Luminescent Host Molecules for Multisite Recognition of Polyphosphate Anions

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The search for new drugs often begins with high-throughput screening of lead compounds followed by determination of mode of action of the potential drug and measurements of selectivity and potency. Many drugs act by inhibiting enzyme activity, therefore, to increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical companies have robust, affordable assays to measure enzyme activity accurately. Enzymes that consume and produce nucleoside polyphosphate anions represent a major target in cancer drug discovery. This will enable the best drug candidates to be identified before they enter expensive animal and human testing.The majority of existing enzyme assays require expensive, unstable antibodies or chemically modified reagents, which are time consuming to prepare and require special care in handling. The high cost of these reagents and time required to validate the assays places a significant strain on the drug development process. In addition, these assays are restricted to single 'end-point' measurements, which limits our understanding of the mode of action of a new drug. This increases the risk of a drug candidate passing the early development stages, only to fail at a later, more expensive stage. In order to increase productivity earlier in the drug discovery process, a low-cost method for real-time monitoring of enzyme activity is urgently needed.The aim of this project is to develop molecular probes that bind reversibly to specific nucleoside polyphosphate anions (e.g. adenosine diphosphate) that are produced during pharmaceutically important enzyme reactions. Upon binding to the anion, the probe will provide a luminescent signal that precisely indicates the activity of the enzyme in real-time. The probes will be used to directly measure the production of polyphosphate anions common to several important enzyme classes (kinases, GTPases, glycosyltransferases), eliminating the need for expensive antibodies or chemically modified reagents. The proposed molecular probes have enormous potential to reduce the cost and time required to conduct high-throughput screening assays. They will provide a vital step towards the rapid, accurate determination of enzyme kinetics and mechanism. This will enable better selection and validation of new drug candidates at an early stage in drug discovery, reducing effort pursuing compounds destined to fail in the more lengthy and costly phases of animal and human testing.