Medicine - Enzyme Inhibitors 2 - Suicide Inactivators And Transition State Analogies Flashcards
Describe the use of 5-flourouracil as a suicide inhibitor
5-flourouracil is converted in the body to the fluorinated analogue of 2’-deoxyuridine monophase (FdUMP)
This suicide inactivator is used to inhibit the enzyme thymidylate synthase, which is involved in DNA biosynthesis
Define suicide inactivator
Agents which are converted to irreversible inhibitors by the enzyme-catalysed reaction
Not active until it is bound to the active site (chemically)
React with target enzyme once contact is formed
What is the mode of interactions of suicide inactivators within an enzyme?
What is the effect of increasing the substrate?
At the enzyme active site these inhibitors form a permanent complex with an enzyme via formation of covalent bonds with certain amino acids in the active site of an enzyme
Consequently, they cannot be displaced by the addition of excess substrate
Therefore, they are irreversible, non-competitive enzyme inhibitors
Diagram/series of reactions for suicide inactivators
E+I⇌(inhibitor binds)EI(normal catalytic reaction starts at t active site⇌E…I (reactive species generated) and forms E-I (inactivated enzyme)
NB: enzyme treats the inhibitor as if it is a “normal” substrate/substance
Mechanism-based inhibitors depend on…
Depend on the specific mechanism of catalysis of a particular target enzyme
Advantages of mechanism-based enzyme inhibitors
- Absolute specificity: they depend upon substrate-like binding recognition and upon specific mechanism of catalysis in the active site of a particular enzyme
- High efficiency of enzyme inhibition (covalent complex)
What is Tienilic acid?
Drug marked as a diuretic agent
Suicide inactivator
Withdrawn due to interaction with cytochrome P450 enzymes in the liver, where it acts as a suicide substrate
What helps design powerful inhibitors?
Understanding of enzyme mechanism of reaction helps to design more powerful inhibitors
Transition state analogues
It is possible to design inhibitors that bing extremely strongly to the active site.
One approach to achieve this is to design a drug that resembles the transition state of the substrate in the active site
BUT
Drug design can be based on reaction intermediates which are closer in character to transition states than substrates or products
Define transition state
High energy, transient species
Cannot be isolated or synthesised
What are transition state analogues designed to mimic?
How strongly do they bind?
The transition state of the enzyme-catalysed reaction
They are more likely to bind more strongly than drugs mimicking the substrate or product
Penicillins are what type of enzyme inhibitor?
Is there an alternative theory?
Penicillins are irreversible suicide inhibitors of transpeptidase
Penicillin becomes covalently linked to the enzyme’s active site
Alternative theory: Penicillin mimics D-Ala-D-Ala
How does penicillin resistance occur?
Via beta-lactamases
These enzymes inactivate penicillins by opening beta-lactam rings
This allows bacteria to become resistant to penicillin
Beta-lactamase mechanism of action:
Mechanism of action for beta-lactamases is identical to the mechanism of inhibition of the target enzyme (i.e.transpeptidase)
But the product is removed effectively fro the lactamase active site
How can you overcome penicillin resistance?
Use clavulanic acid
What is Clavulanic acid?
-weak, unimportant antibacterial activity
BUT
-Powerful irreversible suicide inhibitor of beta-lactamase (enzyme that causes bacterial resistance to penicillins)
-used as a guard drug to ampicillin
Where does clavulanic acid come from
Isolated from
Streptomyces clavoligenus
Augmentin=
Ampicillin+clavulanic acid
Allows less ampicillin per dose and increased activity spectrum
Timentin=
Ticarcillin + clavulanic acid
Why should a transition state be bound more strongly to an enzyme that a substrate or product?
It is ONLY HYPOTHESIS:
- it is proposed that the binding interactions between an enzyme and a substate are optimal during the transition state of the enzyme-catalysed reaction
- this is a reasonable proposition since the speed and effectiveness of a catalysed reaction is crucially dependent on how much the catalyst stabilises the transition state
How alter the transition state to make the reaction occur more easily and make the enzyme more effective?
The more stable the transition state, the easier the reaaction will occur and the more effective the enzyme will be as a catalyst
Therefore
It is more important that enzymes form their strongest interactions with ‘guest molecules’ at the transition state of the reaction, rather than the substrate or the product
Strong interactions with substrate or product would be…
Detrimental because it would result in slow “turnover” with substrate and product spending too much time in the active site
Name an enzyme of a transition state inhibitor and describe how it works
Renin inhibitors (Antihypertensives)
Renin is a protease enzymes which is responsible for hydrolysing a specific peptide bond in the protein angiotensinogen
This converts angiotensinogen into angiotensin I, which then (via an ACE enzyme) is converted into angiotensin II
Angiotensin II is a vasoconstrictor and constricts blood vessels, thus raises blood pressure. Renin blocks synthesis of of angiotensin I and thus, angiotensin II
Describe how renin works
Renin contains two aspartyl residues and a bridging water in the active site
The first stage involves formation of a tetrahedral intermediate via processing through a high energy transition state
The two aspartyl resides are involved in the enzyme-catalysed reaction and the tetrahedral intermediate is involved.
What is Aliskiren? What does it do?
Aliskiren is a renin inhibitor
- it contains a hydroxyethylene transition state mimic
- it therefore mimic the tetrahedral geometry of the usual reaction intermediate when renin catalyses angiotensinogen
- it mimics one of the hydroxyl groups (binding group)
- it is stable, no leaving group present
Tetrahedral geometry of transition states is in what enzymes?
Statins
Renin
ACE inhibitors
Protease inhibitors
Is transition-state inhibition usually reversible or irreversible?
-Reversible inhibition, unless very tight binding, as no covalent bond formed
Criteria for transition-state inhibition?
- Reversible (unless v.tight binding)
- Close structural resemblance to postulated transition state
- Tighter binding to the enzyme than substrate binding
- often seen as competitive inhibition as active site is targeted and increase in substrate concentration may displace the inhibitor. This may mean that the transition state analogue forces the enzyme to adopt a new conformation
- sometimes inhibition is slow (minutes to hours) as conformation is crucial
Are transition state inhibitors competitive or non-competitive
Often seen as competitive as active site is targeted
Increase in substrate concentration may displace the inhibitor
This may mean that the transition state analogue forces the enzyme to adopt a new conformation
