Aspects of two components of the cardiovascular system will be investigated at the molecular level using multinuclear NMR methods, with the long term aim of understanding the structure and mechanism of action of these systems. This in turn will aid in design of more selective and potent pharmaceutical agents for their regulation, that also have fewer unwanted side effects. (i) Heparin is extensively used as an anticoagulant. It expresses this activity through interaction with antithrombin III, an inhibitor of several blood clotting proteins. Precise structural studies of the heparin-antithrombin III interaction have been hindered by the heterogeneous nature of heparin. It is proposed to prepare low molecular weight, high affinity heparin, homogeneous by size and charge and characterise these oligosaccharides by 1 H and 13 C NMR. The molecular interaction of these with antithrombin III will then be investigated by NMR methods and correlated with antifactor Xa and anti-thrombin activity. Development of low molecular weight highly active fragments of heparin that stimulate antithrombin III against only one coagulation protease may provide the antithrombotic properties of crude heparin without the unwanted problem of hemmorhage. Heparin also stimulates phagocytosis through interaction with fibronectin. Binding of heparin resides within small domains of fibronectin, which will be isolated and their interaction with heparin characterised by high resolution NMR. (ii) Angiotensin II is a potent vasoconstricter produced by the action of angiotensin converting enzyme (ACE) on the decapeptide angiotensin I. ACE is thus important in blood pressure homeostasis. Design of ACE inhibitors is therefore an important problem, whose solution is made empirical by the absence of a structure for the active site of the enzyme. Instead, comparison is made to carboxypeptidase A. It is proposed to obtain Cd113 and P31 NMR data on phospho- and thio-inhibitor binding to native zinc and Cd113-substituted carboxypeptidase A and to the dipeptidase thermolysin and use these details of metal-inhibitor interaction as a basis for comparative studies with ACE. For this substantial quantities of ACE will be isolated by affinity chromatography. With known crystal structures for both carboxypeptidase A and thermolysin, comparative NMR data for all three enzymes should provide a firmer basis for proposal of an active site structure and mode of inhibitor interaction for ACE.
Showing the most recent 10 out of 17 publications
