ACE-I

Question 1

What is ACE?

Answer 1

Angiotensin converting enzyme

-is a zinc (zn2+) enzyme that digests proteins and peptides:

• Cleaves last two residues (His.Leu) of angiotensin I to form angiotensin II
• cleaves bradykinin by removing the last two residues (Phe. Arg) forming an inactive peptide

Question 2

What is angiotensinogen?

Answer 2

A 452 amino acid residue peptide/ globulin formed in the liver

Is the precursor molecule to angiotensin 1

Is a prohormone with the ending -ogen- a protein that is processed by the body to form the active molecule

Question 3

What is angiotensin 1?

Answer 3

A 10 residue peptide/ active molecule
Vasconstrictor, causes an elevation in BP
Is cleaved by ACE to form angiotensin II

Question 4

What is bradykinin?

Answer 4

Is an inflammatory mediator that causes blood vessels to dilate, causing a fall in blood pressure

Question 5

What is angiotensin II?

Answer 5

An 8 residue peptide
A more potent vasoconstrictor
Induces the retention of Na and water in the kidneys
Both pathways raise the BP

Question 6

To lower blood pressure, why is ACE a better target than renin?

Answer 6

If ACE is overactive it forms the vasoconstrictor angiotensin II AND inactivates the vasodilator bradykinin causing an increase in blood pressure on both accounts.

Question 7

How does the MOA of ACE enzyme compare with carboxypeptidases?

Answer 7

Carboxypeptidases in the intestines digest proteins and peptides in the duodenum.

ACE hydrolyses the C- terminal dipeptide whereas carboxypeptidase cleaves off one amino acid

The 3D structure of ACE was originally unknown so inferences were made by analogy with carboxypeptidase.

Question 8

How was an inhibitor of ACE designed?

Answer 8

In banana plantations, brazillian pit vipers bit workers of whom suffered a lowering of BP and died shortly after.

Found that nonapeptide toxin (teprotide) in the venom caused the catastrophic fall in BP.

Teprotide is a peptide and the idea of making a peptide into an oral medicine was not well recieved as it would get digested easily and cannot pass through membranes very well.

Question 9

How did they make inferences from carboxypeptidase to make an inhibitor?

Answer 9

Treated carboxypeptidase with a pentapeptide. The enzyme was inhibited to a degree to slow it down.

Carboxypeptidase C showed there was a zinc ion in the active site held in place by interactions with a protein.

Zn2+ acts a lewis acid and catalyses the cleaving- carbonyl group in amide bond will get removed.

Question 10

What was the first inhibitor that was made where its structure was inferred by carboxypeptidase?

Answer 10

succinyl peptide

Had a carboxyl group at both ends.

Double negative charges gave the molecule poor membrane absorption and low bioavailability.

Question 11

What was the first drug that came onto the market to treat BP?

Answer 11

Captopril

• has a carboxyl group at one end to interact with the protein (ACE)
• the thiol group of captopril is able to interact with Zn2+ of ACE via lone pairs as electronegativity on S is sufficient to give a good interaction with zinc

First drug to be designed in a rationale way using structure of a peptide/protein to guide the process.

Question 12

What is the inhibitory dissociation constant of captopril?

Answer 12

Ki = 10^-9M

Is in the nanomolar range

Question 13

What are the side effects of captopril?

Answer 13

Caused through inactivation of bradykinin

• rashes
• loss of taste
• airway stimulation leading to dry cough and sinusitis

Question 14

How is the pharmacophore constructed?

Answer 14

To design other molecules, place dummy atoms in place of the enzyme

Use 3 points in space at anchor points to investigate low energy stable forms of different molecules

Explore changes in conformation/ map out pharmacophore

Question 15

What was the next molecule that was designed based on captopril?

Answer 15

Enalaprilat - A TRANSITION STATE ANALOGUE

Question 16

Cons of enalaprilat?

Answer 16

Poor absorption when administered orally

Double negative charged molecules do not get absorbed well.

Therefore, is administered as an inactive pro drug ENALAPRIL (ethyl ester) which is hydrolysed to the active drug in vivo

(one of the carboxyl groups is protected with an ethyl ester)

very potent inhibitor/ has subnanmolar activity

Question 17

When designing inhibitors, what state is the best to mimick- substrate, product or transition state?

Answer 17

transition state

Is half way between the substrate down to the enzyme and the product being formed by the enzyme.

It is meta-stable.

Copying a meta stable structure is not done easily especially with a covalent molecule.

To copy a molecule, need for it to be stable, otherwise would not be able to isolate it or purify it.

Question 18

Why does it not matter that a rubbish copy of the transition state is made?

Answer 18

as the affinity of the transition state to the enzyme is so high, making a rubbish attempt at copying the transition state will still get a good inhibitor/ potent drug

Question 19

Give an example of a transition state analgoue.

Answer 19

enalaprilat

Question 20

Human structure of the testicular ACE?

Answer 20

ACE structure in humans is membrane bound.

ACE enzyme in testicles was also found to be membrane bound but was much smaller. Cleaving it, allows one to purify, crystallise and identify the structure of the testicular ACE.

Question 21

Name the interactions of the tACE-lisinopril complex

Answer 21

• postively charged lysine interacts with a carboxyl group on the end
• proline group is present
• carbonyl group after the proline hydrogen bonds with 2 histidines
• NH3+ interacts with 2 negatively charged groups- aspartic acid and glutamic acid
• carboxyl group interacts with zinc enzyme of ACE
  (needs to be protected otherwise would get poor absorption problems seen with two negatively charged groups)

Question 22

What is the top prescribed ACE-I?

Answer 22

ramipril

2. 5mg OD (initial dose)
3. 5-20mg OD/BD

Dosing decreases with better design an potency of inhibitors. Less drug intake results in fewer side effects.

Question 23

Actions of ACE-I

Answer 23

• inhibits conversion of angiotensin I to angiotensin II
• dilates arteries
• reduces blood volume
• reduces afterload, cardiac work
• improves tissue perfusion to increase stroke volume and CO
• reduce oedema/ fluid retention
• dilates veins
• diminishes pulmonary congestion, oedema, preload
• reduce ventricular filling pressure
• reduce cardiac wall stress and ischaemia
• delay abnormal cardiac hypertrophy and fibrosis