Enzyme inhibition 2: suicide inactivators and transition state analogues

Question 1

What are the 2 types of mechanism based inhibitors?

Answer 1

Suicide inactivators
Transition state analogues

They rely on SPECIFIC enzymatic reaction catalysed by target enzyme that we would like to damage or down regulate

Question 2

What are the advantages of mechanism-based inhibitors?

Answer 2

Practically absolute specificity -> they not only recognise their target enzyme and active centre based on close resemblance of substrate structure, but their activity actually depends on an enzyme – enzyme converts them into active site.
They don’t work against anything other than the target enzyme
High efficiency of enzyme inhibition  very tight covalent complex within the active centre of the enzyme

Question 3

What is the mode of interaction with an enzyme for suicide inhibitors ?

Answer 3

They act at the enzyme active site  they rely on enzymatic mechanism of action
They form A PERMANENT complex with an enzyme (covalent bonds with amino acids in active site)
THEREFORE, they cannot be displaced by a substrate with a higher concentration
NON-COMPETITIVE AND IRREVERSIBLE

Question 4

What is the mode of inhibition for a suicide inhibitor ?

Answer 4

E + I -> EI
I = Suicide inhibitor
Before interaction with enzyme, the suicide inhibitor is latent (inactivated), then when complex is formed because the inhibitor resembles nitrile substrate structure and the enzyme treats it as a NORMAL SUBSTRATE, the normal catalytic reaction starts which converts the inhibitor into reactive species:
E…..I*
I* = highly reactive activated suicide inhibitor
Then, the activated suicide inib interacts immediately with enzyme active centre and inactivates the enzyme:
E-I
The enzyme ‘commits suicide’

Question 5

When do suicide inhibitors react?

Answer 5

ONLY when the inhibitor is in the active centre - highly selective

Question 6

Examples of suicide inhibitors ?

Answer 6

5-FLUOROURACIL - anticancer drug
The van der waals radius of F is similar to H
SO its possible to replace an H atom in a substrate by an F atom (in URACIL)
Then its recognised as a normal uracil but is actually a suicide inactivator and is used to inhibit enzyme Thymidylate Synthase, which is involved in DNA biosynthesis (reacts with amino acids)
Thymidylate synthase is good target for anticancer drug therapy

Question 7

Is the suicide inhibitor active at all times?

Answer 7

NO - it is latent until activated by the enzyme itself

Question 8

What are some pharmaceutical drawbacks to suicide inhibition?

Answer 8

Inhibitor structure may still bind to the other cell components and could be wasted  substrate may share active site with other proteins or cells.
If enzyme is produced in the cell as an inactive pre-cursor, then there is no point to design a suicide inhibitor as no reaction will happen

Question 9

What is the detrimental effect of some suicide inhibitors ?

Answer 9

Can cause toxicity due to their high reactivity (e.g. Tienilic acid)

Question 10

What is Tienilic acid?

Answer 10

Developed as a diuretic agent 1979
BUT found out that it produced toxicity especially associated with the liver  withdrawn due to interaction with cytochrome P450 enzymes
It was acting as a SUICIDE INHIBITOR on P450 enzymes (mimicked the substrate)

Question 11

Are penicillins suicide inhibitors?

Answer 11

Not acting as suicide inhibitors in humans but do in BACTERIA - beta lactam essential
Suppresses the synthesis of bacterial cell wall.
It inhibits a bacterial enzyme - transpeptidase which is involved in cell wall synthesis (reacts with nucleophillic groups and attacks electrophillic groups)
Beta-lactam ring is involved in the mechanism of inhibition
Penicillin becomes covalently linked to the enzymes active site leading to irreversible inhibition
Kills bacteria -
Transpeptidase enzyme then becomes inactive so peptide chains wanting to react and catalysed cannot react as the active centre is already occupied (it is BLOCKED) so water molecules can’t react either.  damaged FOREVER

Question 12

How do bacteria gain resistance to penicillin?

Answer 12

The presence of b-lactamase enzyme is the most important mechanism by which bacteria gain resistance to penicillins.
Enzymes inactive penicillins by opening b-lactam rings, allowing bacteria to be resistant to penicillin.
MOA for lactamases is the same to the MOinhibition for the target enzyme (i.e. transpeptidase)
But product is removed efficiently from the lactamase active site

Question 13

What is clavulanic acid?

Answer 13

Irreversible suicide inhibitor of beta-lactamase

Used as a guard drug for ampicillin

Question 14

What is Augmentin?

Answer 14

ampicillin + clavulanic acid  ampicillin inhibits transpeptidase, whereas clavulanic acid protects penicillin from beta-lactamases
Allows less ampicillin per dose and increased activity spectrum

Question 15

What is timentin?

Answer 15

ticarcillin + clavulanic acid (as a guard)

Question 16

What is clavulanic acids mechanism of action?

Answer 16

Clavulanic acid comes into beta-lactamase active centre and reacts with nucleophilic group and forms covalent bonds (TWICE) – INACTIVATES THE ENZYME

Question 17

What are transition state analogues?

Answer 17

Drugs designed to mimic the transition state of an enzyme-catalysed reaction.
They’re more likely to bind more strongly than drugs mimicking the substrate or product
Transitions states are high energy (VERY REACTIVE) therefore difficult to isolate substrate or synthesise
Transition-state inhibitor mimics the stereochemistry and binding properties of the reaction intermediate, but is chemically stable

Question 18

Why should a transition state be bound more strongly to an enzyme than a substrate or product?

Answer 18

 Speed and effectiveness of a catalysed reaction is crucially dependent on how much the catalyst stabilises the transition state (reasonable proposal)
 The more stable the transition state, the easier the reaction will occur and the more effective the enzyme will be as a catalyst
 THEREFORE it is more important that enzymes have their strongest interactions with guest molecules at the transition state of the reaction, rather than to the substrate at the beginning of the process, or the product at the end
 Strong interactions with the substrate or product, turnover would be slow and spend too much time in the active site

Question 19

Example of transition-state inhibitors ?

Answer 19

Renin inhibitors

Question 20

Describe the angiotensinogen reaction

Answer 20

Protease enzyme which hydrolyse specific peptide bonds in the protein Angiotensinogen
This converts Angiotensinogen to angiotensin 1 followed by further reaction to form angiotensin 2
Important as angiotensin 2 constricts blood vessels and raises blood pressure

Question 21

What do renin inhibitors do?

Answer 21

Renin inhibitors blocks synthesis of angiotensin 1 and 2

Question 22

Reaction mechanism for Renin inhibitors ?

Answer 22

Renin contains 2 aspartyl residues in the active centre which form water bridges and attack the amide group in the peptide and a tetrahedral intermediate is formed via proceeding through a high energy transition state.

Question 23

Examples of renin inhibitors?

Answer 23

Aliskiren, Statins, ACE inhibitors, protease inhibitors

Question 24

Is transition-state inhibition competitive?

Answer 24

YES - doesnt form covalent bonds

usually reversible