Download Enzyme Inhibition and Drug Action

Enzyme Inhibition and Drug Action
•Malfunction of enzyme
•Introduction of enzyme by microorganism
Disease
Inhibition of enzyme - Interesting but difficult drug strategy
O
NH
OH
N
N
Inhib. of enzymes from microorganisms
H2N
N
CO2H
N
H
N
CO2H
Antibact. sulfonamide
O
R
S NH
O
H2N
H2N
H2N
OH
- Enzyme only in microorg.
- Different structure of enzyme,
human and microorg
OH
OH
N
N
O
Folic acid
O
N
N
H
From food
Higher animals
N
H
Dehydropteridinsyre
PABA
O
O
OH
N
H2N
N
H
N
NH
N
H
N
H
Tetrahydrofolic acid
Trimetoprim
OCH3
N
H2N
N
N
N
H
N
H
Dehydrofolic acid
NH2
Essintial processes,
animals and bacteria
N
N
H2N
CO2H
NH
OH
CO2H Folatreduktase
OCH3
OCH3
CO2H
CO2H
Enzyme inhibition
k3
k1
[ E-S ]
E + S
[ E-P ]
E + P
E: Enzyme
S: Substrate
P: Product
k2
Two last steps ≈irreversible, E-S to E-P rate limiting
Reaction velocity, V=k3 [E-S]
Rate of form. ES: k1[E][S]
Rate of decomp. ES: (k1 + k3)[E][S]
Assume steady state ([E-S] doesn’t change)
k1[E][S] = (k1 + k3)[E][S]
[E-S] =
[E] [S]
(k2 + k3) / k1
[E-S] =
Michaelis const.: KM = (k2 + k3) / k1
[E] = [E tot] - [E-S]
[E] [S]
KM
[E-S] =
([Etot] - [E-S] [S]
KM
[E-S] =
[Etot] [S]
[S] + KM
k3
k1
[ E-S ]
E + S
[ E-P ]
E + P
E: Enzyme
S: Substrate
P: Product
k2
[E-S] =
[Etot] [S]
V=k3 [E-S]
[S] + KM
V =
k3[Etot] [S]
[S] + KM
V =
Vmax [S]
[S] + KM
Vmax: All enzyme sites occupied by S
[S]
[S]>>KM,
Michaelis Menten eq.
Vmax=k3 [Etot]
≈1
[S] + KM
V
Vmax
Vmax : 2
KM
[S]
V
V =
Vmax
Vmax [S]
Michaelis Menten eq.
[S] + KM
Vmax : 2
KM
[S]
1
V
1
V
=
KM
Vmax
1
[S]
+
1
Vmax
Slope =
Lineweaver-Burk eq.
-1
KM
Measure rate at different [S]:
Determine KM and Vmax
KM
Vmax
1
Vmax
1
[S]
Reversible and irreversible enzyme inhibitors
E + 1
[ E-I ]
E + 1
Reversible inhibition
•Competitive
•Non-competitive
E +S
[ E-S ]
[ E-I ]
If covalently bond to enzyme, bond relatively easily be broken
i.e. hydrol. of ester
P
E + I
P
[ E-I ]
1
With inhibitor
No inhib.
V
Binding to the same site
Inhib. can be reversed by increasing [S]
Vmax unchanged
KM increase
Structural resemblance S and I
Designed I drugs - Antimetabolites
Slope =
-1
KM
KM
Vmax
1
Vmax
1
[S]
O
Ex. antimetabolites
N
N
H2N
Antibact. sulfonamide
H2N
O
R
S NH
O
OH
PABA
N
CO2H
N
H
N
CO2H
O
H2N
N
N
Folic acid
OH
OH
N
O
H2N
NH
OH
N
H
From food
Higher animals
N
H
Dehydropteridinsyre
O
NH
OH
N
N
Essintial processes,
animals and bacteria
H2N
N
CO2H
N
H
N
H
CO2H
O
O
Me
HN
O
HO
N
O
HO
Me
HN
HIV
Reverse transcriptase
O
RNA chain
HO
N
O
AZT
OH
Thymidin
N
N
N
OH
Ex. transition state analogs
NH2
N
N
N
N
HO
Adenosine
deaminase
N
HN
OH
N
N
HO
O
HO
N
HN
N
N
HO
O
HO
O
H2N OH
OH
O
HO
OH
Inosine
Adenine
sp3
OH
N
HN
N
HO
O
HO
OH
Coformycin
Also metab. of anticancer / antiviral
adenine deriv.
Reversible inhibition
•Competitive
•Non-competitive
E +S
P
[ E-S ]
+I
[E-S-I]
E + I
[ E-I ]
+S
1
Binding to different sites
Inhib. can be reversed by increasing [S]
Vmax decrease
KM unchanged
Inhib. and substrate very different structures
Difficult to design inhib.
With inhibitor
No inhib.
V
Slope =
-1
KM
KM
Vmax
1
Vmax
1
[S]
Irreversible enzyme inhibitors
E + 1
[ E-I ]
Often covalent bonds between E and I
Enzyme is permanently modified and inactivated
•Affinity labels and active site directed irreversible inhibitors
•Mechanism based irreversible enzyme inactivators
Structural recemblance with substrate
Electrophilic - alkylate nucleophilic subst in enzyme active site
Low selectivity - generally highly toxic
•Affinity labels and active site directed irreversible inhibitors
•Mechanism based irreversible enzyme inactivators
Suicide substrate - kcat inhibitors - Trojan horse inhib. - latent alkylating agent
≈ Pro-drug, must be activated by the enzyme
Penicillins are cleaved by β-lactamase
O
OH
O
N
O
R
N
H
H
O
β-Lactamase
O
S
R
OH
HN
N
H
H
S
Clavulanic acid irreversibly inhibits β-lactamase
OH
O
O
OH
N
H
Nu
O
O
O
O
H
H
H
O
O
O
HN
H
OH
OH
OH
O
OH
HN
H
O
OH
Nu
Nu
O
O
O
OH
HN
HH
O
Nu
OH