Genetic engineering is being used in an attempt to reduce the toxicity of a promising drug, writes Dick Ahlstrom
A researcher at University College Dublin is turning a very useful drug with bad side effects into a very useful drug with fewer side effects.
"The antifungal antibiotic amphotericin B is one of the most notoriously toxic drugs used in clinical medicine," states Centre for Synthesis and Chemical Biology and UCD Conway investigator Dr Patrick Caffrey.
The drug can damage kidneys, the heart and the brain. Yet amphotericin B is powerfully active against most fungal pathogens, he says. Importantly, resistance to amphotericin B has been slow to emerge despite being in use for 50 years.
Based in the School of Biomolecular and Biomedical Science, his goal is to work from the original drug to produce analogues with lower levels of toxicity while retaining its therapeutic benefits.
He has long experience in the biosynthesis of naturally occurring substances, starting as a post doctoral student at Cambridge in 1989. "Many of the drugs used in modern medicine are derived from living plants, bacteria and other life forms," he explains.
Amphotericin B is a complex natural product obtained from a bacterium called Streptomyces nodosus. It is grown up in fermentation cultures and the active substance is then extracted and purified.
Its toxicity can be reduced by chemical means but Caffrey decided to modify what S nodosus produced by engineering the original organism.
The first stage is to clone the genes that produce the antifungal substance, he explains. "From the DNA sequence it is possible to work out the biochemical pathway that leads to the antifungal. About 150 kilobases of chromosomal DNA is dedicated to amphotericin production," he says. "Sequence analysis revealed that over 90 enzymes are involved in the biosynthetic pathway."
Some of the enzymes were systematically knocked out to produce different versions of amphotericin. These in turn were analysed by Dr Dilip Rai of the Centre's mass spectrometry facility at UCD.
A detailed structural analysis was provided in collaboration with Dr Bernard Rawlings of the Department of Chemistry at the University of Leicester. Toxicity tests were also conducted using human and animal tissues.
The great advantage of producing analogues in this way is once a useful analogue is found it is much simpler to produce, says Caffrey. "The first fermentation gives you what you want." These compounds are very difficult to make using conventional chemical synthesis.
The most promising new analogues show antifungal activity at least as good as the original, but also a "dramatic reduction" in toxicity, says Caffrey. Work is now in progress to scale up fermentation and extraction of these compounds.
"One of our problems is the yields are low compared with the original compound," he says. "In addition the group is devising new biosynthetic methods for improving other natural products, including antibacterial and antitumour compounds."
The Centre for Synthesis and Chemical Biology is a collaboration in the chemical sciences between University College Dublin, Trinity College Dublin and the Royal College of Surgeons of Ireland. It was created using €26 million from the Higher Education Authority's Programme for Research in Third Level Institutions.
Funding for Caffrey's work came from the EU's FP5 programme and from the Higher Education Authority.