THE USE OF X-RAY CRYSTAL STRUCTURES IN DESIGNING POTENT THROMBIN INHIBITORS

Anne Cleasby, Harren Jhoti, Shila Patel, Alan Wonacott, Malcolm Weir

Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Thrombin is a key component of the blood coagulation cascade, playing a central part in thrombosis and hemostasis. In addition to its role as a procoagulant in which it cleaves fibrinogen to form fibrin, thus initiating clot formation, it also activates factors V, VIII , XIII and protein C and is an important controlling factor for blood coagulation. Because of these activities, thrombin has attracted a great deal of interest over the past few years, as a target for the prevention of thrombosis.

Thrombin is a trypsin like serine proteinase having the His, Asp Ser catalytic triad, and a specificity for positively charged side chains, such as arginine or lysine in S1.

Our aim has been to identify an active site inhibitor, which is suitable for use in the prevention of venous thrombosis and pulmonary embolism. The desired properties of these potential inhibitor molecules are potency against thrombin, which can be translated into good anticoagulant activity, selectivity over trypsin and other fibrinolytic enzymes, as well as good pharmacological properties such as oral activity and high bioavailability. Knowledge of the structure of thrombin in complex with its inhibitors would help to optimise the first two properties.

A high throughput screen of methanolic extracts from the leaves of the plant, Lantana camara isolated a series of potent thrombin inhibitors, which were identified as 5,5-trans-fused cyclic lactone euphane triterpenes . An example of these is GR133487, which has an IC50 against thrombin of 4nM, and against chymotrypsin of 10nM. It has some activity against other serine proteinases.

X-ray crystal structures of both GR133487 and a close analogue, GR133686 complexed with thrombin, were determined and showed the binding modes of these compounds in the thrombin active site. These results, together with biochemical data, have helped to explain the mode of inhibition of these compounds, and have yielded information which could form the basis of exploitation of this new class of thrombin inhibitors in the drug discovery process.