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Bioassay-guided fractionation of the methanol extract of the Australian sponge Neopetrosia exigua led to the isolation of exiguaquinol (2), a new pentacyclic hydroquinone that inhibited Helicobacter pylori glutamate racemase (MurI) with an IC(50) of 4.4 microM. Its structure and relative configuration were assigned on the basis of spectroscopic data. Exiguaquinol (2), bearing a novel pentacyclic ring skeleton, is the first natural product to show inhibition of H. pylori MurI. Its protein-ligand modeling is also discussed.

Fourteen natural products, known to inhibit other proteins of the Zincin-like fold class, were screened for inhibition of the Zincin-like fold metalloprotease thermolysin using mass spectrometry. Fourier Transform Mass Spectrometry was successful in identifying actinonin, a known inhibitor of astacin and stromelysin, to be an inhibitor of thermolysin. Molecular modelling studies have shown that specificity within the Zincin-like fold is determined by Protein Fold Topology.

Natural products have withstood the test of time as therapeutics, but new lead-generation strategies have focussed away from natural products. A new approach that uses natural-product recognition to drive an understanding of biological space might provide an impetus for renewed focus on natural-product starting points. Protein fold topology (PFT) has been shown to be an underlying factor for natural-product recognition. An investigation of natural product inhibitors of the Zincin-like fold has demonstrated their capacity also to inhibit targets of different fold types. Analysis of crystal structure complexes for natural products cocrystallised within different fold types has shown similarity at the PFT level. Two new PFT(T) (where subscript T denotes PFT shared between therapeutic targets) relationships have been established: the Zincin-like- metallohydrolase/oxidoreductase PFT(T) and the Zincin-like-phosphorylase/hydrolase PFT(T). The PFT relationship between a natural product's biosynthetic enzyme and therapeutic target, and now between different fold targets of the same natural product, suggests that PFT is the simplest descriptor of biological space. This fundamental factor for recognition could facilitate a rational approach to drug development guided by natural products.

The relationship between a natural product's biosynthetic enzyme and its therapeutic target is unknown. The concept of protein fold topologies, as a determining factor in recognition, has been developed through molecular modeling techniques. We have shown that biosynthetic enzymes and the therapeutic targets of three classes of natural products that inhibit protein kinases share a common protein fold topology (PFT) and cavity recognition points despite having different fold type classifications. The clinical agent flavopiridol would have been identified by this new approach.

The effects of a single 50 mg intramuscular injection of the anabolic steroid stanozolol (Stromba) on fibrinolysis, blood coagulation and lipids was evaluated in 12 healthy male volunteers. Significantly increased plasminogen activator levels (p less than 0.05) was noted 24 hours following the injection and these remained elevated for one week. Plasminogen levels increased significantly by day two (p less than 0.01) and remained elevated for three weeks. HDL cholesterol fell (p less than 0.01) and both total and LDL cholesterol increased (p less than 0.05) when measured one month post injection. Stanozolol appears to have therapeutic potential as an activator of the fibrinolytic system when given by intramuscular injection.

There is an increased frequency of arterial thrombosis in cigarette smokers. The changes in blood coagulation seen in these subjects have been studied by many workers but results have not always been in agreement. We wished to study the effects of acute smoking on platelet behaviour, fibrinolysis and haemorheology in ten habitual smokers, and to compare these results with non-smoking controls. Results show that the smoking group had higher plasma fibrinogen (p less than 0.04), lower plasminogen (p less than 0.02) and plasminogen activator (p less than 0.05), and higher plasma viscosity (p less than 0.003). The changes seen in cigarette smokers after smoking three cigarettes were an increase in the rate of platelet aggregation to ADP (p less than 0.02), an increase in alpha 2M, (p less than 0.02), and factor VIII RAG (p less than 0.05). Plasma viscosity was decreased (p less than 0.02) as was red cell deformability (p less than 0.02). We confirm an increased tendency to hypercoagulability in smokers compared to controls which becomes more pronounced immediately after smoking three cigarettes.

Blood viscosity and several haemostatic factors were measured in 42 patients with long-standing retinal vein occlusion and 33 control subjects. Blood viscosity, haematocrit, plasma viscosity, fibrinogen, fibrinopeptide A, and beta-thromboglobulin were increased in the 20 subjects with capillary nonperfusion or new vessels, but not in the 22 subjects without these complications. Patients with nonperfusion or new vessels also had a lower platelet count than patients without complications. Increased levels of factor VIII antigen and decreased levels of antithrombin III were found in the retinal vein occlusion group as a whole. These findings suggest that blood viscosity, platelets, and coagulation may be involved in retinal vein occlusion and its vascular complications.

Fibrinolytic shutdown may be important in the development of postoperative deep vein thrombosis (DVT). We have previously shown that stanozolol 50 mg, given intramuscularly 24 hr before surgery, prevents the decrease in plasminogen activator activity (PA) seen on the first postoperative day in patients at high risk of DVT. To investigate the role of fibrinolytic shutdown in causation of DVT, sixty patients were randomized in a double-blind controlled trial to receive stanozolol or placebo intramuscularly, and DVT was detected by leg scanning and confirmed by venography. Scan positive DVT occurred in 11 of 31 placebo patients (35%) and 12 of 29 who received stanozolol (41%). A significant decrease in PA was confirmed in the placebo group, while stanozolol caused a significant increase in PA on the first postoperative day. Patients in either group who did not develop DVT showed minimal changes in PA. We conclude that prevention of fibrinolytic shutdown by this regimen of stanozolol does not prevent postoperative DVT, and that further studies are required to clarify the relationship of postoperative fibrinolysis and DVT.

Clinical features were noted and routine and non-routine laboratory variables were measured before elective major gastrointestinal surgery in 63 patients aged 40 years or more. Deep-vein thrombosis (DVT), detected by routine 125I-fibrinogen leg scanning, developed in 21 patients. Five clinical variables but no laboratory variables were significantly associated with DVT: age; percent mean weight for age, sex, and height (%MW); presence of varicose veins; cigarette-smoking; and sex. The most useful discriminant index of these variables was age in years plus 1.3 x %MW. The index was validated prospectively in a further 41 patients, in 18 of whom DVT developed. The value of the index in selective prophylaxis was then assessed in a further 40 patients, of whom 24 (60%) with high-risk index received low-dose heparin (5000 units twice daily). DVT developed in 4 of the 40 patients, an incidence of 10% compared with the incidence of 37.5% (39 of 104) in the earlier studies with no prophylaxis.

The effects of a single pre-operative intramuscular injection of the anabolic steroid stanozolol (Stromba, 50 mg) on fibrinolysis and blood viscosity were evaluated in 14 patients at high risk of post-operative deep venous thrombosis. A control group of 13 high risk patients was also studied. The mean level of plasma plasminogen activator activity decreased significantly (p < 0.05) on the first post-operative day in the control group. In contrast, the fibrinolytic activator activity showed a non-significant rise on the first postoperative day in the stanozolol treated group. The difference in plasminogen activator levels on the first post-operative day between treated and control groups was significant (p < 0.05). Plasma plasminogen levels on the first post-operative day increased from preoperative levels in the treated group (p < 0.01), but not in the control group. The prevention of fibrinolytic shut-down and stimulation of plasminogen levels by a single pre-operative injection suggests that trials of intramuscular stanozolol in the prevention of postoperative deep venous thrombosis are indicated.

Blood rheology and several haemostatic factors were studied in patients with type II hyperlipoproteinaemia (HLP) and matched controls. HLP patients had increased blood viscosity (p less than 0.01), the mean level being 18% higher at a low shear-rate (0.94 s-1) and 13% higher at a high shear-rate (94 s-1). The increased viscosity was due partly to a raised haematocrit (p less than 0.05), and partly to increased plasma viscosity (p less than 0.01) associated with increased plasma fibrinogen (p less than 0.02). Red cell deformability was normal, and viscosity was unrelated to either lipid or lipoprotein concentrations. Levels of the major fibrinolytic inhibitor. alpha 2-antiplasmin, measured by both functional and immunological techniques were higher in HLP patients (mean increase 30-32%). Plasminogen activator levels were normal in HLP patients and the ratio of fibrinolytic inhibitor to activator was therefore increased. Plasminogen concentrations were also increased. Levels of factor VIII activity and antigen, antithrombin III and anti-factor Xa activity, alpha 2-macroglobulin, platelet count, and platelet aggregation by adenosine diphosphate and adrenaline did not differ significantly in HLP patients and controls. These results suggest that the premature arterial disease associated with HLP may be related to increased blood viscosity, which reduces arterial blood flow, and increased alpha 2-antiplasmin, the major inhibitor of fibrinolysis.