Sunday, 15 April 2012

Under the Microscope: Kinesin Motors and Cancer.

This post was written first for the Birkbeck College events blog and is re-posted here with permission.

There are many different types of cancer but each is caused by the failure of a cell to control its normal healthy cell division.  Uncontrolled proliferation produces a cluster of cancerous cells, a tumour, often the first indicator of many cancer types.  Several prevalent cancer drugs target microtubules, which are used by the cell to orchestrate the intricate ballet of cell division, but disabling this machinery provokes various unpleasant associated side effects.  In this lecture, which was part of Science Week, Dr Carolyn Moores, of the Department of Biological Sciences at Birkbeck shared some exciting recent developments in her work which pave the way for new cancer drugs which are less toxic to the rest of the body.
To understand the size of the machinery under discussion, if a human body were amplified to fill the whole of Buckingham Palace, each cell would be approximately the size of a single grain of sand.  These cells need to replicate, both to grow and to repair normal wear and tear and this replication is a delicate and highly regulated process.  Dr Moores showed a video of a dividing cell, taken from a remarkable online library of cellular images.  This process starts with the separation of the chromosomes, the familiar four legged bearers of our DNA, of which there are 23 pairs in humans.  The chromosomes arrange themselves in the centre of a spindle-like framework, which then retracts in opposite directions, separating each chromosome into halves and grouping them into two new nuclei centres, ready for the division of the rest of the cell. 

An image of mitosis showing the microtubules (in green) in spindle formation and chromosomes (in blue) about to be divided

The dynamic spindle framework at the heart of this incredibly accurate mechanism is primarily composed of microtubules along with associated proteins including members of the kinesin family of molecular motors, which organise them.  Microtubules are made up of pairs of tubulin molecules, or dimers, each of which has a polar structure.  The dimers bind to each other both longitudinally, with opposites attracting so that the overall polarity is maintained, and laterally so that long thin stable cylinders are formed.  These cylinders can grow and shrink with great flexibility and at all lengths the cylindrical structure provides a frame which can withstand the tension required in pulling chromosomes apart.  Drugs that block the dynamics of microtubules can therefore block the ability of cells to proliferate, which is why they are used in chemotherapy.  Unfortunately, microtubules are also critical in healthy cell repair, as well as providing frameworks essential for cellular structure, organelle positioning and vital transport networks within the cell.
Kinesins are highly attractive as potential targets since each appears to operate primarily in support of one of the major microtubule functions, in which case an inhibitor could be designed to attack the cell’s ability to divide without affecting its other vital processes.
Kinesin proteins comprise several domains, one of which is the motor domain, responsible for the protein’s movement.   This motor binds to a microtubule and uses it as a track with a directionality given by its polarity.  It also binds ATP, the universal cellular fuel, which provides the energy required to move along the track.  This has been particularly well studied in kinesin 1, whose function is to transport cargo along the microtubules.  In a mechanism that Dr Moores compared to a child walking on his hands, each unit of cargo is transported by a linked pair of kinesin 1 molecules.  The molecules alternate so that one will bind to the microtubule and the energy source, ATP, and then its partner will displace it so that the motor effectively steps hand over hand along the microtubule track.  Structural studies of kinesin 1 bound to a microtubule show that a small linker region of each kinesin reacts with the polarity of the track to point and presumably inform the direction of travel.
Dr Moores’ group are studying kinesin 5, which combines into oligomers of four molecules and forms crosslinks between microtubules.  This has been shown to be essential to cell division in humans.  The structural studies have involved cryo electron microscopy which has given a 3D model of the motor domain of kinesin 5 bound to a microtubule, both binding ATP and without ATP present.  Electron microscopy is a technique much like ordinary microscopy except that an electron beam is used instead of visual light and this gives images at a molecular level.  This technique is covered in detail in the TSMB course, one of the options following PPS for students working towards an MSc.   I have included the link although current PPS students will not have access to the course material.  The fact that a fast freezing method is employed is extremely useful since biological samples are effectively viewed in solution, as they are in their natural state.  By fitting x-ray crystallography models, which give atomic level detail of smaller molecular configurations, into the 3D cryo electron microscopy models, an enormous level of detail is obtained.
Drugs that target kinesin 5 are currently in clinical trials and appear to be successful so far.  It would appear that the drugs interact with an on/off switch elucidated by Dr Moores’ team but at the moment the precise function of the on/off switch is not known.  PDB 1Q0B shows an example of an inhibitor bound to kinesin 5 at the loop which functions as a switch.  Work continues in this rewarding area of study with the aim of understanding the purpose of the on/off switch and consequently being able to design future cancer drugs which have even higher specificity and consequently better outcomes.
Dr Moores has written a section of the TSMB course covering structural investigation of kinesin 5.  Again, unfortunately, current PPS students will not yet have access to this link.  The piece is taken from the last section of TSMB, which showcases the application of the many different structural techniques covered in the course to the study of a wide variety of proteins.
More details of Dr Carolyn Moores’ work are available at

Thursday, 12 April 2012

Commemorating World TB Day: Drugs from Plants

This post was written first for the Birkbeck College events blog and is re-posted here with permission.

World TB Day is held on 24 March every year, to mark the day in 1882 when Robert Koch, one of the fathers of microbiology, first announced that he had discovered the cause of tuberculosis (TB) – the bacterium now known as Mycobacterium tuberculosis (link to the TB proteome page in PPS section 5). Over 125 years since its discovery, and despite billions of dollars of investment in drug discovery, this bacterium is still a killer. The World Health Organisation estimates that about two billion people are infected with latent tuberculosis; in 2010, the last year for which full figures are available, over eight million people became ill with active tuberculosis, and 1.4 million people died from the disease. Two factors help make TB particularly deadly: it often occurs in people infected with the HIV virus, where it is one of the major causes of death, and drug resistant forms are becoming more common. In January 2012, Nature reported the identification in India of so-called “totally drug resistant” (TDR) tuberculosis, resistant to all anti-TB drugs in general use.

 Image of Mycobacterium tuberculosis bacteria
Photo credit: Janice Carr, Centers for Disease Control and Prevention, USA

In 2012 at Birkbeck, World TB Day coincided with the start of the College’s annual Science Week. Dr Sanjib Bhakta, head of the Mycobacteria Research Laboratory in the Department of Biological Sciences, organised a well-attended symposium on tuberculosis and its treatment. Besides two scientific presentations, the symposium featured a short video, Tuberculosis: The Real Story, highlighting the views of people affected by TB in the UK, and a panel discussion led by the grassroots volunteer organisation Results UK on some of the political challenges raised by tuberculosis.

Both science lectures focused on plants as a source of potential new drugs for tuberculosis. Professor Franz Bucar from the University of Graz in Austria highlighted the extreme chemical diversity of compounds that could be extracted from plants, particularly as compared to those found in the average synthetic compound library. Plants have always existed alongside their own microbial pathogens and have evolved natural antibiotics to protect themselves. Our ancestors, before the dawn of scientific medicine, used plant extracts to treat infectious disease, often quite successfully. The sub-discipline of ethnomedicine involves “mining” these traditional or historical remedies for pure chemicals that can be developed as, or modified into, drugs.

Bucar described a European herb, elecampane or Inula helenium, which is known to have been used to treat lung disease in the sixteenth century. He explained how a complex mixture of natural products derived from this plant had been tested against mycobacteria. Compounds found to have anti-mycobacterial activity were extracted and purified. Other plants have also yielded useful lead compounds; extracts of bark from a small tree with the Latin name of Berchemia discolor have even been shown to inhibit multi-drug resistant strains of Mycobacterium tuberculosis at useful concentrations.

Discovering antibacterial products in plant extracts, however, is only a first step towards drug discovery. Even when natural products like these compounds are found to be selective for bacterial over human cells, it is necessary to discover their mechanism of action; to modify them to optimize their activity; and, since plant sources are often scarce and extraction processes costly, to determine methods of synthesizing them in the laboratory.

The second scientific presentation was given by Dr. Bhakta himself and described current work in Birkbeck’s Mycobacteria Research Laboratory in searching for potential drugs for TB. These are needed not only to combat resistant forms of the bacteria but to improve current treatment regimens for “standard”, drug-sensitive TB. This requires a combination of four drugs to be taken for two months followed by two drugs for another four months, and many patients, particularly poorer and less well educated ones, fail to complete such a long and complex regimen. This in turn can lead to the development of further resistant strains.

Ideally, new drugs are required that target proteins not targeted by existing drugs, as resistance will be harder to develop. Mycobacteria have extremely complex cell walls, unlike those of other types of bacteria; they are essential for the bacteria to survive, and the enzymes used to synthesise them have no equivalents in mammalian genomes. These enzymes, therefore, have many of the characteristics of excellent drug targets.  Bhakta and his group have been exploring ways to inhibit the synthesis of the peptidoglycan that is one of the most important constituents of that cell wall. This molecule has been described as the bacterium’s “Achilles heel”, but no drugs targeting its synthesis have yet entered the clinic.

Mycobacteria synthesise peptidoglycan via a series of enzymes known as ligases, each of which adds a new link to the growing peptidoglycan chain. Bhaka’s group has focused on one of these ligases, termed MurE. This enzyme is essential for the bacterium to survive and it is conserved in all Mycobacterium tuberculosis strains. Working in collaboration with Professor Nick Keep, also in the Department of Biology, Bhakta solved the structure of MurE (PDB 2XTA) and showed it to have an active site that could in theory, at least, be occupied, and blocked, by a relatively small, “drug-like” molecule. He and his co-workers are now searching libraries of natural products for compounds that might inhibit this enzyme. They have identified promising MurE inhibitors from plants endemic to both Colombia and China, and are synthesizing analogues of these compounds for further testing.

It is unlikely that the next generation of anti-tuberculosis drugs will include any unchanged natural products. It is extremely likely, however, that natural products will yield the “scaffolds” on which these desperately needed drugs may be built, and perhaps one of these will be generated from within Bucar’s or Bhakta’s groups.