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Cardiovascular Diseases & Rational Drug Design 2002

(3 Lectures: Dr Illingworth)

  1. Basic cardiovascular physiology and pathology: what you need to know and where to find information on the control of heart rate, cardiac output, blood pressure, blood volume, ionic composition, renin / angiotensin system, vascular endothelium, regulation of tissue perfusion, hypertension, dislipidaemias, atherosclerosis, blood clotting, ischaemic heart disease, cardiomyopathies, cardiac arrhythmias and cardiac failure.
  2. Cardiovascular drugs: inotropic agents, b blockers, calcium antagonists, organic nitrates, anti-arrhythmics, ACE inhibitors, ATII (=AT1) antagonists, diuretics, cholesterol lowering drugs, clot-busters, anti-coagulants, anti-platelet drugs.
  3. Examples and opportunities for rational drug design in relation to renin, angiotensin, aldosterone, cytokines, vasoactive peptides and other cardiovascular targets.

Basic Cardiovascular Physiology

  1. The right side of the heart (pumping blood to the lungs) is a low pressure pump, while the left side of the heart (serving the rest of the body) operates at a much higher blood pressure. This is reflected in the chamber dimensions and wall thickness.
  2. The Starling mechanism. If the heart chambers are distended with blood, the ensuing beat is much more forceful than if the chambers were initially empty. It is essential that hearts should respond in this way, since otherwise Laplace's Law would make cardiac pumping impossible if the ventricles ever became overfull. The molecular explanation is that calcium ion release from the sarcoplasmic reticulum is much greater when the SR is mechanically stretched. This should be considered in conjunction with the next point.
  3. About 70% of the blood volume resides in the great veins, which have muscular walls. Contraction of venous smooth muscle, or an expansion in blood volume, raises central venous pressures and transfers some of this reserve blood supply into the heart chambers, stretching the heart and greatly increasing the cardiac output. Ventricular filling is often decribed as the PRELOAD applied to the heart.
  4. Most of the resistance to blood flow arises from smooth muscle compressing the walls of the arterial tree. The main effects are in the small arterioles, and in the pre-capillary sphincters which ration blood flow into the capillary beds. The aortic pressure depends on the cardiac output and the peripheral vascular resistance, and is often called the AFTERLOAD applied to the heart.
  5. Local control of blood flow relies mainly on nitric oxide and endothelin produced by vascular endothelial cells and adenosine released by ischaemic tissues. Capillary loops seem to be folded back on themselves so as to bring the inflow and outflow vessels into close juxtaposition, and thereby facilitate this local signalling.
  6. Local blood flow regulation helps to keep the cellular oxygen concentration low. Oxygen is a very toxic substance, and cells are easily damaged by high concentrations. Local regulation directs the limited cardiac output to the areas where it is most needed.
  7. Blood osmolarity is monitored by osmoreceptor cells in the hypothalamus which control overall water balance through the pituitary peptide vasopressin (= anti-diuretic hormone, or ADH). These sensors are also connected to the behavioural systems controlling thirst and salt craving. Stretch receptors in the right atrium and the arterial tree monitor total blood volume, and this neurally transmitted information is also used to modulate ADH release.
  8. There is interaction between the control of blood volume and blood osmolarity. Stretching the right atrium stimulates the release of atrial natriuretic peptide (ANP) from the atrium, which promotes sodium excretion by the kidney, ultimately reducing blood volume and salt content. Ventricles produce brain natriuretic peptide or BNP. Conversely, low salt concentration in the distal convoluted tubule, or inadequate perfusion of the kidney, both stimulate release of renin into the bloodstream. This short half-life protease cleaves circulating angiotensinogen producing angiotensin I. Subsequent proteolysis by the lung endothelium yields angiotensin II which has a powerful vasoconstrictor effect on arteriolar smooth muscle. This raises systemic blood pressure, restores kidney perfusion, and stimulates release of the salt-retaining hormone aldosterone from the adrenal cortex, which promotes renal sodium retention and potassium loss.
  9. In many tissues there is a local angiotensin metabolism superimposed on the whole body mechanisms described above. Angiotensin is also concerned with tissue growth.
  10. The whole ensemble is monitored and regulated by the autonomic nervous system. There are blood pressure receptors (baroreceptors ) in the great veins and in the aorta and the arterial tree. The baroreceptor signals are integrated with information on body position from the system controlling voluntary movements, and with information from chemical sensors, ultimately regulating renin production, cardiac contractility and arterial and venous smooth muscle tone. The physical position of the body is important, because the hydrostatic pressures associated with an upright posture are greater than arterial blood pressures, and very much greater than the pressure in the veins.

The cardiac cycle: Each beat is initiated by spontaneous depolarisation of pacemaker cells in the sino-atrial (SA) node. These cells trigger the neighbouring atrial cells by direct electrical contacts and a wave of depolarisation spreads out over the atria, eventually exciting the atrio-ventricular (AV) node. Contraction of the atria precedes the ventricles, forcing extra blood into the ventricles and eliciting the Starling response. The electrical signal from the AV node is carried to the ventricles by specialised conducting tissue (Purkinje fibres) in the interventricular septum, formed from modified cardiac muscle cells. The heart does not require innervation in order to beat, but the autonomic nerve supply can vary the force and frequency of cardiac contraction.

Control of cardiac output: Aortic output varies over a twenty-fold range between sleeping and vigorous exercise, and is also increased during pregnancy. The Starling mechanism is very important, but in addition circulating adrenalin, and noradrenalin released by cardiac nerve terminals, have inotropic effects, enhancing cardiac contractility via cyclic AMP and protein kinase A. Increased contractility means that the same peak systolic pressure can be achieved with a lower end diastolic pressure, i.e. with a lower degree of ventricular stretch. Increased contractility does not necessarily produce a rise in arterial pressure, but may produce a fall in the central venous pressure. Ion channel phosphorylation increases calcium entry during the action potential, calcium release from the SR during systole (contraction), and calcium uptake by the SR during diastole (relaxation). [Direct electrical connections between adjacent cardiac muscle cells preclude the progressive fibre recruitment seen in voluntary muscles: in the heart every muscle cell depolarises every beat.] Catecholamines from the sympathetic nerves acting on cardiac b1 adrenoceptors make the beats more rapid and forceful, while acetylcholine from the parasympathetic nerves acting on muscarinic M2 receptors has the opposite effect.

Congestive heart failure (CHF): This term is often misunderstood. It does NOT mean the heart has stopped beating, it means that output is insufficient to meet the needs of the body. The autonomic nervous system detects the inadequate tissue perfusion, and reacts as though the blood volume were too low. It leads to vasoconstriction, salt and water retention, reduced parasympathetic activity, increased sympathetic activity and cytokine production. The low inherent cardiac contractility requires excessive venous pressures to maintain ventricular output through the Starling mechanism. Permanently high venous pressures are a serious problem: if the systemic venous pressures are high (right side failure) it may cause disabling lower limb oedema, while if the pulmonary venous pressures are raised (left side failure) it will cause lung congestion (fluid accumulation) breathlessness (dyspnoea) and coughing. Both sides of the heart may be affected at the same time.

The haemodynamic improvements sought during therapy are: decreased pulmonary capillary wedge pressure, decreased systemic vascular resistance, decreased mean right atrial pressure and decreased pulmonary artery pressure, while improving cardiac index, stroke volume and classical heart failure symptoms such as dyspnoea (breathlessness) and swollen ankles. [The cardiac index is the output in litres/minute divided by the body surface area in square metres.]

Traditional therapy was based on diuretics, vasodilators and inotropic agents, which relieved symptoms and improved cardiovascular status, without improving overall mortality! The later introduction of loop diuretics (frusemide), potassium-sparing diuretics (amiloride) aldosterone antagonists (spironolactone), b-blockers, and particularly ACE inhibitors and angiotensin receptor antagonists has greatly reduced the morbidity and mortality associated with CHF. [Trap for the unwary: angiotensin II binds to AT1 receptors (type 1 angiotensin II receptors).]

Remmea & Swedbergb (2002) Comprehensive guidelines for the diagnosis and treatment of chronic heart failure. Europ. J. Heart Failure 4(1), 11-22.

Angina: This is a pain in the chest caused by insufficient blood flow through the coronary arteries, leading to cardiac ischaemia. It may be a chronic condition persisting for many years, and often exacerbated by excitement, smoking and over-eating. Reduced coronary blood flow sometimes results from coronary artery spasm, but most commonly follows atherosclerotic damage to the blood vessel endothelial lining. This inflammatory process leads to blood clot formation and obstruction of the vessel lumen. See Staels (2002) Nature 417, 699 - 701 for a recent brief review. Angina may be associated with other manifestations of atherosclerosis, such as strokes and intermittent claudication in skeletal muscle. Prolonged or severe angina may progress to necrosis of cardiac cells, release of the cell contents into the bloodstream and myocardial infarction (MI).

Cardiac oxygen demand correlates closely with systolic blood pressure (or more strictly with the area under the ventricular pressure * time curve), but does not increase so markedly with the volume of blood actually pumped by the heart. [This is because myosin ATP hydrolysis does not depend on whether the muscle actually succeeds in shortening.] The treatment is therefore to reduce the cardiac afterload (the mean aortic pressure) with drugs which dilate the systemic arterioles. The resulting fall in systemic blood pressure allows the left ventricle to empty more completely and reduces the overall cardiac work load. This brings the cardiac oxygen supply and demand into better balance. The benefit may be less than desired because the treatment necessarily reduces the coronary perfusion pressure and this may reduce the coronary blood flow.

Nitroglycerine tablets dissolved under the tongue remain the classical treatment for anginal attacks. The drug is rapidly absorbed through the buccal epithelium, and is metabolised in the tissues to nitric oxide, which has vasodilator and hypotensive effects. [Hypotension was noted many years ago among workers in munitions factories who absorbed organic nitrates through their skin.] A variety of polyol nitrates, calcium channel antagonists and b adrenergic blocking agents may also be used to achieve the desired effect. A new treatment for unstable angina without infarction is abciximab - a chimeric mouse-human monoclonal antibody directed against the platelet glycoprotein IIb/IIIa fibrinogen receptor. This treatment interferes with blood clotting, and delays the extension of the arterial plaque.

medical condition

angina pectoris

congestive heart failure

physiological problem

cardiac oxygen demand exceeds supply

low contractility (more rarely, atrial fibrillation) causes high venous blood pressure

treatment strategy

reduce cardiac work output, try to improve oxygen supply

increase cardiac volume output, reduce cardiac workload, reduce venous congestion

drug 1

nitroglycerine causes arterial vasodilation, lowers afterload

ACE inhibitors or AT1 blockers produce arterial vasodilation

drug 2

b-blocker (e.g. metoprolol) but care needed in diabetics

diuretics (e.g. furosemide plus amiloride) remove excess fluid

drug 3

calcium channel blocker (e.g. nifedipine or verapamil)

b-blockers (e.g. carvedilol) in hemodynamically stable patients

drug 4

lipid lowering and anti-clotting drugs, aspirin or clopidogrel

digitalis (especially for atrial fibrillation, or severe failure)

Myocardial infarction: The heart is uniquely sensitive to lack of oxygen because it operates a highly aerobic, lipid-based metabolism, with up to 95% arteriovenous oxygen extraction. Many tissues have arterio-venous shunts in their capillary network and extract only about 30% of the available oxygen in arterial blood. The high cardiac oxygen extraction may arise because the capillaries are squeezed during systole, so that blood flow only occurs during diastole.

If the coronary arteries are partially or completely blocked, then downstream muscle cells may die, releasing their enzymes into the extracellular space. The enzymes continue to leak into the bloodstream over several hours. This release is non-specific but can be useful in the diagnosis of suspected myocardial infarction from peripheral blood samples. Not all heart attacks are painful enough to be immediately recognised, although many are. Other signs of myocardial infarction are changes in the ECG, and under favourable conditions these can show the precise location of the damage. A really big transmural infarct carries the risk that the ventricle may eventually rupture with disastrous results, but normally the most serious risks are cardiogenic shock and ventricular fibrillation provoked by the abnormal electrical properties of partially ischaemic cells at the edge of the infarct. If the patient survives the first few days the damaged area is invaded by macrophages and fibroblasts. The cells are replaced by scar tissue, leaving the heart usable, but permanently weakened. Infarcts affecting the cardiac impulse conducting system may produce partial or complete heart block, where synchronisation is lost between atria and ventricles. This can be treated with an artificial pacemaker.

The objectives of MI therapy are to prevent potentially fatal ventricular arrhythmias and to minimise the infarct size. The morphine initially prescribed for pain relief and sedation also has valuable haemodynamic effects, reducing preload (end diastolic pressure) through venodilation and afterload (peak systolic pressure) through arteriolar dilation. Aspirin (anti-platelet action) and heparin (anti-coagulant) are given as early as possible to minimise further clot enlargement. The top priority is to unblock the damaged coronary artery, either by emergency surgery, or by intravenous or (better) intracoronary administration of a clot-dissolving enzyme such as tissue plasminogen activator, streptokinase or urokinase. There are enormous benefits in starting this treatment as early as possible, ideally within 60 minutes.

In the longer term, ACE inhibitors will reduce the cardiac work load, possibly in conjunction with long-acting nitrates. Other therapy is aimed at removing risk factors: stopping smoking, cholesterol reduction using HMG-CoA reductase inhibitors (lovastatin), long-term aspirin therapy, exercise and hormone replacement therapy in post-menopausal women. b-blockers may also be useful, especially in non-diabetic patients.

Recent experiments in rodents suggest that it may be possible to repair the damaged heart muscle either using embryonic cardiomyocytes, or with stem cells from bone marrow after cytokine treatment. It has already been shown that the new cardiac muscle cells are functional and electrically coupled to the remainder of the myocardium. At present the cytokine treatment must start before the myocardial lesion, which is a clinically unrealistic scenario. If this work can be successfully extended to humans and started after the event rather than before, then it is likely become the preferred therapy.

Arrhythmias: These are disturbances in the normal sequential pattern of cardiac activiation, (sinus rhythm) triggered by the sino-atrial node. They range from the occasional ventricular ectopic beat (often provoked by tiredness or caffeine in otherwise healthy people) to the completely disorganised activity seen in ventricular fibrillation, which is fatal within minutes. Between these two extremes there exists a bewildering variety of abnormal behaviour, and an equally bewildering number of drugs to treat it. All of them modify the pattern of ion channel opening and closing during the cardiac action potential, usually with the hope of narrowing the window during which a further unwanted action potential can be triggered. Controlled clinical trials have shown that several antiarrhythmic drugs may actually make matters worse. A better result can often be obtained by cardioversion (electric shock treatment) and / or permanent pacing. Current practice is not to treat cardiac arrhythmias with drugs unless the condition is life-threatening, and nothing else seems likely to work.

Myocarditis: Inflammation of the heart which may arise through a wide variety of causes, including staphylococci, streptococci and other bacteria, Coxsackie B virus, rickettsiae (scrub typhus) and Trypanosoma cruzi (Chagas disease). The symptoms include fatigue, weakness, tachycardia, leucocytosis, arrhythmias, cardiomegaly (cardiac enlargement) and mitral valve incompetence leading eventually to heart failure. It is suspected that viral infections may be more common than was previously recognised, and may pre-dispose to dilated cardiomyopathy (see below). Myocarditis is usually an acute condition: the patients either die or get better.

Cardiomyopathies: This diverse group of serious diseases are the most common reason for cardiac transplantation. They are all characterised by long-term heart failure and cardiomegaly, usually with little inflammation. Recognised classes include

i) Nutritional: e.g. cobalt-induced and alcohol-induced cardiomyopathies.

ii) Hypertrophic cardiomyopathy: the ventricles are enlarged with unusually thick walls. This may be caused by outflow obstruction, but genetic factors may also be important.

iii) Dilated cardiomyopathy: the ventricles are grossly enlarged with unusually thin walls. The disease affects all races, but is particularly common in black males. About 75% of patients are believed to suffer from an auto-immune condition, which may be precipitated by a previous Coxsackie virus infection. Circulating antibodies may be internalised using receptor-mediated endocytosis. Myosin, b-receptors and the mitochondrial adenine nucleotide transporter have all been suggested as possible auto-antigens. A variety of inherited factors can be identified in the remaining patients, including both dominant and recessive mutations affecting the dystrophin, tropomodulin and connexin-40 genes. There are huge individual variations in the age of onset, time course and severity of the disease even when a genetic mechanism has been identified.

Felix et al (2002)Removal of cardiodepressant antibodies in dilated cardiomyopathy by immunoadsorption J. Am. Coll. Cardiol. 39(4), 646-652.

The treatment of dilated cardiomyopathy resembles that for heart failure in general, except that there may be more emphasis on maintaining adequate cardiac contractility using catecholamine analogue infusions (dopamine or dobutamine), providing that the coronary arteries are intact. There is a serious risk of pulmonary embolism as a result of venostasis, and anti-coagulants may be indicated.

See cardiomyopathy website.

medical condition

myocardial infarction

dilated cardiomyopathy

physiological problem

partially blocked coronary artery dead muscle in ventricle wall

heart failure, low contractility, extreme dilation

treatment strategy

clear obstruction, relieve pain and reduce cardiac work load

reduce venous congestion maintain adequate output

drug 1

aspirin, heparin, morphine and proteases to dissolve the clot

diuretics (e.g. furosemide plus amiloride) remove excess fluid

drug 2

ACE inhibitors or AT1 block to produce arterial vasodilation

ACE inhibitors to produce arterial vasodilation

drug 3

b-blocker (e.g. atenolol) but care needed in diabetics

catecholamines (e.g. dopamine) to increase contractility

drug 4

lovastatin to control blood cholesterol long term

 

Blood clotting and anti-coagulants: The cardiovascular system requires a delicate balance between excessive clotting causing obstructions, and leaks following a failure to clot. In most patients this balance is more or less correct, and controlled clinical trials have only supported the prophylactic use of anti-coagulants in two particular circumstances: (1) aspirin appears to give real protection against myocardial infarction, and (2) warfarin and heparin are of benefit in the management of deep vein thrombosis among those at risk. There is, however, a very clear benefit from speedy attempts to dissolve the clot blocking the coronary artery through the use of injected proteases during or immediately after a heart attack.

The coagulation system uses proteolytic cascades to amplify a tiny initial stimulus and allow the formation of substantial clots. Most of the required proteins are secreted by the liver into the bloodstream as inactive precursors, which are subsequently cleaved to yield the active clotting factors. Several of these proteins (factors II, VII, IX and X, and the inhibitory proteins C and S) undergo a specialised post-translational modification in the liver microsomal fraction, which converts glutamate residues near the amino termini into g-carboxyglutamate. These modified residues provide high-affinity Ca ++ binding sites which are essential for the assembly of a functional clotting complex. The reaction requires oxygen and carbon dioxide and is catalysed by a vitamin K dependent carboxylase which produces vitamin K epoxide. Warfarin blocks this process by inhibiting the first stage of the NADPH-dependent reductase system that recycles the epoxide back to the fully reduced, hydroquinone form of vitamin K.

There are two routes for the activation of the clotting system. The intrinsic pathway is normally activated by contact with collagen from damaged blood vessels, but any negatively charged surface will suffice. Kaolin (clay) is used to artificially activate the pathway for the measurement of the activated partial thromboplastin time - a clinical test used to monitor the activity of this part of the clotting cascade. Clotting may alternatively be activated via the extrinsic pathway , which requires a tissue factor from the surface of extravascular cells. The final stages of both pathways are common, and involve the proteolytic activation of thrombin which then initiates the formation of a fibrin clot. A transglutaminase reaction catalysed by factor XIIIa then cross links the fibrin monomers. A greatly over-simplified version of these events is shown in the diagram below:

The intrinsic pathway normally requires platelet activation in order to assemble a tenase complex involving factors VIIIa, IXa and X. The activation process uses the IP3 signalling pathway, and involves degranulation and myosin l.c. kinase activation in order to change the platelet shape and allow them to adhere. Phospholipase A2 activation leads to the formation of thromboxane A2 which promotes further platelet aggregation via a positive feedback system. Aspirin inhibits the cyclooxygenase involved in thromboxane biosynthesis. Endothelial cells synthesize prostacyclin PGI2 , which inhibits platelet aggregation. The anti-coagulant heparin activates the inhibitor antithrombin III, which deactivates several of the plasma clotting factors, including thrombin. Clot dissolution requires a further protease, plasmin, which is incorporated into the forming clot as an inactive precursor, plasminogen. The "clot buster" enzymes tPA and streptokinase are used to activate this internal fibrinolytic mechanism.

Check list of common cardiac drugs

Drugs

Main effects

Mechanism

Sites of action

abciximab

anticoagulant stops platelet activation

monoclonal antibody to fibrinogen receptors

platelets

amiloride (combination with frusemide is frumil)

potassium sparing diuretic

plasmalemma sodium & chloride channels

kidney (distal tubules)

amiodarone

class III anti-arrhythmic

prolongs action potential duration

myocardium

aspirin

anticoagulant stops platelet activation

COX inhibitor, blocks TXA2 synthesis

platelets

atropine (sometimes used to stop vagus bradycardia)

parasympatholytic, increases heart rate

blocks muscarinic AcCh receptors

pacemaker cells (sino-atrial node)

captopril

reduces arterial blood pressure

ACE inhibitor

relaxes vascular smooth muscle

clopidogrel

anticoagulant stops platelet activation

blocks ADP receptor

platelets

digitalis and ouabain

increase cardiac contractility, delay AV node triggering

block Na / K ATPase raising intracellular sodium, then calcium

all tissues, but the Na/Ca exchanger is mainly in heart

dipyridamole (often used for X-ray imaging)

coronary vasodilation

inhibition of adenosine uptake

coronary vasculature

furosemide (= frusemide)

diuretic

plasmalemma sodium & chloride channels

kidney (loop of Henle)

isoprenaline (and other adrenaline analogues)

increase cardiac contractility

beta agonist raises cyclic AMP

many tissues

losartan

reduces arterial blood pressure

angiotensin AT1 receptor blockade

relaxes vascular smooth muscle

lovastatin

reduces blood cholesterol levels

HMG-CoA reductase inhibitor

liver

morphine

pain relief (mainly)

opiate receptors

brain

nitroglycerine (and many other organic nitrates)

reduce cardiac work load

metabolised to NO

relaxes vascular smooth muscle

propranolol

reduces cardiac contractility, class II anti-arrhythmic

beta blocker lowers cyclic AMP

many tissues

quinidine, novocaine and other local anaesthetics

class I anti-arrhythmics

delay recovery of sarcolemma sodium channels after AP

myocardium

spironolactone (usually added to other diuretics)

reduces diuretic potassium losses

aldosterone antagonist

kidney (distal tubules)

urokinase (streptokinase is cheaper but antigenic)

dissolves blood clots (fibrinolytic)

activates plasminogen to plasmin (protease)

blood clots

verapamil, nifedipine and other dihydropyridines

reduce cardiac work load, class IV anti-arrhythmic

block sarcolemma calcium channels

myocardium; relax vascular smooth muscle

warfarin

anticoagulant

vit. K antagonist

blocks g-carboxy glutamate synthesis

liver

Captopril and rational drug design

Cushman & Ondetti (1999) Design of angiotensin converting enzyme inhibitors Nature Medicine 5, 1110-1112.

Khalil et al (2001) A remarkable medical story: Benefits of angiotensin-converting enzyme inhibitors in cardiac patients J. Am. Coll. Cardiol. 37(7), 1757-1764.

Opie & Kowolik (1995) The discovery of captopril: from large animals to small molecules. Cardiovasc. Res. 30, 18-25.

Discovery of new drugs started as a random process which depended on chance observations of natural products. These provided the first drug leads, which were exploited by pharmaceutical chemists to produce the earliest synthetic drugs. Progress was haphazard and initially very slow, but by the 1960's a sufficient range of compounds had been synthesised for scientists to correlate structure and activity in a systematic fashion.

Quantitative structure-activity relationships (QSAR) correlate the biological properties of the potential drug with systematic structural variations (Free Wilson analysis) or with molecular properties such as lipophilicity, polarisability and stereochemistry (Hansch analysis). Both techniques are essentially multivariate statistical methods that indicate promising directions for further chemical modification. Development is cyclical: the new compounds are compared with their predecessors and a family of promising compounds evolves in the desired direction.

It isn't just the effect on the intended target that must be optimised. Toxicity, biological half-life, and ease of administration are equally important factors, and research on ADME (absorption, distribution, metabolism and excretion) of the new drugs must proceed in parallel with the main development effort.

The development of X-ray crystallography led to an increasing appreciation of the three dimensional relationships between the ligand and its target protein, while advances in synthetic organic chemistry have lead to a growing automation and acceleration of the drug development process. The task is not easy because of the considerable flexibility of both drug and the target molecule, and the continuing uncertainty about their active conformations in aqueous solution.

The angiotensin converting enzyme (ACE) inhibitor captopril which was developed around 1975 is regarded as a major turning point in the drug development process. Captopril was the first drug designed to block a particular target protein, and has subsequently become the preferred therapy for hypertension and congestive heart failure.

Blood pressure range (mm Hg)

Category

Diastolic

 

<85

Normal blood pressure

85-89

High normal BP

90-104

Mild hypertension

105-114

Moderate hypertension

>114

Severe hypertension

Systolic (when diastolic <90)

 

<140

Normal

140-159

Borderline systolic hypertension

>159

Isolated systolic hypertension

About 22% of the American population are reckoned to be hypertensive. Of these, over half are not receiving therapy, and the treatment is not completely successful in about half of those on medication. Hypertension is a major risk factor for the development of cardiovascular diseases, and remains an important area of pharmaceutical research.

The link between renal disease and hypertension was appreciated by a few scientists during the nineteenth century, and in 1898 Tigerstedt & Bergman showed that renal extracts contained a substance (renin) that could provoke hypertension when injected into dogs. In 1934 Goldblatt demonstrated that renal ischaemia produced hypertension, and a few years later it was realised that renal ischaemia was a powerful stimulus for renin release.

Renin was partially purified in the 1940's and recognised to be an enzyme that acted on a protein already present in the blood to produce the actual pressor substance that was named angiotensin. It was subsequently realised that a second enzyme present mainly in the lungs converted angiotensin I into the more effective angiotensin II. It was also realised that the same system inactivates bradykinin, a nonapeptide involved in the inflammatory response. Bradykinin relaxes vascular smooth muscle (causing vasodilation) but it also causes intense contractions of visceral smooth muscle. Several of these components were characterised in the late 1960's by John Vane and coworkers.

Vane persuaded Cushman and Ondetti at the Squibb Institute to study the angiotensin system, and drew their attention to Brazilian work on Pit Viper venom, which potentiates the action of bradykinin and contains natural peptide inhibitors of angiotensin converting enzyme. One of these was developed into an anti-hypertensive drug teprotide, which could only be given by injection because it was inactivated in the gut.

There was no X-ray data on angiotensin converting enzyme, but Cushman and Ondetti recognised its similarity to another zinc-containing enzyme, carboxypeptidase, for which a partial structure was available. They devised a simple model of the active centre and started a systematic search for inhibitors, using the quick spectrophotometric assay for ACE that Cushman had developed.

Click here to see the structure of human carboxypeptidase which provided a model for the structure of angiotensin converting enzyme [ACE] and the design of captopril.

Measuring inhibitor potency:

Ki - This is similar to Km, but for inhibitors.

Plotting 1/velocity against [inhibitor] (NB not 1/[inhibitor]) often yields linear graphs from which the Ki can be estimated.

IC50

The IC50 can be estimated from a plot of velocity against log[inhibitor]. The IC50 is the inhibitor concentration where the velocity is cut by half. At low substrate concentrations, Ki~IC50

Active site models

This is a representation of angiotensin I bound to the active centre.

This is a representation of captopril bound to the active centre.

Drug development

Side effects:

Alternative ACE inhibitors lack the sulphydryl group

It is now recognised that there are at least three potential drug targets within the renin angiotensin system:

Here is the X-ray structure of human renin binding a transition state analog inhibitor that is not initially visible. The structure shown below was originally reported by Rahuel et al (1991) J. Struct. Biol. 107, 227. [Protein Data Bank code 1RNE.]

This is an interactive display using the CHIME software. Rotate the molecule by dragging it with the left mouse button. Change its appearance by clicking the image with the right mouse button to show the structure more clearly. Click HERE for a brief reminder of the main CHIME commands, or HERE for a full tutorial.

Using the right mouse button, set "Display" to cartoons. Click again and set "Color" to structure. Click again and set "Select" via "hetero" to ligand. Click again and set "Display" via "spacefill" to Van der Waals radii.

You should now be able to see the inhibtor in the active site.

Rotate the ensemble with the left button, and experiment with different views to see how the protein grips its ligands. Try to relate these images to the more recent paper on renin inhibition by Rahuel et al in the reading list below.


In addition to the systemic effects of angiotensin, there exist local renin angiotensin systems within many tissues that are involved in growth and microvascular regulation. Crackower et al (2002) Nature 417, 822 - 828 report on a second angiotensin converting enzyme, ACE2 which is a carboxypeptidase that removes single amino acids from both angiotensin I and angiotensin II. ACE2 apparently reduces the concentration of angiotensin II and antagonises the actions of ACE. Gene knockout mice lacking ACE2 have superficially normal blood pressure, but develop severe cardiac abnormalities, that are not seen in double knockout animals, lacking both ACE and ACE2. These investigators suspect that this is due to the unrestrained local action of ACE within cardiac muscle.

Rational drug design: the modern development cycle.


New cardiac drugs:

Heart disease is common and the potential rewards for drug companies are high, so research continues on a broad front. All stages of the disease process are under attack, ranging from hypertension, diabetes, blood lipids, atherosclerosis, thrombosis, clot dissolution, and support for the failing heart. Heart failure is the most intractable problem: all forms of heart disease may ultimately lead to heart failure, and currently 50% of patients with severe heart failure will be dead within 2 years.

Cost-effectiveness is also an important issue. Gaspoz et al (2002) NEJM 346, 1800-1806 have pointed out that if appropriate treatment were offered to all eligible patients, the platelet inhibitor clopidogrel is currently ten times more expensive than aspirin therapy per quality-adjusted year of life gained.

Advances in basic research have identified many additional targets for the drug development cycle. Particular attention has focused on the signalling mechanisms that maintain normal blood pressure and tissue perfusion in healthy subjects.

Vasoactive peptides (all of these use seven transmembrane helix receptors)

peptide name produced by actions

adrenomedullin

everywhere

vasodilation

angiotensin

renin, ACE (proteases)

vasoconstriction

atrial natriuretic

right atrium stretch

increased water and sodium losses by kidney

B-natriuretic

ventricular muscle

as above

C-natriuretic

vascular endothelium

as above

bradykinin

kallikrein (protease)

vasodilation, increased vascular permeability

endothelin

vascular endothelium

vasoconstriction, bronchoconstriction

vasopressin

posterior pituitary

increases renal water retention

VIP

parasympathetic nerves

vasodilation (salivary glands)


other vasoactive substances

name produced by actions

adenosine

all working tissues

vasodilation

adrenaline

adrenal medulla

vasodilation (b) predominates

aldosterone

adrenal cortex

sodium retention by kidney

histamine

mast cells

both constriction & dilation (receptors)

leukotriene LTC4

leukocytes

vasoconstriction, bronchoconstriction

noradrenaline

sympathetic nerves

vasoconstriction (a) predominates

nitric oxide

vascular endothelium

vasodilator via cGMP

prostacyclin PGI2

vascular endothelium

vasodilation, prevents clotting

prostaglandin PGE1 / E2

macrophages

both constriction and dilation (receptors)

thromboxane TXA2

platelets

vasoconstriction, promotes clotting


New drugs now being developed for heart failure include selective aldosterone receptor antagonists, calcium sensitizers, cytokine inhibitors, endothelin receptor antagonists, growth hormone releasers, natriuretic peptides, neutral endopeptidase inhibitors, vasopeptidase inhibitors and vasopressin antagonists.

drug name

type

actions

levosimendan

calcium sensitizer

enhances troponin Ca++ binding & opens vascular ATP-sensitive K+ channels

 

cytokine inhibitor

anti-inflammatory

bosentan

endothelin receptor antagonist

 

ghrelin

growth hormone releaser

 

nesiritide

natriuretic peptide

increases salt and water excretion

thiorphan

neutral endopeptidase inhibitor

blocks degradation of bradykinin & ANP

eplerenone

selective aldosterone receptor antagonist (SARA)

blocks aldosterone actions in kidney and in cardiac muscle (anti-fibrosis)

omapatrilat

vasopeptidase inhibitor

blocks both angiotesin II formation, and the degradation of bradykinin and ANP

 

vasopressin antagonist

blocks V2 receptors in collecting ducts


Adrenomedullin is a potent vasodilator that reduces blood pressure while increasing cardiac output. Gene knockout shows that it is essential for normal cardiovascular development. It is produced by many tissues, including heart and vascular smooth muscle when stimulated by pro-inflammatory cytokines such as TNF-a, and it exerts a protective effect in animal models of toxic shock. It probably acts via nitric oxide and cAMP, but the principal signalling system varies between tissues and species. There is a collection of reviews in Microscopy Research and Technique, volume 57 issues 1 and 2, March and April 2002.

Aldosterone is produced by the adrenal cortex when stimulated by angiotensin II. It causes sodium and water retention by the kidney, and a urinary potassium losses. It is also regulates salt resorption in the colon and sweat glands, and promotes collagen synthesis in cardiac muscle leading to myocardial fibrosis. Spironolactone is a non-specific aldosterone antagonist that has been in use for several years, but it also binds to sex steroid receptors leading to unwanted side effects. It is commonly added to other drug regimes. Eplerenone is a more selective compound that is currently in phase III clinical trials.

Coats (2001) Exciting new drugs on the horizon - Eplerenone, a selective aldosterone receptor antagonist (SARA) Int. J. Cardiol.80(1), 1-4.

Weber (2001) Aldosterone in Congestive Heart Failure NEJM 345(23), 1689-1697. Ask the library help desk for the username and password.

Bradykinin is a vasodilator peptide released from clotting factor XII by a tissue protease called kallikrein. It plays little role in the regulation of normal blood flow, but becomes more important following injury or infection.

Kaplan et al (2002) Pathways for bradykinin formation and inflammatory disease J. Allergy & Clinical Immunology 109(2) 195-209. This excellent article is available electronically, but only via OVID. 'Orrid OVID. You can get to it (with patience & persistence) using the library catalogue. The library will shortly switch to a more convenient source.

Brain natriuretic peptide (BNP, nesiritide) has recently been approved for clinical use. This peptide is released naturally from overstretched ventricular muscle.

Tremblay et al (2001) Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases Mol. Cell. Biochem. 230(1-2), 31 - 47. [Write down the reference, click the link and then select the article from the publishers' website.]

Calcium sensitisers allegedly increase the efficiency of cardiac muscle by modifying calcium binding to troponin and they are claimed to increase cardiac output without increasing oxygen consumption. There is dispute about their mode of action, and levosimendan also activates the ATP sensitive potassium channel in vascular smooth muscle plasmalemma, leading to an increase in coronary flow. These compounds are only just starting clinical trials and it will be several years before their efficacy can be properly assessed.

Kaheinen et al (2001) Levosimendan Increases Diastolic Coronary Flow in Isolated Guinea-Pig Heart by Opening ATP-Sensitive Potassium Channels J. Cardiovasc. Pharmacol. 37(4), 367 - 374. [Write down the reference, click the link and then select Contents on the publishers' website.]

Pro-inflammatory cytokines (TNF-a, IL-1b, IL-6) and chemokines (MCP-1 & IL-8) are produced by macrophages and many other cell types. They are raised in CHF, and there is increased expression within the failing myocardium. They are implicated in the pathogenesis of dilated cardiomyopathy (DCM). These peptides act on the hypothalamus to increase body temperature, reduce food intake and result in the mobilisation of energy and protein stores. They stimulate the liver to secrete C-reactive protein and mannan-binding lectin as part of the acute phase response. Various anti-cytokine trials have so far yielded disappointing results.

Adamopoulos et al (2001) A glossary of circulating cytokines in chronic heart failure Europ. J. Heart Failure 3(5), 517-526.

Blum & Miller (2001) Pathophysiological role of cytokines in congestive heart failure Ann. Rev. Medicine 52, 15 - 27.

Damas et al (2001) Cytokines as new treatment targets in chronic heart failure Current controlled trials in cardiovascular medicine 2(6), 271-277.

Feldman et al (2000) The role of tumor necrosis factor in the pathophysiology of heart failure J. Am. Coll. Cardiol. 35(3), 537 - 544.

Hasegawa (2001) Neurohormonal Regulation of Myocardial Cell Apoptosis During the Development of Heart Failure J. Cell. Physiol. 186(1), 11-18.

Kotler (2000) Cachexia Ann. Intern. Med. 133(8), 622-634.

Silverberg (2001) The pathological consequences of anaemia Clin. Lab. Haem. 23, 1-6

Valen et al (2001) Nuclear Factor Kappa-B and the Heart J. Am. Coll. Cardiol. 38(2), 307-314.

Endothelin is a potent vasoconstrictor and smooth muscle mitogen secreted by endothelial cells lining the blood vessel walls. The endothelin system is activated in several disease states including hypertension and heart failure. There are two classes of endothelin receptor: ETAR located mainly on vascular smooth muscle cells and ETBR located mainly on the endothelial cells themselves. ETAR signaling causes both vasoconstriction and myoproliferation. ETBR signals vasodilation when expressed on endothelial cells but vasoconstriction when expressed on smooth muscle cells. Selective and non-selective endothelin receptor antagonists (ETRA) have been developed. Pre-clinical studies have shown limited effects on hypertension, but these drugs have an excellent ability to prevent end organ damage

Kedzierski1 & Yanagisawa (2001) Endothelin system: the double-edged sword in health and disease Annu. Rev. Pharmacol. Toxicol. 41, 851-76.

Growth hormone is an experimental drug for the treatment of CHF and a growth hormone releasing peptide from stomach also appears to be effective. Physical exercise and sleep are physiological stimuli for growth hormone release, and it is also produced in response to fasting. Many of the actions of growth hormone are mediated indirectly by locally-produced insulin-like growth factors. So far most of this work has been carried out with animals and as yet (April 2002) there are no results available from large scale clinical trials.

Napoli (2002) Growth Hormone Corrects Vascular Dysfunction in Patients With Chronic Heart Failure J. Am. Coll. Cardiol. 39(1), 90 - 95.

Nagaya et al (2001) Hemodynamic, renal, and hormonal effects of ghrelin infusion in patients with chronic heart failure. J. Clin. Endocrinol. Metab. 86(12), 5854 - 5859.

Vasopeptidase inhibitors simultaneously inhibit both neutral endopeptidase and angiotensin-converting enzyme (ACE). Neutral endopeptidase is responsible for the breakdown of both bradykinin and the natriuretic peptides, so inhibition of this enzyme should produce valuable clinical effects. Early results have been very promising and omapatrilat, the most studied drug in this class, is currently in phase II clinical trials.

Corti et al (2001) Vasopeptidase inhibitors Circulation 104(15), 1856-1862.

Weber (2001) Vasopeptidase inhibitors Lancet 358(9292), 1525-1532. [There are some printing mistakes in this paper, see "Department of error" link.]

Vasopressin is a powerful vasoconstrictor that also activates water uptake from the kidney collecting ducts, producing a concentrated urine and retaining water within the body. There are three classes of vasopressin receptors. V1 receptors are found in the vascular system, V2 receptors are on the kidney collecting ducts and V3 receptors are in the posterior pituitary. Both V1 and V2 are targets for drug development work. Vasopressin antagonists are only just starting clinical trials, but have already been shown to produce a powerful aquaretic effect in human volunteers. They displace vasopressin from V2 receptors in the renal collecting ducts, leading to a large increase in water (but not salt) excretion. In subjects given free access to water, plasma osmolarity is unchanged.

Thibonnier et al (2001) The Basic and Clinical Pharmacology of Nonpeptide Vasopressin Receptor Antagonists Annu. Rev. Pharmacol. Toxicol. 41, 175202.

Surgical Interventions:

These lectures have concentrated on new cardiovascular drugs, but there have also been important advances in surgical techniques. Revascularisation and stenting can yield considerable benefits in ischaemic heart failure, although there is a tendency for coronary occlusion to re-appear where the underlying metabolic defects are still in existence. In addition, the MIRACLE trial [Hare (2002) Cardiac-Resynchronization Therapy for Heart Failure NEJM 346, 1902-1905] indicates that bi-ventricular pacing may be of benefit in cardiomyopathic heart failure. Encouraging results have also been obtained with mechanical devices to assist the acutely failing heart. In some cases patients who were being prepared for transplantation have recovered sufficient function to continue with their own hearts after weaning from the mechanical pump. See, for example, Farrar et al (2002) Long-term follow-up of thoratec ventricular assist device bridge-to-recovery patients successfully removed from support after recovery of ventricular function J. Heart & Lung Transplantation 21(5), 516-521.

Reading list:

Textbooks: Within reason, it doesn't matter which text books you consult for the basic cardiovascular physiology and pharmacology. The Health Sciences Library has multiple copies of the following titles, but there are numerous alternative books that are equally as good as those listed here. Opie & Gersh is exceptionally good, and worth pursuing.

Kumar & Clark Clinical Medicine 4th edn. Saunders; 1998 [heart disease]

Lodish et al. Molecular Cell Biology 4th edn., Freeman; 2000 [muscle contraction]

Opie & Gersh Drugs for the Heart 5th edn. Saunders; 2001 [highly recommended, but make sure you get the fifth edition!]

Rang, Dale & Ritter Pharmacology 4th edn. Churchill Livingstone; 1999 [cardiac drugs]

Tortora & Grabowski Principles of Anatomy & Physiology 9th edn. Wiley; 2000 [physiological mechanisms]


Original Papers & Reviews: In addition to the papers listed above, almost all of the following papers are available on-line through Leeds University Library. There are nearly 1000 pages in total, and we don't expect you to read them all. Use the collection to prepare a couple of topics in detail for the assessment and to check specific points if you need clarification.

Adams et al (2000) Atherogenic lipids and endothelial dysfunction: mechanisms in the genesis of ischemic syndromes Annu. Rev. Med. 51, 149-167.

Bennett (2001) Novel platelet inhibitors Annu. Rev. Med. 52, 161-84.

Böger & Bode-Böger (2001) The clinical pharmacology of L-arginine Annu. Rev. Pharmacol. Toxicol. 41, 79-99.

Briggs et al (1996) Computational science: new horizons and relevance to pharmaceutical design Trends in Cardiovascular Medicine 6(6), 198-203.

Brown et al (1998) Angiotensin-converting enzyme inhibitors Circulation 97(14), 1411-1420.

Carlson & McCammon (2000) Accommodating Protein Flexibility in Computational Drug Design Molecular Pharmacology 57, 213218.

Chew & Moliterno (2000) A critical appraisal of platelet glycoprotein IIb/IIIa inhibition J. Am. Coll. Cardiol. 36(7), 2028-2035.

Cooke & Dzau (1997) Nitric oxide synthase: role in the genesis of vascular disease Annu. Rev. Med. 48, 489-509.

Cramer et al (1999) Prospective identification of biologically active structures by topomer shape similarity searching J. Med. Chem. 42, 3919-3933.

Debouck & Metcalf (2000) The impact of genomics on drug discovery Annu. Rev. Pharmacol. Toxicol. 40, 193-208.

Doughty & Sharpe (1997) Beta-adrenergic blocking agents in the treatment of congestive heart failure: mechanisms and clinical results Annu. Rev. Med. 48, 103-14.

Farber (1999) New approaches to rational drug design Pharmacology & Therapeutics 84, 327332

Finn & Kavraki (1999) Computational Approaches to Drug Design Algorithmica 25, 347371. [this paper is about the maths]

Flower (1999) Modelling G-protein-coupled receptors for drug design Biochimica et Biophysica Acta 1422, 207-234.

Garbers & Dubois (1999) The molecular basis of hypertension Annu. Rev. Biochem. 68, 127-155.

Harvey & Dufton (1999) The therapeutic potential for targetting potassium channels: are dendrotoxins a suitable basis for drug design? Perspectives in Drug Discovery and Design 15/16, 281-294.

Hennekens (1997) Aspirin in the treatment and prevention of cardiovascular disease Annu. Rev. Public Health 18, 37-49.

Hobbs et al (1999) Inhibition of nitric oxide synthase as a potential therapeutic target Annu. Rev. Pharmacol. Toxicol. 39, 191-220.

Hockerman et al (1997) Molecular determinants of drug binding and action on L-type calcium channels Annu. Rev. Pharmacol. Toxicol. 37, 361-96.

Hook & Means (2001) Ca2+/CAM-dependent kinases: from activation to function Annu. Rev. Pharmacol. Toxicol. 41, 471-505.

Hu & Ertl (1999) Potential role of mixed ACE and neutral endopeptidase inhibitor in the treatment of heart failure Cardiovascular Res. 41, 503-505.

Huang (2000) The cardiovascular effects of PFRF amide and PFR(Tic) amide, a possible agonist and antagonist of neuropeptide FF (NPFF) Peptides 21, 205-210.

Iqbal et al (1998) Synthesis, Rotamer Orientation, and Calcium Channel Modulation Activities of Alkyl and 2-Phenethyl 1,4-Dihydro-2,6-dimethyl-3-nitro-4-(3- or 6-substituted-2-pyridyl)-5-pyridinecarboxylates J. Med. Chem. 41, 1827-1837. [write down the reference so that you can click on "back issues"]

Joseph-McCarthy (1999) Computational approaches to structure-based ligand design Pharmacology & Therapeutics 84, 179191.

Kubini (1997) QSAR and 3D QSAR in drug design Part 1: methodology Drug Discovery Today 2(11), 457-467.

Kubini (1997) QSAR and 3D QSAR in drug design Part 2: applications and problems Drug Discovery Today 2(12), 538-546.

Linden (2001) Molecular approach to adenosine receptors: receptor-mediated mechanisms of tissue protection Annu. Rev. Pharmacol. Toxicol. 41, 775-87.

Lloyd-Jones & Bloch (1996) The vascular biology of nitric oxide and its role in atherogenesis Annu. Rev. Med. 47, 365-75.

Marrone et al (1997) Structure-based drug design: computational advances Annu. Rev. Pharmacol. Toxicol. 37, 71-90.

Matsusaka & Ichikawa (1997) Biological functions of angiotensin and its receptors Annu. Rev. Physiol. 59, 395-412.

Mavromoustakos et al (1999) An effort to understand the molecular basis of hypertension through the study of conformational analysis of losartan and sarmesin using a combination of nuclear magnetic resonance spectroscopy and theoretical calculations J. Med. Chem. 42, 1714-1722. [write down the reference so that you can click on "back issues"]

Ohlstein et al (2000) Drug discovery in the next millennium Annu. Rev. Pharmacol. Toxicol. 40, 177-91.

Perola (2000) Successful virtual screening of a chemical database for farnesyltransferase inhibitor leads J. Med. Chem. 43, 401-408. [write down the reference so that you can click on "back issues"]

Pfeffer (1995) Left ventricular remodeling after acute myocardial infarction Annu. Rev. Med. 46, 455-66.

Rahuel et al (2000) Structure-based drug design: the discovery of novel nonpeptide orally active inhibitors of human renin Chemistry & Biology 7, 493-504.

Ramesh et al (1998) Synthesis and Calcium Channel-Modulating Effects of Alkyl (or Cycloalkyl) 1,4-Dihydro-2,6-dimethyl-3-nitro-4-pyridyl-5-pyridinecarboxylate Racemates and Enantiomers J. Med. Chem. 41, 509-514. [write down the reference so that you can click on "back issues"]

Ray et al (2000) Endothelin-receptor antagonists: current and future perspectives Drug Discovery Today 5(10), 455-464.

Sadoshima & Izumo (1997) The cellular and molecular response of cardiac myocytes to mechanical stress Annu. Rev. Physiol. 59, 551-71.

Samson (1999) Adrenomedullin and the control of fluid and electrolyte homeostasis Annu. Rev. Physiol.61, 363-89.

Smith et al (2000) Cyclooxygenases: structural, cellular, and molecular biology Annu. Rev. Biochem. 69, 145-82.

Steinberg & Brunton (2001) Compartmentation of G-protein-coupled signaling pathways in cardiac myocytes Annu. Rev. Pharmacol. Toxicol. 41, 751-73.

Thibault et al (1999) Regulation of natriuretic peptide secretion by the heart Annu. Rev. Physiol. 61, 193-217.

Wagner et al (2000) Decoy oligodeoxynucleotide characterization of transcription factors controlling endothelin-B receptor expression in vascular smooth muscle cells Mol. Pharmacol. 58(6), 1333-1340.

Zimmerman & Dunham (1997) Tissue renin-angiotensin system: a site of drug action? Annu. Rev. Pharmacol. Toxicol. 37, 53-69.