Part of WCAIR’s target based screening strategy
The cells of animals need many different products to carry out their functions. Each product is made by a process within the cell which resembles a production line in a factory. The cell’s production lines are made up of many different molecular machines (themselves made by other production lines within the cell). These molecular machines are called enzymes. Enzymes are important proteins that build or break down other cellular proteins and other products, altering how cells work. Because cells need functioning enzymes to survive they are good targets for anti-infective medicines. However, parasites can contain the same cellular processes as human cells. So scientists need to design the medicine to selectively inhibit the action of the parasite’s enzyme and not the patient’s enzyme.
When we are screening against a known enzyme target we create a test (assay) based on the process the enzyme carries out in the cell. We then use the assay to identify whether compounds in our screening collection can stop the process and measure how well hit compounds achieve this. Traditional enzyme assays have limitations. The assays rely on a ‘reporter’ substance (often a change in colour or amount of fluorescent light produced) to show the scientists how well the process has worked. Because the reporters are themselves chemical compounds some of the compounds we are testing can interfere with the read out making it unreliable.
We are now developing assays to run on the Agilent RapidFire instrument. The RapidFire instrument washes and prepares enzyme test samples very quickly (in seconds) for measuring using mass spectrometry. This is a technique that identifies individual molecules based on their mass and charge [ Read how a mass spectrometer works on the site explainthatstuff ]. The quality of data and capacity – we can run more than 15,000 tests in an individual run – allows us to quickly understand whether a chemical we have made is on the road to being a useful medicine or needs further alteration to improve activity or selectivity. Because the RapidFire contains a robot to carry out the steps the scientists can set up a run overnight (while they sleep!) and come in the next morning to analyse the results.
The advantages of the RapidFire are:
- Increased sensitivity
- Reduced enzyme consumption
- Increase signal to background noise
- No requirement for coupled assays
- Cheaper than many commercial fluorescence assays
Over the last year we have successfully moved some of our existing enzyme assays from traditional reporter kits to the RapidFire. Going forward the RapidFire will be our first choice for enzyme assays (there are some situations where it cannot be used) and more assays will be validated on the RapidFIre. In addition, we will look at new applications within the Mode of Action team and DMPK.