Screening of Natural Products
Over the last 25 years, nearly 50% of all new chemical entities have originated from natural products. Australia’s Eskitis Institute for Cell and Molecular Therapies (www.eskitis.org.au) focuses on the development of drugs from natural products against a range of cell-signaling targets. It holds a library of 42,000 archived biota samples originating from Queensland, China, Tasmania, and Papua New Guinea, and has been undertaking a natural product discovery collaboration with AstraZeneca (www.astrazeneca.com).
“Screening natural product extracts is far from trivial,” comments Michael Jobling, Ph.D., a research fellow in the lead discovery biology group. “Because solubility is an issue, we have to repeatedly re-optimize experimental conditions. At the same time, we have to work with limited samples. Thus, for our screening purposes, the detection system has to be extremely sensitive and able to perform under homogeneous reaction conditions.”
One of the projects studies heparanase (Hpa), an enzyme that cleaves to heparan sulphate proteoglycan (HSPG), a major component of extracellular matrix, vital for its structural integrity. Degradation of HSPGs by heparanase facilitates entry of tumor cells into the tissues. Inhibition of Hpa expression in emerging tumor cells may prevent tumor invasion and metastasis.
“The classical assay for Hpa activity measures the release of the fluorescent label from immobilized HSPG substrate,” continues Dr. Jobling. “This is a multistep procedure that takes up to 72 hours to complete.” It also requires large quantities of natural extracts for screening. “We switched to an HTRF-based assay because it is a one-tube, one-hour procedure, and it requires less material.”
The HTRF heparanase assay is based on HSPG substrate labeled with biotin on one end and Eu-cryptate on the other end. Streptavidin-XL665 binds to the substrate, causing energy transfer and a fluorescent signal. When an active enzyme cleaves to the substrate, the biotinylated end is released, and the emission is reduced. Dr. Jobling adds, “The only caveat is that sometimes the cleavage occurs on the wrong end of Eu-cryptate, resulting in incomplete inhibition of the signal.”
HTRF assay works in a homogeneous format, when all components of the assay remain in the well at the time of readout. The acceptor fluorescence could be affected by media variability in each well. A correction method accounting for well-to-well variations is based on measuring the ratio of fluorescent signals of the acceptor (665 nm) to Eu-cryptate (620 nm). Since the emission of Eu-cryptate is affected in the same proportion as the acceptor, the ratio of fluorescent signals is reflective of the interaction studied.
“We found, however, that natural libraries are full of nuisance compounds affecting 620 nm fluorescence, and resulting in false-positives,” says Dr. Jobling. “To remove the artefacts we simply looked at the average 620 baseline across the whole screening campaign. Any sample that showed a 620 reading outside of 80 percent to 150 percent of the average was discounted from the final hit list.”