Transition state analogs as inhibitors:
HIV Protease
Creighton: chapter 9.2-9.3
Goals:
Introduction to enzyme structure and inhibition
Case study: Inhibition of HIV protease
• Introduction:
• Why is HIV protease a good drug target?
• Why is it difficult to design an anti-protease drug?
• What is an enzyme
• What is a protease
• Mechanism of HIV protease
• Structure of HIV protease
• Evolution of aspartyl proteases
• Design of transition state analog inhibitors
Why is Human Immunodeficiency Virus (HIV) protease
a good drug target?
According to the UNAID 4th Global Report (2004):
35.7 million adults, 2.1 million children are infected with HIV
In 2003, 700,000 children under 14 years of age became infected
Sub-Saharan Africa has the greatest infection rate:
- 10% of the world’s population with 60% of the HIV cases
- 26.6 million people with HIV
US: - 800,000 to 900,000 people with HIV
- 40,000 new infections per year
1
Role of HIV protease in the lifecycle of the virus
Role of HIV protease in the lifecycle of the virus
Problems in developing drugs against HIV
1) Resistance mutations:
Mutation rate is very high: 1 per 1000 to 10,000 bases
106 times higher than in humans.
High because HIV reverse transcriptase does not “proofread”
2) Cost: $1,000-$2,000 per month
2
HIV proteolytic cut sites
A protease inhibitor has to be 98% effective
to prevent viral infectivity
Ribbon diagram of HIV protease bound to JE-2147
flaps
substrate
binding
pocket
active site Asp’s
dimer domain
3
Ribbon diagram of HIV protease bound to JE-2147
JE-2147 is a transition state analog with Ki = 40 pM
Uncatalyzed biological reactions can be very slow
The depth of chemical time and the power of enzymes as catalysts
R Wolfenden and MJ Snider
Acc. Chem. Res. (2001) 34: 938-945
Uncatalyzed biological reactions can be very slow
The depth of chemical time and the power of enzymes as catalysts
R Wolfenden and MJ Snider
Acc. Chem. Res. (2001) 34: 938-945
4
Enzymes accelerate reactions.
The amount of acceleration depends on the enzyme.
ADC arginine decarboxylase
ODC orotidine 5’-phosphate
decarboxylase
STN staphylococcal nuclease
GLU swee potato β-amylase
FUM fumarase
MAN mandelate racemase
PEP carboxypeptidase B
CDA E. coli cytidine deaminase
KSI ketosteroid isomerase
CMU chorismate mutase
CAN carbonic anhydrase
The depth of chemical time and the power of enzymes as catalysts
R Wolfenden and MJ Snider
Acc. Chem. Res. (2001) 34: 938-945
A catalyst lowers the energy
of the transition state.
The transition state
S
knon
P
free energy
S
transition state: highest
energy intermediate.
Determines the rate of the reaction
S – substrate
P - product
S
P
reaction coordinate
5
Thermodynamics of the transition state
S
S
P
assumes rapid equilibrium
between substrate and
transition state:
K =[S ]
[S]
S
free energy
Treat like equilibrium:
∆G
∆G = - RT ln K
S
P
∆Greaction
reaction coordinate
catalyst: a substance that accelerates the rate of a reaction
without itself being permanently altered.
Rate enhancement results from stabilizing the transition state
free energy
uncatalyzed
S
Sc
∆∆G‡
catalyzed
S
P
reaction coordinate
A catalyst does not change the overall equilibrium
of a reaction
free energy
uncatalyzed
S
Sc
catalyzed
S
P
∆Greaction
remains
unchanged
reaction coordinate
6
Enzymes are designed to stabilize the transition state
more than the substrate
∆GES < ∆GS
S
free energy
ES
∆GS
∆GES
S
uncatalyzed
catalyzed
∆Greaction
unchanged
ES
P
EP
reaction coordinate
Enzymes increase reaction rates by
stabilizing the transition state
Uncatalyzed reaction
S
P
simple enzymatic mechanism
ES + S
ES
EP + P
more complex mechanism
kcat
k1
ES + S
ν
K
ES
ES
EP + P
What is a protease?
7
What is a protease?
Proteases cleave the peptide bond.
What is a protease?
Proteases cleave the peptide bond.
Resonance structures of peptide bond
Families of proteases
• Serine proteinases - trypsin, chymotrypsin
• Cysteine or thiol proteinases - bromelain (pineapple),
papain (papaya latex)
• Aspartic (acid) proteinases - pepsin (gastric stomach)
• Metallo-proteinase - collagenase (Clostridia), keratinase
Convergent evolution
8
The best understood proteolytic mechanism is for
the Serine Protease family, eg trypsin:
peptide
binding
sites
protease
subsites
1) polypeptide substrate binds
noncovalently with side chains
in the binding pockets
Tetrahedral transition state
Asp
Asp
H is
H is
O
Ser
C
Ser
C
O
O
H
O
H
CH2
N
N
H
CH2
N
N
O
O
H
oxyanion
hole
R
R
R
1
R
1
N
C
sp3
sp2
H
N
C
H
O
O
O
Ser
The activate site Ser is activated
through Asp and His (the catalytic
triad). Ser donates a proton to
histidine ring, Asp stabilizes
the protonation of the His.
C
||
OH
stabilize
Gly
Tetrahedral transition state: the
activated Ser attacks the carbonyl
of the peptide bond forming a
tetrahedral intermediate.
Acyl intermediate
A sp
Asp
H is
H is
Ser
C
O
O
H
CH2
N
N
O
Ser
C
O
O
H
CH2
N
N
O
H
H
R
R
1
N
H
R
R
C
1
O
N
H
C
O
Cleavage yields an acyl-enzyme intermediate, N-terminal part of
the substrate is covalently bound to the enzyme
9
The active site water
Asp
His
Ser
C
O
O
Transfers a proton of the water
to His. The hydroxyl links to the
acyl intermediate to form the
second transition state.
Asp
H
His
CH2
N
Ser
C
N
H2 O
O
R1 NH2
H
O
H
O
O
H
CH 2
N
R
N
O
H
C
R
O
O
C
H
Water binds to the enzyme
in place of the departed
polypeptide
O
Asp
His
Ser
C
O
O
H
CH 2
N
N
The proton is transferred from
His back to Ser. The remaining
peptide is released and the
enzyme returns to original state.
H
O
R
O
H
C
O
Mechanism of Aspartic Proteases
• Active site residues - 2 Asp’s
• Mechanism is still debated.
• No acyl intermediate
• Catalytic nucleophile is a water molecule
• Active site Asps work together in general acid-base catalysts
• The pKa values of the Asp residues are crucial
• One Asp has a relatively low pKa,
the other has a relatively high pKa
Titration of glutamic acid
O
||
C—OH
|
CH2
|
CH2
|
NH3+--Cα—C00-
protonated
carboyxlate
equivalents OH added
O
||
C—O|
CH2
|
CH2
|
NH3+--Cα—C00-
unprotonated
carboxylate
neutral pH
10
Titration of Glycine
amino group
equivalents OH- added
carboxylate group
R
H3N+ – Cα – COOH
H
R
R
H3N+ – Cα – COO-
H2N – Cα – COO-
H
acidic pH
H
neutral pH
basic pH
Evidence For General Acid-Base
Mechanism----Bell Shaped pH profile
Evidence For General Acid-Base
Mechanism----Bell Shaped pH profile
pKa 3.5
pKa 5.5
11
Most accepted mechanism of aspartic proteases
oxyanion
hole
Deprotonated Asp acts as
general base, accepting a
proton from HOH forming
OH- in the transition state
Protonation of scissile
nitrogen
tetrahedral
intermediate
Other Asp acts as
general acid, donating
a proton, facilitating formation
of tetrahedral intermediate
Breakdown of tetrahedral
intermediate
Proposed concerted mechanism of Aspartyl proteases
Concerted attack of scissile peptide bond by active site Asp
and active site water. No tetrahedral intermediate, but
a tetrahedral transition state.
12
Architecture of HIV protease
monomer 1
monomer 2
C
N
Structure of HIV protease and its relation to activity
flaps
substrate
binding
pocket
active site Asp’s
dimer domain
Binding of JE-2147 to the active site of HIV protease
Ki ~ 40 pM
13
Active site of HIV protease
Evolution of aspartyl proteases
Most mammalian aspartic proteases are monomeric, with a
tertiary structure consisting of two lobes (N-terminal and Cterminal) with approximate two-fold symmetry
HIV-1 protease is a homodimer – the evolutionary pre-cursor
14
If you want to design an inhibitor that mimics a natural
reactant, should it mimic S, P or S ?
∆GES < ∆GS
S
free energy
ES
∆GS
∆GES
S
uncatalyzed
catalyzed
∆Greaction
unchanged
ES
P
EP
reaction coordinate
HIV Protease inhibitors
What does a transition state look like for HIV protease?
Two-step mechanism of aspartic proteases
tetrahedral intermediate
15
What does a transition state look like for HIV protease?
Proposed concerted mechanism of Aspartyl proteases
FDA approved anti-protease drugs:
filling the binding pockets
FDA approved anti-protease drugs:
filling the binding pockets
16
FDA approved anti-protease drugs:
filling the binding pockets
Broad substrate specificity
8 distinct sites cleaved by HIV protease
Protein dynamics:
problem with “rational” drug design:
• rigid body motions
• overall flexibility
• side chain flexibility
17
Comparison of “open” and “closed” protease structures
liganded HIV protease
unliganded SIV protease
Energetic barrier to substrate binding
ES + S
ES
ES
EP + P
ES
free energy
E +S
E +S
ES
P
EP
reaction coordinate
Dynamics and flexibility of HIV Protease:
concerted motions
anisotropic B-factors
increased movements in beta sheet
dimer domain upon ligand binding
18
HIV resistance mutations:
an added barrier to drug design
mutation rate: each possible point mutations
occurs 104 to 105 times per day for every base of viral DNA
19