Microtubules are long protein filaments that form an important part of the cell cytoskeleton. They are formed from polymers of a protein called tubulin - first tubulin forms dimers, each consisting of one alpha and one beta subunit, and then these dimers polymerise head-to-tail to form structures called protofilaments. The microtubules are hollow tubes formed from assemblies of parallel protofilaments. Several proteins collectively known as microtubule associated proteins (MAPs) associate with these microtubules and stabilise - or occasionally destabilise - different forms.
Doublecortin is a MAP that stabilises the most commonly found type of microtubule, which is composed from thirteen protofilaments. Mutations in both proteins that destabilise the interaction between them can cause devastating neurological diseases. A recent paper by Carolyn Moores, an electron microscopist working at Birkbeck, has highlighted details of the interaction between these proteins.
You can read a summary of this paper here (PDF format). This is one of a series of summaries of high impact papers by Birkbeck scientists that I have written for the website of the Institute of Structural and Molecular Biology, which links researchers in these disciplines at Birkbeck with those at UCL.
If you have time once you have read the summary you may explore the structures of these proteins further by downloading the atomic coordinate files that were "docked" into the electron density in this paper from the PDB. The PDB entry used for the alpha-beta tubulin dimer structure is 1JFF and the one used for the doublecortin domain that is bound to tubulin is 1MJD.
Thursday, 25 November 2010
Friday, 5 November 2010
Protein Structure Highlighted in Birkbeck Science Week
At the end of October Birkbeck College hosted its first Science Week, featuring lectures from distinguished researchers in all science departments. Two talks by members of the Department of Biological Sciences highlighted the beauty and importance of protein structure: the topic of the PPS course. Both lecturers came originally from North America; both moved to Birkbeck about twenty years ago; and both have achieved very significant honours as researchers in structural biology since.
Proteins, DNA and the Components of Life
Professor Bonnie Wallace’s research focuses on the structural analysis of proteins and peptides bound to membranes, and particularly on those involved in ion transport and cell signalling. Her talk in Science Week was entitled “Proteins, DNA and the Components of Life”. She began by describing proteins and nucleic acids as large molecules – “macromolecules” – that are essential components of all living cells. They are both polymers of smaller units; as you should know by now, proteins are polymers of amino acids and nucleic acids similarly are polymers of nucleotides. Amino acids combine as “beads on a string” in many different ways to form the vast array of known proteins; the functional – structural, metabolic or transport among many others – of each is determined by its three-dimensional structure. The techniques used for determining protein (and DNA) structure, including X-ray diffraction, which have developed from fairly crude beginnings about fifty years ago, now allow even large structures to be determined quickly. The section of Professor Wallace’s talk that introduced basic protein structure was illustrated with a physical model of a protein from the 1970s that was then compared with modern molecular graphics representations of protein structures. “Cartoon”, “ball and stick” and “space-filling” protein models, as well as stereo views that can give the illusion of three-dimensionality, are all useful for different purposes.
DNA molecules also are linear polymers, and these – particularly in eukaryotic cells – are extremely long and need to be packed very tightly if they are to fit into the tiny cell nuclei. The packing mechanism involves interactions between the DNA molecule and proteins called histones, to form a structure resembling “beads on a string” that condenses further to form fibres and eventually chromosomes.
The final part of Professor Wallace’s talk concerned the evolutionary differences between DNA in different individuals and in different species, celebrating the tenth anniversary of the publication of the first draft of the human genome. Genomics has provided a useful correction to human arrogance by revealing that many organisms have larger, if not necessarily more complex, genomes than ours. More importantly, differences between bacterial and human cells, between cancer cells and normal ones, and between dysfunctional (mutated) and normal versions of disease-linked proteins, are enabling us to design and develop drugs against a wide range of diseases. Important drugs for infectious diseases including influenza and HIV have been designed using “structure-based” techniques. Professor Wallace ended her talk by describing some of her group’s work on the structure of a protein that is embedded in a cell membrane and provides a channel for sodium ions to pass into cells. Anti-epileptic drugs such as lamotrigine work by binding to and blocking these channels, which will be discussed in detail in the section on Membrane Proteins towards the end of the PPS course.
Model for the structure of a human sodium channel
The Arms Race between Man and Pathogen
Professor Helen Saibil studies proteins using electron microscopy, which can determine the structure of very large proteins and of protein complexes embedded in cell membranes. Her talk, intriguingly titled “The Arms Race between Man and Pathogen”, focused on two pieces of her group’s research into proteins that punch holes, or pores, in membranes. These independent studies led to the discovery of an unexpected similarity between a group of bacterial proteins that cause damage during infection and one of human (and other mammalian) proteins that help protect against viral infection and blood cancers.
Pore-forming protein toxins are “weapons” that bacteria can use in order to break holes in the cells of their human or other animal hosts. They are used by a wide range of bacteria, including some very dangerous ones; one example is Streptococcus pneumoniae, which, as its name implies, causes pneumonia. Unusually, these proteins can exist in two forms, one that is water-soluble and another that is membrane-embedded. The change between these forms requires a considerable rearrangement of the protein’s shape, with one hydrophobic (“water-hating”) segment that is located deep in the core of the soluble protein uncurling to form a “beta-hairpin” structure that embeds in the membrane.
In 2005, Professor Saibil’s group used electron microscopy to determine the structures of two forms of the pore forming toxin from S. pneumoniae, pneumolysin, that together illustrate its mechanism of action. Initially, the protein associates into ring-shaped “pre-pores” of about forty monomers that sit on the membrane surface; the pore itself is formed when part of each monomer changes shape and burrows deep into the membrane. They were able to fit the more detailed X-ray crystal structure of a related protein as a soluble monomer into the shape of the pre-pore protein, and show the type of rearrangement that would be necessary for part of that protein to “collapse” into the membrane.
Another protein that has been studied by Professor Saibil and her colleagues recently is perforin, which is secreted by some lymphocytes (white blood cells that form an important part of the immune system) and which also punches holes in membranes: this time, in the membranes of infected or cancerous cells. This allows the lymphocyte to deliver toxins to the affected cell that trigger the process of apoptosis, or programmed cell death. People with a defective form of this protein suffer from severe viral infections and have a greatly increased risk of blood cancers.
There is no detectable similarity between the sequence of perforin and that of pneumolysin. However, when the first structure of a human perforin-like molecule was discovered in 2007, researchers were surprised to discover that its fold was similar to that of the bacterial protein. It became clear that human and bacterial pore-forming proteins must have evolved from a common ancestor. Protein structures are more tightly conserved than gene and protein sequences, and the structures of distantly related proteins are often much more clearly similar than their sequences.
Hoping to understand more about the mechanism of this protein, Professor Saibil’s group then used electron microscopy to solve the structure of perforin assemblies in contact with membranes. They observed pore structures that were similar, but not identical, to those of pneumolysin. In particular, the perforin structures indicated a much smaller conformational change between non-bound and membrane bound forms than the one seen with pneumolysin. Also, the perforin molecule seemed to be arranged “inside out” in the pore structure as compared to that of pneumolysin, an observation that the group was able to verify with biochemical tests. This finding was very unexpected, but turned out to be consistent with a twist between two parts of the pneumolysin molecule that does not occur in perforin. Both molecules have “arms” that extend down into the membrane to form the sides of the pore, but perforin’s arms are much longer and do not require as big a conformational change to reach down into the membrane.
View of the cryo-EM reconstruction of a perforin pore, with the front half cut away to show how modelled perforin molecules fit into the EM electron density.
The grey surface is the EM map and the subunits are coloured by domain / subregion, as follows. N-terminal region, blue; central beta sheet, red; C2 domain, yellow; modelled beta hairpins (pore lining), orange; EGF domain, green; C-terminal region, magenta. The membrane is seen as a double layer of grey electron density at the base of the pore.
This story of two related proteins, one on each side of the battle between man and pathogen, features many aspects of protein structure research that are covered during the PPS course. These include the beta-hairpin structure, the interatomic forces that determine the way proteins fold, and the structures of membrane proteins. If, however, you have been inspired to learn more about the way electron microscopy is used to study protein structure, you should aim to take our second-year MSc module, Techniques in Structural Molecular Biology.
Tuesday, 5 October 2010
Welcome - and Birkbeck Science Week
Welcome PPS 2010-11 students to the PPS blog!
I have run this blog in some recent years to help students understand how what they study relates to current research in protein structure and in areas related to it such as drug design and development. This year, I will be posting accounts of lectures, conferences and new papers in the areas covered by the course, and link them to the scientific literature more widely. Comments are welcomed and if any of you would like to have a go at blogging I can set you up as an author.
We have a weekly seminar programme shared between Biological Sciences at Birkbeck and the other departments in the Institute of Structural and Molecular Biology, and later in the course I will occasionally post reports of these seminars when they seem particularly relevant to the course material. There is a theme for the seminars each term, and this term (Autumn 2010) this is "Cellular and Structural Biology of Infection".
Before then, however, I will be blogging from Birkbeck Science Week at the end of October. There will be two lectures then that are very relevant for this course, both given by senior professors in the Department of Biological Sciences: Prof. Helen Saibil on the structures of bacterial protein toxins and Prof. Bonnie Wallace on proteins and DNA. Do check back on this blog afterwards as I hope that will give you an idea of both the beauty and the elegance of our subject. Or - if you are within reach of London on either of those days - do come and hear what they have to say! Science Week lectures are free and open to all, although registration is required.
Enjoy the blog - and do let us know what you think of it!
Clare Sansom
I have run this blog in some recent years to help students understand how what they study relates to current research in protein structure and in areas related to it such as drug design and development. This year, I will be posting accounts of lectures, conferences and new papers in the areas covered by the course, and link them to the scientific literature more widely. Comments are welcomed and if any of you would like to have a go at blogging I can set you up as an author.
We have a weekly seminar programme shared between Biological Sciences at Birkbeck and the other departments in the Institute of Structural and Molecular Biology, and later in the course I will occasionally post reports of these seminars when they seem particularly relevant to the course material. There is a theme for the seminars each term, and this term (Autumn 2010) this is "Cellular and Structural Biology of Infection".
Before then, however, I will be blogging from Birkbeck Science Week at the end of October. There will be two lectures then that are very relevant for this course, both given by senior professors in the Department of Biological Sciences: Prof. Helen Saibil on the structures of bacterial protein toxins and Prof. Bonnie Wallace on proteins and DNA. Do check back on this blog afterwards as I hope that will give you an idea of both the beauty and the elegance of our subject. Or - if you are within reach of London on either of those days - do come and hear what they have to say! Science Week lectures are free and open to all, although registration is required.
Enjoy the blog - and do let us know what you think of it!
Clare Sansom
Monday, 29 June 2009
Fragment-Based Screening and Drug Design
The last Monday seminar of the Summer term at Birkbeck was given by Dr Rob van Montfort of the Institute of Cancer Research. Van Montfort, a former postdoc in the School of Crystallography here, spent six years in industry, at the biotech company Astex Therapeutics before joining the ICR two years ago. There, he is developing high throughput, structure-based drug screening techniques and using them to design novel compounds as candidate anti-cancer drugs.
Van Montfort first described the problem that techniques such as his have been designed to solve: that of attrition in drug discovery. Not only does it often take well over 10 years to move a potential drug compound "from the bench to the bedside", but the attrition rate is immense and not sustainable. A typical drug discovery programme will involve the testing of millions of compounds, and result in, say, half a dozen candidates for Phase I clinical trials and, if all goes well, a single registered drug. And even if a compound reaches the clinic, it may well not recoup the millions of dollars that have been spent on its development.
Drug companies and adacemic groups have been turning to novel technologies to help address this problem and reduce the time and cost of drug development. The technique de Montfort described, fragment-based screening, is one of these. It was originally developed by Wim Hol's group at the University of Groningen, the Netherlands, and developed for commercial use by companies including Astex and Plexxikon.
Historically, X-ray crystallography has been used mainly in the mid stages of drug development, in modify promising lead compounds into drug candidates. Now - thanks largely to high-throughput crystallography or structural genomics programmes - X-ray technology has improved to the point where it can be used at a much earlier stage. In fragment-based screening, groups of small molecules taken from a "fragment library" are soaked into protein crystals and the resulting structures examined by X-ray crystallography to see which fragments have bound to which parts of the protein's ligand-binding sites. These fragment hits generally bind very weakly but may be "joined together", if they bind into different pockets within the binding site, and modified further to generate tight-binding inhibitors and, eventually, candidate drugs.
De Montfort went on to describe a few published case studies of Astex' protein drug targets, including the P38a MAP kinase. This kinase is involved in cellular responses to stress, and its inhibitors may be therapeutically useful in a variety of inflammatory and auto-immune diseases. Fragment-based screening identified a lipophilic fragment that bound into the selectivity pocket of this kinase (Hartshorn et al. (2005), J. Med. Chem. 48, 403-413; PDB 1W7H) and modified it to produce a larger, tight-binding inhibitor (Gill et al. (2005), J. Med. Chem. 48, 414-426; PDB 1W82 & 7 others).
Thrombin, a serine protease involved in blood coagulation, is an important therapeutic target for stroke and deep vein thrombosis (DVT). Van Montfort was involved in the development of thrombin inhibitors, a process that was particularly hard because the thrombin active site is charged, and the charged compounds that would be expected to bind there are unlikely to work as oral drugs. Howard et al. (2006) published a fragment-based screen against thrombin using a library of uncharged compounds, finding small fragments that bound to one pocket within the substrate-binding site and larger ones that bound to a different one (J. Med. Chem. 49, 1346-55; PDB 1WBG). Combining the chemistry of these fragments into a single, larger molecule produced a potent series of uncharged, non-peptide inhibitors with structures that had not previously been seen in protease inhibitors.
Kinases are signalling proteins that control many biochemical and physiological processes, including the cell cycle, and cell-cycle kinases are very important as targets for anti-cancer drugs. Both Astex and the ICR have extensive programmes developing kinase inhibitors, and thehe two organisations have collaborated on the discovery of compounds that inhibit protein kinase B (see e.g. Saxty et al. (2007), J. Med. Chem. 50, 2293-6; PDB 2UW3).
Van Montfort has now set up a fragment-based screening lab at the ICR and developed a library of over 1800 fragments. His group is investigating potential protein targets for drugs against cancer, including kinases.
All the journal references in this blog post are in the Journal of Medicinal Chemistry, which should be available via the Birkbeck e-library.
Van Montfort first described the problem that techniques such as his have been designed to solve: that of attrition in drug discovery. Not only does it often take well over 10 years to move a potential drug compound "from the bench to the bedside", but the attrition rate is immense and not sustainable. A typical drug discovery programme will involve the testing of millions of compounds, and result in, say, half a dozen candidates for Phase I clinical trials and, if all goes well, a single registered drug. And even if a compound reaches the clinic, it may well not recoup the millions of dollars that have been spent on its development.
Drug companies and adacemic groups have been turning to novel technologies to help address this problem and reduce the time and cost of drug development. The technique de Montfort described, fragment-based screening, is one of these. It was originally developed by Wim Hol's group at the University of Groningen, the Netherlands, and developed for commercial use by companies including Astex and Plexxikon.
Historically, X-ray crystallography has been used mainly in the mid stages of drug development, in modify promising lead compounds into drug candidates. Now - thanks largely to high-throughput crystallography or structural genomics programmes - X-ray technology has improved to the point where it can be used at a much earlier stage. In fragment-based screening, groups of small molecules taken from a "fragment library" are soaked into protein crystals and the resulting structures examined by X-ray crystallography to see which fragments have bound to which parts of the protein's ligand-binding sites. These fragment hits generally bind very weakly but may be "joined together", if they bind into different pockets within the binding site, and modified further to generate tight-binding inhibitors and, eventually, candidate drugs.
De Montfort went on to describe a few published case studies of Astex' protein drug targets, including the P38a MAP kinase. This kinase is involved in cellular responses to stress, and its inhibitors may be therapeutically useful in a variety of inflammatory and auto-immune diseases. Fragment-based screening identified a lipophilic fragment that bound into the selectivity pocket of this kinase (Hartshorn et al. (2005), J. Med. Chem. 48, 403-413; PDB 1W7H) and modified it to produce a larger, tight-binding inhibitor (Gill et al. (2005), J. Med. Chem. 48, 414-426; PDB 1W82 & 7 others).
Thrombin, a serine protease involved in blood coagulation, is an important therapeutic target for stroke and deep vein thrombosis (DVT). Van Montfort was involved in the development of thrombin inhibitors, a process that was particularly hard because the thrombin active site is charged, and the charged compounds that would be expected to bind there are unlikely to work as oral drugs. Howard et al. (2006) published a fragment-based screen against thrombin using a library of uncharged compounds, finding small fragments that bound to one pocket within the substrate-binding site and larger ones that bound to a different one (J. Med. Chem. 49, 1346-55; PDB 1WBG). Combining the chemistry of these fragments into a single, larger molecule produced a potent series of uncharged, non-peptide inhibitors with structures that had not previously been seen in protease inhibitors.
Kinases are signalling proteins that control many biochemical and physiological processes, including the cell cycle, and cell-cycle kinases are very important as targets for anti-cancer drugs. Both Astex and the ICR have extensive programmes developing kinase inhibitors, and thehe two organisations have collaborated on the discovery of compounds that inhibit protein kinase B (see e.g. Saxty et al. (2007), J. Med. Chem. 50, 2293-6; PDB 2UW3).
Van Montfort has now set up a fragment-based screening lab at the ICR and developed a library of over 1800 fragments. His group is investigating potential protein targets for drugs against cancer, including kinases.
All the journal references in this blog post are in the Journal of Medicinal Chemistry, which should be available via the Birkbeck e-library.
Thursday, 18 June 2009
Analytical Ultracentrifugation: Structures of Unstructured Proteins
Dr David Scott of the National Centre for Macromolecular Hydrodynamics, based at the University of Nottingham, has been visiting Birkbeck for a few days to give a course. He also gave a seminar in which he explained one of the biophysical techniques used in the Centre, analytical centrifugation, and how it is used to help determine something of the "structure" inherent in unstructured, or partially structured, proteins.
The analytical ultracentrifuge was invented by Theodor Svedberg in 1923; three years later, he won the Nobel Prize in Chemistry for research using it. It is simply a centrifuge that spins very fast (from about 1,000 - 60,000 rpm); the sample being spun is monitored optically over a period of time. The normal settling of particles in solution under gravity (sedimentation) is speeded up by spinning in a centrifuge, which essentially replaces the gravitational force by a centrifugal force. The speed of sedimentation depends on the masses and shapes of the particles involved. The maths is far too complex to be described here.
One of the first uses of ultracentrifugation was in the determination of molecular mass. Sedimentation times are measured in Svedberg units (S); 1S is exactly equivalent to 10-13 seconds. These times are related rather inexactly to molecular mass and often used to characterise large proteins and protein complexes; you have come across these in the PPS course in our discussion of ribosomal subunits. The small subunit of the Thermus thermophilus ribosome, illustrated there, is described as "30S".
Ultracentrifugation is now used routinely to determine whether a sample is homogeneous; if it is, all particles will have the same mass and shape, and therefore the same sedimentation time. A plot of sedimentation velocity for a sample can show whether the solution is homogenous or heterogenous, and whether protein is aggregated (in which case, aggregates will consist of different numbers of molecules and have different masses). Sedimentation equilibration experiments, which investigate the final steady state where sedimentation is balanced by diffusion, can be used to determine ligand binding and chemical reactions.
Scott has used ultracentrifugation and other biophysical techniques to investigate the structures of unstructured regions of a bacterial DNA-binding protein, KorB. This protein has a DNA-binding domain and a dimerisation domain each with a known structure, and it is known to interact with RNA polymerase as well as with DNA. Other parts of the protein, however, are only known to be "intrinsically unstructured". A combination of ultracentrifugation with other techniques useful for studying unstructured proteins, including circular dichroism and small angle X-ray scattering, were used to investigate the range of structures adopted by these unstructured regions. Results so far indicate that KorB forms a range of relatively compact structures when isolated in solution but that the unstructured regions extend when it binds to a related protein, KorA.
Analytical ultracentrifugation, CD spectroscopy and other biophysical techniques will be described in much more detail in the second year MSc module, Techniques in Structural Molecular Biology (TSMB).
The analytical ultracentrifuge was invented by Theodor Svedberg in 1923; three years later, he won the Nobel Prize in Chemistry for research using it. It is simply a centrifuge that spins very fast (from about 1,000 - 60,000 rpm); the sample being spun is monitored optically over a period of time. The normal settling of particles in solution under gravity (sedimentation) is speeded up by spinning in a centrifuge, which essentially replaces the gravitational force by a centrifugal force. The speed of sedimentation depends on the masses and shapes of the particles involved. The maths is far too complex to be described here.
One of the first uses of ultracentrifugation was in the determination of molecular mass. Sedimentation times are measured in Svedberg units (S); 1S is exactly equivalent to 10-13 seconds. These times are related rather inexactly to molecular mass and often used to characterise large proteins and protein complexes; you have come across these in the PPS course in our discussion of ribosomal subunits. The small subunit of the Thermus thermophilus ribosome, illustrated there, is described as "30S".
Ultracentrifugation is now used routinely to determine whether a sample is homogeneous; if it is, all particles will have the same mass and shape, and therefore the same sedimentation time. A plot of sedimentation velocity for a sample can show whether the solution is homogenous or heterogenous, and whether protein is aggregated (in which case, aggregates will consist of different numbers of molecules and have different masses). Sedimentation equilibration experiments, which investigate the final steady state where sedimentation is balanced by diffusion, can be used to determine ligand binding and chemical reactions.
Scott has used ultracentrifugation and other biophysical techniques to investigate the structures of unstructured regions of a bacterial DNA-binding protein, KorB. This protein has a DNA-binding domain and a dimerisation domain each with a known structure, and it is known to interact with RNA polymerase as well as with DNA. Other parts of the protein, however, are only known to be "intrinsically unstructured". A combination of ultracentrifugation with other techniques useful for studying unstructured proteins, including circular dichroism and small angle X-ray scattering, were used to investigate the range of structures adopted by these unstructured regions. Results so far indicate that KorB forms a range of relatively compact structures when isolated in solution but that the unstructured regions extend when it binds to a related protein, KorA.
Analytical ultracentrifugation, CD spectroscopy and other biophysical techniques will be described in much more detail in the second year MSc module, Techniques in Structural Molecular Biology (TSMB).
Targeting Mycolic Acid Biosynthesis - Towards New TB Drugs
The Department of Biology and Chemistry at Birkbeck recently hosted as a seminar speaker Dr Geoff Coxon, from the University of Strathclyde, Glasgow. Dr Coxon described a series of compounds that his group had synthesised, some of which are promising candidate drugs against tuberculosis. These are inhibitors of the enzyme beta-ketoacyl-ACP synthase (otherwise known as FabH) with a novel chemical scaffold.
Mycobacterium tuberculosis now infects over a third of the world's population, and tuberculosis kills two million people a year. The threat of tuberculosis is growing largely because of two reasons: a synergy between HIV and TB infections, and the growth of drug resistant strains of the bacterium. Multi-drug resistant (MDR) TB is defined as TB that is resistant to the two most commonly used anti-TB drugs, and extensively drug resistant (XDR) TB as TB that is resistant to four drugs including a fluoroquinolone. The latter is particularly hard to treat. More information on this is available from the TB Alliance.
In order to combat drug resistance it is essential to keep introducing new drugs, and preferably drugs with novel targets and mechanisms. Ideally, a drug should be active against active, replicating TB and persistent TB, which can resist treatment by remaining dormant in macrophages. Much work in anti-TB drug development has focused on enzymes involved in synthesising the very complex cell wall of the bacteria.
The genome sequence of M. tuberculosis (Camus et al., 2002, and material in PPS section 6) revealed a number of enzymes involved in cell wall synthesis. Coxon's group is focusing on the synthesis of one cell wall component, mycolic acids. These are long chain, 2-alkyl 3-hydroxyl fatty acids with between 60 and 80 atoms in their hydrocarbon chains. Two enzyme systems are involved in their synthesis: FAS-1 synthesises the main chain and FAS-2 adds the alpha branch. FAS-2 is found only in plants and mycobacteria.
The FAS-2 system includes a large number of enzymes, all involved in the complex, cyclic process of synthesising a long lipid chain. Inhibiting any of these enzymes will prevent the synthesis of the mature long chain mycolic acids. Coxon's group has been targeting one key enzyme in this process, known as FabH, which connects the FAS-1 and FAS-2 systems. The reaction it catalyses is an extension of the lipid chain by two carbon units.
This enzyme is a homodimer with a tunnel-shaped active site; the chain extension takes place after the substrate has moved into the tunnel. Its structure was first solved in 2001 by Scarsdale and co-workers (PDB code 1HZP). Coxon and his co-workers started their search for a specific inhibitor of this enzyme from the natural antibiotic, thiolactomycin (TLM). This, however, is a relatively weak inhibitor and a chiral compound that is extremely challenging to synthesise. He used fragment-based chemical libraries to develop a range of likely inhibitors with similar skeletons but that would be simpler to synthesise.
Some of the first compounds investigated were better inhibitors of the enzyme but not active against the whole FAS-2 system or M. tuberculosis itself. It appeared that another enzyme, known as KasA, may sometimes take the place of FabH if that is inhibited. They have now produced a series of compounds based on a 2-aminothiazole-4-carboxylate scaffold that includes some very active ones. However, the compounds in this series that are the most potent inhibitors of the enzyme are not the most active against the intact bacterium, and vice versa - there are complex interactions going on that are not yet entirely understood. It is likely, even, that FabH is not their most important protein target.
A good medicine against tuberculosis must be active against both resistant and dormant forms of the bacterium, with few side effects and few interactions with other drugs, and preferably orally available, and it must be cheap, and therefore easy, to synthesise. These 2-aminothiazole-4-carboxylates are easily synthesised and some are effective against M. tuberculosis in vivo, but much more work is needed to determine whether they will fulfil the other criteria.
See Al-Balas, Q. et al. (2009) PLoS ONE 4(5) (open access) for more information about this work.
Dr Coxon also works in TB Drug Discovery UK, an alliance of scientists involved in developing new treatments for tuberculosis.
Mycobacterium tuberculosis now infects over a third of the world's population, and tuberculosis kills two million people a year. The threat of tuberculosis is growing largely because of two reasons: a synergy between HIV and TB infections, and the growth of drug resistant strains of the bacterium. Multi-drug resistant (MDR) TB is defined as TB that is resistant to the two most commonly used anti-TB drugs, and extensively drug resistant (XDR) TB as TB that is resistant to four drugs including a fluoroquinolone. The latter is particularly hard to treat. More information on this is available from the TB Alliance.
In order to combat drug resistance it is essential to keep introducing new drugs, and preferably drugs with novel targets and mechanisms. Ideally, a drug should be active against active, replicating TB and persistent TB, which can resist treatment by remaining dormant in macrophages. Much work in anti-TB drug development has focused on enzymes involved in synthesising the very complex cell wall of the bacteria.
The genome sequence of M. tuberculosis (Camus et al., 2002, and material in PPS section 6) revealed a number of enzymes involved in cell wall synthesis. Coxon's group is focusing on the synthesis of one cell wall component, mycolic acids. These are long chain, 2-alkyl 3-hydroxyl fatty acids with between 60 and 80 atoms in their hydrocarbon chains. Two enzyme systems are involved in their synthesis: FAS-1 synthesises the main chain and FAS-2 adds the alpha branch. FAS-2 is found only in plants and mycobacteria.
The FAS-2 system includes a large number of enzymes, all involved in the complex, cyclic process of synthesising a long lipid chain. Inhibiting any of these enzymes will prevent the synthesis of the mature long chain mycolic acids. Coxon's group has been targeting one key enzyme in this process, known as FabH, which connects the FAS-1 and FAS-2 systems. The reaction it catalyses is an extension of the lipid chain by two carbon units.
This enzyme is a homodimer with a tunnel-shaped active site; the chain extension takes place after the substrate has moved into the tunnel. Its structure was first solved in 2001 by Scarsdale and co-workers (PDB code 1HZP). Coxon and his co-workers started their search for a specific inhibitor of this enzyme from the natural antibiotic, thiolactomycin (TLM). This, however, is a relatively weak inhibitor and a chiral compound that is extremely challenging to synthesise. He used fragment-based chemical libraries to develop a range of likely inhibitors with similar skeletons but that would be simpler to synthesise.
Some of the first compounds investigated were better inhibitors of the enzyme but not active against the whole FAS-2 system or M. tuberculosis itself. It appeared that another enzyme, known as KasA, may sometimes take the place of FabH if that is inhibited. They have now produced a series of compounds based on a 2-aminothiazole-4-carboxylate scaffold that includes some very active ones. However, the compounds in this series that are the most potent inhibitors of the enzyme are not the most active against the intact bacterium, and vice versa - there are complex interactions going on that are not yet entirely understood. It is likely, even, that FabH is not their most important protein target.
A good medicine against tuberculosis must be active against both resistant and dormant forms of the bacterium, with few side effects and few interactions with other drugs, and preferably orally available, and it must be cheap, and therefore easy, to synthesise. These 2-aminothiazole-4-carboxylates are easily synthesised and some are effective against M. tuberculosis in vivo, but much more work is needed to determine whether they will fulfil the other criteria.
See Al-Balas, Q. et al. (2009) PLoS ONE 4(5) (open access) for more information about this work.
Dr Coxon also works in TB Drug Discovery UK, an alliance of scientists involved in developing new treatments for tuberculosis.
Monday, 9 March 2009
Structure of the trypanosome microtubule cytoskeleton
Twice a term, the School of Crystallography hosts a seminar for the whole of the Institute of Structural and Molecular Biology, which consists of research departments in related disciplines from both Birkbeck and neighbouring University College London. Tom Blundell's (see previous post) was an ISMB seminar; today, we heard one from Professor Keith Gull of the Sir William Gunn School of Pathology, University of Oxford.
Much of Keith Gull's work concerns the single-celled parasite Trypanosoma brucei, which is endemic in large parts of Africa and causes African trypanosomiasis, otherwise known as sleeping sickness. Diseases like this one attract relatively little research funding even though they are important causes of morbidity and mortality in many poor countries; they are classed as neglected tropical diseases. Keith and his colleagues in Oxford are studying many aspects of the molecular biology and genetics of this parasite.
The trypanosome cell surface is covered by a dense coat made up of very many copies of a single protein, called variable surface glycoprotein (VSG). A glycoprotein is a protein with carbohydrate (sugar) groups attached to one or more amino acid sidechains; the word "variable" is used because there are about 1000 variants of this protein. Each of these variants is encoded by a different gene, so the VSG genes account for about 10% of the trypanosome's genome. However, only one of these is expressed at any time, so the trypanosome coat (almost) always consists of multiple copies of a single protein. (There will be times when two proteins are present because the expressed variant is in the process of changing.) Although the sequences of VSG proteins differ considerably, their structures are very similar: they are anchored to the cell membrane and the large extracellular domain consists of an antiparallel coiled coil of alpha helices (see PDB file 2vsg).
Most of Keith's talk, however, concerned the structure of the trypanosome cytoskeleton, which consists of microtubules. Very basically, these are composed of polymers of a protein called tubulin and they are involved in maintaining the structure of components of many types of cells. Keith and his group have used the technique of electron tomography - a type of transmission electron microscopy - to obtain exquisite pictures - if at much lower than atomic resolution - of the structure of this cytoskeleton and begin to understand its function and role in trypanosomal cell division. Structures of these multi-protein complexes are not - yet - available in the PDB.
Trypanosomes have flagella - whip-like structures attached to the cells that can propel them through the host bloodstream. Flagella are found in many cell types, and if they have receptors bound they can also be used to sense cells' surroundings. The trypanosome flagellum is attached to the cell body via a filament and this point of attachment is within a pocket that, interestingly, is the end point for all vesicular traffic within the cell. This means that this pocket needs to contain a large number of proteins vital to the parasite's survival, including its transferrin receptor and haemoglobin receptor. Imaging the structure of the trypanosome cytoskeleton, particularly in the pocket where the flagellum attaches, has also given insight into the rather unusual process through which this cell conducts mitosis (cell division).
Much of Keith Gull's work concerns the single-celled parasite Trypanosoma brucei, which is endemic in large parts of Africa and causes African trypanosomiasis, otherwise known as sleeping sickness. Diseases like this one attract relatively little research funding even though they are important causes of morbidity and mortality in many poor countries; they are classed as neglected tropical diseases. Keith and his colleagues in Oxford are studying many aspects of the molecular biology and genetics of this parasite.
The trypanosome cell surface is covered by a dense coat made up of very many copies of a single protein, called variable surface glycoprotein (VSG). A glycoprotein is a protein with carbohydrate (sugar) groups attached to one or more amino acid sidechains; the word "variable" is used because there are about 1000 variants of this protein. Each of these variants is encoded by a different gene, so the VSG genes account for about 10% of the trypanosome's genome. However, only one of these is expressed at any time, so the trypanosome coat (almost) always consists of multiple copies of a single protein. (There will be times when two proteins are present because the expressed variant is in the process of changing.) Although the sequences of VSG proteins differ considerably, their structures are very similar: they are anchored to the cell membrane and the large extracellular domain consists of an antiparallel coiled coil of alpha helices (see PDB file 2vsg).
Most of Keith's talk, however, concerned the structure of the trypanosome cytoskeleton, which consists of microtubules. Very basically, these are composed of polymers of a protein called tubulin and they are involved in maintaining the structure of components of many types of cells. Keith and his group have used the technique of electron tomography - a type of transmission electron microscopy - to obtain exquisite pictures - if at much lower than atomic resolution - of the structure of this cytoskeleton and begin to understand its function and role in trypanosomal cell division. Structures of these multi-protein complexes are not - yet - available in the PDB.
Trypanosomes have flagella - whip-like structures attached to the cells that can propel them through the host bloodstream. Flagella are found in many cell types, and if they have receptors bound they can also be used to sense cells' surroundings. The trypanosome flagellum is attached to the cell body via a filament and this point of attachment is within a pocket that, interestingly, is the end point for all vesicular traffic within the cell. This means that this pocket needs to contain a large number of proteins vital to the parasite's survival, including its transferrin receptor and haemoglobin receptor. Imaging the structure of the trypanosome cytoskeleton, particularly in the pocket where the flagellum attaches, has also given insight into the rather unusual process through which this cell conducts mitosis (cell division).
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