Enzyme Inhibition and Drug Action

•Malfunction of enzyme Disease

•Introduction of enzyme by microorganism

Inhibition of enzyme - Interesting but difficult drug strategy

OH NH
N CO2H
N N
Inhib. of enzymes from microorganisms H
H2N N N
Antibact. sulfonamide CO2H
O R O Folic acid
H2N S NH
OH OH
O
N
N N
O H From food
H2N H2N N N Higher animals
OH H
- Enzyme only in microorg. PABA Dehydropteridinsyre
- Different structure of enzyme, O O
human and microorg OH NH OH NH
H
CO2H Folat- CO2H
N N
N N reduktase N N
H H
H2N N N H2N N N
H H CO2H
CO2H
Tetrahydrofolic acid Trimetoprim Dehydrofolic acid
NH2
OCH3
N
Essintial processes,
animals and bacteria H2N N OCH3
OCH3
Enzyme inhibition

k3 E: Enzyme
k1 S: Substrate
E + S [ E-S ] [ E-P ] E + P 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] = Michaelis const.: KM = (k2 + k3) / k1
(k2 + k3) / k1

[E] [S] [E] = [E tot] - [E-S]

[E-S] =
KM

([Etot] - [E-S] [S] [Etot] [S]

[E-S] = [E-S] =
KM [S] + KM
 k3 E: Enzyme
k1
S: Substrate
E + S [ E-S ] [ E-P ] E + P P: Product
k2

[Etot] [S]
[E-S] = V=k3 [E-S]
[S] + KM

k3[Etot] [S] Vmax: All enzyme sites occupied by S

V =
[S]
[S] + KM ≈1
[S]>>KM, Vmax=k3 [Etot]
[S] + KM

Vmax [S]
V = Michaelis Menten eq.
[S] + KM V

Vmax

Vmax : 2

KM [S]
 V

Vmax
Vmax [S]
V = Michaelis Menten eq.
[S] + KM
Vmax : 2

KM [S]

1
V

KM KM
1 1 1 Slope =
= + Lineweaver-Burk eq. Vmax
V Vmax [S] Vmax

-1
1
KM
Vmax
Measure rate at different [S]:
Determine KM and Vmax 1
[S]
Reversible and irreversible enzyme inhibitors
E + 1 [ E-I ] E + 1 [ E-I ]

Reversible inhibition If covalently bond to enzyme, bond relatively easily be broken

•Competitive i.e. hydrol. of ester
•Non-competitive

E +S [ E-S ] P E + I [ E-I ] P

1 With inhibitor
No inhib.
V

KM
Slope =
Binding to the same site Vmax
Inhib. can be reversed by increasing [S]
-1
Vmax unchanged KM
1
Vmax
KM increase
1
[S]
Structural resemblance S and I
Designed I drugs - Antimetabolites
 O
Ex. antimetabolites OH NH
N CO2H
N N
H
H2N N N
Antibact. sulfonamide CO2H
O R O Folic acid
H2N S NH
OH OH
O
N
N N
O H From food
H2N H2N N N Higher animals
OH H
PABA Dehydropteridinsyre

OH NH
N CO2H
Essintial processes, N N
H
animals and bacteria H2N N N
H CO2H

O
O
Me
Me HN
HN
HIV O N
O N
Reverse transcriptase HO
HO RNA chain O
O
AZT
N OH
HO OH
N
N
Thymidin
 Ex. transition state analogs

NH2 H2N OH O
N HN N HN N
N Adenosine
N deaminase N N
Also metab. of anticancer / antiviral
N N N
HO HO HO adeninederiv.
O O O

HO OH HO OH HO OH

Adenine Inosine

sp3

OH
N
HN
N
HO
O

HO OH

Coformycin
Reversible inhibition
•Competitive
•Non-competitive

E +S [ E-S ] P
+I

[E-S-I]

E + I [ E-I ] +S

With inhibitor
1
No inhib.
V
Binding to different sites
Inhib. can be reversed by increasing [S] KM
Slope =
Vmax decrease Vmax
KM unchanged -1
1
KM
Vmax

Inhib. and substrate very different structures 1

Difficult to design inhib. [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

Pe nicillins are cle ave d by β-lactam ase

O OH
O OH
O N β-Lactam ase O
HN
O
R N S S
H H R N H
H

Clavulanic acid irreversibly inhibits β-lactam ase

OH O O O OH
OH O OH O
O
N O HN H O HN

O OH H O OH
H O OH
H H H Nu
Nu
Nu

O OH
O
O HN
HH O OH
Nu