Enzyme structure may breathe new life into asthma R&D
that plays a pivotal role in the asthma biocascade, providing
information that could lead to more efficient asthma therapies.
The latest research, published online in the journal Nature describes the structure of the human leukotriene C4 synthase that plays a key role in the onset of asthma. According to World Health Organization (WHO) estimates, there are more than 300m asthma sufferers worldwide and 255,000 people died of the condition in 2005. There are several drugs currently on the market for the treatment of asthma that target the leukotriene receptors downstream of the LTC4 synthase such as Merck's Singulair (montelukast). Montelukast blocks the action of leukotriene D4 on the cysteinyl leukotriene receptor CysLT1 in the lungs by binding to it and reducing the constriction caused by leukotriene. However, such drugs do have their limitations and according to Professor Haeggstrom of the Karloinska Institutet in Sweden and lead author of the report, there are other receptors that play a part in this cascade. He therefore believes it is more rational to target the LTC4 enzyme to block the entire production of the bioactive molecules that cause asthma. "LTC4 is the key enzyme for generation of 'slow reacting substance of anaphylaxis', the cysteinyl leukotrienes, which are smooth muscle contracting substances that cause asthma and allergies." said Prof. Haeggstrom. "The enzyme is a trimeric protein with three subunits linked together and there are residues from two of the subunits that form the active site, it has to be an intact trimer to be fully active which is quite an intriguing organisation." To date LTC4 has remained undrugged, but the publication of the structure of the enzyme and its active site should allow pharmaceutical companies to design more effective asthma therapies. The crystallisation of the enzyme was not easy and Prof. Haeggstrom described the project as 'high risk'. "The enzyme is an integral membrane protein and it is a difficult challenge to solubilise the protein and keep it in solution in an active state and is even more difficult to crystallise," he said. "There are only three such human proteins that have been crystallised - it's extremely rare that you manage to crystallise a membrane protein like this." The researchers used a novel sitting drop vapour diffusion technique to crystallise the enzyme over three to four days after it had been amplified using cloning techniques. The X-ray crystal structure was then collected at the European Synchrotron Radiation Facility (ESRF). Prof. Haeggstrom said that the crystallisation techniques used could allow many other membrane proteins to be crystallised and analysed using X-ray crystallography techniques. Many proteins of interest as drug targets are membrane proteins and until now structural information about these proteins was missing making drugs that target them very difficult to design rationally. The research group at the Karolinska Institutet is currently using the techniques they developed during this study to look at the members of the MAPEG (membrane associated proteins in eicosanoid and glutathione metabolism) family of enzymes that are associated with a variety of diseases.
