expertise in structural biology to drug discovery programmes for a wide range of cardiovascular, inflammatory and infectious diseases. The topic of his LSBC presentation was the evolution of structural biology in the pharmaceutical industry as he had experienced it during the last two decades.

The history of structural biology in drug discovery goes back a little longer than that, to the use of NMR by companies such as Abbott and Agouron to determine structure-activity relationships for compound series. The first drugs to be largely designed based on structural principles were the HIV protease inhibitors, which are covered in some depth in both section 5 and section 10 of PPS. The first protease inhibitor to be licensed for treating AIDS was Saquinavir (Invirase™), which entered the clinic in 1995, only ten years after the protease gene was first detected in the newly sequenced HIV genome.

By the mid-1990s, most major pharma companies had specialist structural biology groups. The most important developments since then have been driven by technical improvements in molecular biology, gene cloning and protein purification, and in diffraction technology. Synchrotrons such as the UK’s Diamond Light Source make their facilities available for commercial use at a price that large companies, at least, have no trouble affording. X-ray data collection is now extremely fast and can largely be automated so it has become accessible to non-expert users. Although dedicated structural biology groups in industry are no larger than they were in the 1990s, there are many more researchers there – medicinal chemists and molecular biologists – who spend some of their working life doing structural biology. Arguably, a reasonable knowledge of protein structure is essential for all scientists working in drug design.

But what, exactly, does structural biology contribute to the complex process of drug discovery? Once a likely protein target for a new discovery programme has been identified – an enzyme to be targeted by an inhibitor, for example – knowing its three-dimensional structure can help both in the selection of likely starting molecules or “hits” and in their development into “lead” compounds that are potent enough inhibitors to go forward to the later stages of drug discovery. In the related technique of fragment-based drug discovery, a huge number of very small compounds or fragments are screened against a target and those that bind to different parts of the protein’s active site are selected and linked together to form a larger and theoretically more potent compound. Typically, individual fragments bind to their target so weakly that they can only be identified experimentally rather than through computer modelling. Structural experiments can also be used to probe unexpected ligand-binding interactions, perhaps even identifying previously unknown functional binding sites, and to build selectivity for one member of a large protein family into a drug molecule. Selectivity is essential for the development of safe, non-toxic drugs that bind proteins such as kinases and G-protein coupled receptors (see section 11) that have hundreds of homologs in the human proteome.

Brown then went on to describe some of the work that he had been involved with during his career at Pfizer. Phosphodiesterases are enzymes that catalyse the breaking of phosphodiester bonds; this function is responsible for regulating the concentration of the essential cyclic nucleotide monophosphates in cells, and enzymes in this class are drug targets for a range of diseases. Cyclic GMP-specific phosphodiesterase type 5 or PDE5 is the target of Pfizer’s most famous drug, sildefanil, which is known worldwide as Viagra™ (link is to the Wikipedia entry). Researchers at Pfizer have used structure-based techniques to modify the basic structure of this compound into drugs with the potential to treat very different diseases, including Reynaud’s syndrome (in which the blood supply to the extremities is reduced in cold temperatures) and stroke recovery. The recent structure-led discovery of the mechanism by which another protein in this family, PDE4, regulates cyclic AMP may even lead to the design of drugs for a range of devastating neurological conditions including Alzheimer’s disease and schizophrenia.

Brown summed up his lecture with a series of recommendations for structural biologists working in industry (and by extension, for industrial researchers doing structural biology there). He advised them to get involved in drug discovery programmes at the earliest stage, to use structure to understand mechanisms of action rather than simply to design inhibitors and to collaborate between disciplines and sometimes even across companies. And, overall (and more prosaically), to work as quickly and cheaply as reasonable. These are not bad recommendations for academic researchers, either.