Enzymes as Drug Targets - a Closer Look
Transition state analogs and “suicide substrates”
Enzymes as Drug Targets - a Closer Look
Transition state analogs and “suicide substrates”
Enzymes - Background
What do they do? Enzymes increase the rate of, but do not change
equilibrium position of most reactions that occur in the cell. Catalysts!
S
P (uncatalyzed)
(enzyme catalyzed)
Relative transition-state
stabilization lowers kinetic barrier,
increases rate (k2)!
S
P
Enzymes - Background
(enzyme catalyzed)
How is enzyme activity measured? Using kinetics (measuring rates:
the rate of appearance of product or the rate of disappearance of
substrate)
Enzymes can become saturated
with substrate ([ES] = [E]tot).
Enzymes as Drug Targets - a Closer Look
The majority of enzyme-targeted marketed drugs are related to the
enzyme substrate structure.
Understanding the nature of enzyme catalysis AND the mechanism
of a biochemical reaction can lead to the design of effective drugs
•Substrate and Transition-state structure
•Enzyme mechanism
Enzyme Inhibition by Small Molecules
•What small molecules inhibit enzymes?? Cellular regulators, drugs,
toxic agents
•Inhibitors decrease the effectiveness of the enzyme as a catalyst (so
inhibitors can be drugs; and some enzymes are good targets)
•Inhibitors can be Reversible or Irreversible.
Reversible (competetive, noncompetitive, uncompetetive; kinetics
experiments can distinguish between these modes of inhibition - see
appendix):
Reversible enzyme inhibitors decrease enzyme activity reversibly
Reversible Inhibitors (continued)
To design a reversible competetive inhibitor as a drug, design a
mimic of the substrate or the transition state.
Lecture on Protein targets listed some examples of reversible
inhibitors as drugs
1. Transition state mimic for adenosine deaminase (enzyme
which degrades anticancer drugs)
2. Substrate mimic for dihydropteroate synthase (dihydrofolate
synthesis)
3. Transition state mimic for HMG-CoA reductase (cholesterol
synthesis)
Substrate versus Transition-state analogs: Which approach should
result in the highest affinity drug? Why?
Reversible Inhibitors (continued). The transition state is stabilized
more than the substrate
H B Enzyme
Example 1:
Isopentyl
Diphosphate
isomerase
O
O
HO P O P O
OH OH
:B Enzyme
CH3
H
H H
Enzyme -B:
H
O
O
HO P O P O
OH OH
CH3
H H
H
H
H
Enzyme -B:
O
O
HO P O P O
OH OH
CH3
H
Enzyme -B
Carbocationic Intermediate
Resembles transition state
O
O
HO P O P O
OH OH
:B Enzyme
CH3
N CH3
H
H H
Transition-state analog inhibitor
H
H
H
H
Reversible Inhibitors (continued).
Example 2: Purine nucleoside phosphorylase. Lower activity causes
T-cell immunodeficiency. Potential therapy for T-cell cancer and Tcell autoimmune disorders.
Inhibitors were designed with KD in the nanomolar range
Transition state structure was determined with analogs of substrates:
Reversible Inhibitors (Ex. 2 continued).
Ki = 23pM
Reversible Inhibitors (Ex. 2 continued).
Structures of bovine enzyme target +
Transition-state
Substrate analogs
mimic
Products
These show how the enzyme binds the transition state more strongly
than the substrate.
Reversible Inhibitors (Ex. 2 continued). What about the human
enzyme???
87% homologous to bovine enzyme
Ki ~60pM (weaker binding than bovine enzyme)
Active site structure is completely conserved, so it must have a different
transition state structure (and therefore a different transition state analog)
Ultimate
inhibitor:
Inhibits for
lifetime of cell!!
Reversible Inhibitors (Ex. 2 continued).
Ultimate inhibitor: Inhibits for lifetime of cell!!
If the structure of target enzyme complex revealed additional
potential binding interactions (empty hydrophobic pocket, etc),
an even stronger drug could be designed.
Recap:
•Reversible enzyme inhibitors bind reversibly!
•Competetive inhibitors’ structure should be more similar to
that of the transition state for stronger binding
•Noncompetetive and uncompetetive inhibitors can’t be
“designed”, because they don’t resemble the substrate or
transition state.
Irreversible Inhibitors: Affinity labels, suicide substrates - form
covalent bonds with the enzyme
Affinity labels = molecules that
•Resemble the substrate, so targeted to binding site;
•Contain an electrophilic group (below, or alpha-halo ketones, or
diazoketones) that reacts with a nucleophilic group of the enzyme in
or near the active site to form a covalent bond.
Irreversible Inhibitors - affinity labels (continued)
•Somewhat (or very) toxic because they are so reactive - they
react at other sites than the enzyme binding site.
Ex. 1 Penicillin - resembles acyl D-ala-D-ala and it acylates the
active site serine of transpeptidase. Steric bulk or conformational
changes prevents hydrolysis or transamidation.
Irreversible Inhibitors - affinity label examples (continued)
Ex. 2. TPCK (Tosyl-phenylalanyl-chloromethyl-ketone). Binds to
active site of chymotrypsin (binds Phe, trp). Contains an
electrophilic carbon that forms covalent bond with chymotrypsin
active site histidine.
Big problem - how to avoid reactions with other nucleophiles on
other proteins?
Mask the reactive electrophile until it is in the active site:
Suicide Substrate/Trojan Horse Inhibitor/Mechanism-based Inhibitor!
Irreversible Inhibitors - Suicide substrates
Ex. 1. Halo enol lactones and serine proteases
H
O
O
Br
One reac tive site (elec trophilic ester)
Irreversible Inhibitors - Suicide substrates
Ex. 2. Vigabatrin, an anticonvulsant that inhibits a pyridoxyl
phosphate-dependent enzyme that degrades GABA (neurotransmitter).
H 2N
H 2N
CO2
GABA
Vigabatrin--No reactive
electrophilic center
Part of mechanism for amine
substrates in pyridoxal-dependent
enzymes:
(Intermediate 4.19 can lose H+,
CO2, and may undergo further
reactions).
CO2
Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont)
Normal substrate for aminotransferase: H NH RCO
2
B:
BH
O2C
H
N
O
HO P O
OH
4.19
R
BH
O2C
R
N
H
O
HO P O
OH
OH
N
H
2
H
OH
N
H
O2C
R
N
O
HO P O
OH
H
OH
N
H
One new electrophilic center
Suicide substrate for aminotransferase:
B:
BH
H
N
O
HO P O
OH
H
H 2N
N
H
BH
CO2
CO2
N
H
OH
CO2
O
HO P O
OH
N
H
H
OH
CO2
N
O
HO P O
OH
H
OH
N
H
TWO new electrophilic centers!
Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont)
Reactivity of cationic intermediates: N+ is a good electron “sink,”
making the molecule susceptible to nucleophilic attack. The
nucleophile may be a group on the enzyme, or another molecule
O2C
R
N
O
HO P O
OH
H
OH
N
H
CO2
N
O
HO P O
OH
H
OH
N
H
Michael addition
Nu:
Nu:
N
H
N
H
Irreversible Inhibitors - Suicide substrates Ex. 2 Vigabatrin, (cont)
Normal substrate: final products. Enzyme unchanged and active
O2C
R
N
O
HO P O
OH
NH2
H2O
H
OH
O
HO P O
OH
N
H
OH
O2C
+
R
O
N
H
Suicide substrate: two pathways for products, one which
inactivates the enzyme!
NH2
CO2
N
O
HO P O
OH
N
H
H
OH
H2O
O
HO P O
OH
Enz-Nu:
CO2
OH
+
N
H
O
CO2
Enz-Nu:
N
Active site
nucleophile in
reach of this electrophile
O
HO P O
OH
N
H
Inactivated
enzyme!
H
OH
Recap: Irreversible Inhibitors: affinity labels, suicide substrates
Affinity labels:
•Contain a reactive electrophile that reacts with an enzyme’s
nucleophile to form a covalent (irreversible) bond
•Toxic because the electrophile is too reactive to be specific.
Suicide substrates/mechanism-based inactivators:
•Designed to produce a reactive electrophile only upon binding to
the correct enzyme and undergoing normal catalytic steps
Additional Example 1: JACS 2003, 125 p. 685
Inhibitors of AmpC beta lactamase were developed:
Due to widespread resistance, inhibitors of beta-lactamases are
sought. Clavulanic acid (d) is one inhibitor; ceftazidime (b) is a betalactam that is resistant to beta-lactamases. New substrate analogs “c”
are found to inhibit new broad spectrum beta-lactamases. All have
similar structure: resistance to these are also anticipated.
Additional Example 1 (cont)
Alternate strategy: de novo structure-based design. Have found
novel structures unlike natural substrate that circumvent traditional
resistance mechanisms, but they are weak, with Ki = 25 micromolar.
A third strategy:
Beta-lactamase
intermediate:
Transition state analogs.
The beta-lactam ring is replaced
With the boronic acid; R1 can be
changed to improve affinity.
Investigators focused on “c”:
carboxylate mimics cephalosporin
Carboxylate in transition state.
Ki = 20nM
Additional Example 1 (cont)
Best inhibitor:
O
S
O
B
HO
OH
O-
1nM inhibitor
Stereo view of the
molecule above bound to
the enzyme AmpC
If the Carboxylate is removed,
binding decreases by 30-fold
But now, except for boronic acid,
the molecule looks a bit like a
beta lactam…Will resistance be a
problem?
Additional Example 1 (cont)
Resistance is hardest to develop against analogs that resemble
substrates….A resistant organism must distinguish between inhibitor
and substrate (since it must act on the substrate!).
Transition state analogs do resemble the substrate to some degree…
Additional Example 2 Hepatitis C virus therapy. Target: HCV NS3 protease, a serine
protease that is essential to viral replication.
Serine proteases have a “catalytic triad” of residues in the active site.
Mechanism:
a.
b.
Substrate
binds to
active site
Asp-his
help make
ser a better
Nu.
e. Asp-his make
water a better Nu
that attacks carbonyl
of ester
c. Ser attacks
carbonyl of
amide, forming
a tetrahedral
intermediate
d. Asp-his-H+
helps makes
amine a better
leaving group
(Peptide strand
is broken; one
part is released
from enzyme)
Additional Example 2 (cont)
f. New tetrahedral
intermediate
is formed
g. Asp-his-H+
help make ser
a better
leaving group.
Suicide substrate for a serine protease:
Alpha keto-amide may be attacked by
serine, “trapping” the enzyme
h. Enzyme is back
to original
state. Other
part of peptide
is released.
Additional Example 2 (cont)
Note: No leaving
group attached to
the carbonyl, so
serine -OH will not
cleave the drug.
References
Robertson, J. G. “Mechanistic Basis of Enzyme-Targeted Drugs” Biochemistry, 2005,
44, 5561-5571.
Silverman, R. B. The Organic Chemistry of Drug Design and Drug Action ; Academic
Press: San Diego, CA, 1992
Schramm, V. L. “Enzymatic transition states: thermodynamics, dynamics and analogue
design” Arch. Biochem. Biophys. 2005, 433, 13-26.
Venkatraman, S.; Njoroge, F. G.; Girijavallabhan, V. M.; Madison, V. S.; Yao, N. H.;
Prongay, A. J.; Butkeiwicz, N.; Pichardo, J.“Design and Synthesis of depeptidized
macrocyclic inhibitors of Hepatitis C NS3-4A Protease using structure-based drug
design” J. Med. Chem., 2005, 48, 5088-5091.
Appendix: Enzyme kinetics
No inhibitor:
Simplification of kinetic scheme (by
rapid equilibrium or steady state
approaches) leads to the MichealisMenten equation.
Competetive Inhibition
V
Noncompetetive (mixed)
Uncompetetive Inhibition
Molecular Diversity- Synthetic approaches:
(Note: in synthesis, “target” is the molecule you want to synthesize; in drug discovery,
“target” is the biological macromolecule you want to develop a drug to bind to)
1. Traditional (synthetic target-oriented; know structure of product; one product in one
reaction vessel)
A. Solution phase or Solid phase (beads)
B. Protecting groups used, high yields desirable
C. Parallel (can be solid or solution phase; simultaneous synthesis of many
compounds)
D. Location of active compound in a grid allows determination of structure of
active compound
2. Combinatorial (many different products in one vessel)
A. Use of solid phase, protecting groups, and “mix and split” is most common
synthetic approach
B. Deconvolution or encoding is required to determine structure of active
compound
3. “Chemical structure space” versus “biological structure space”; how to improve
your chances of getting a “hit”?
A. Natural product-guided combichem
B. Diversity-oriented synthesis (smaller libraries of more complex structures that
look more like natural products than the simpler compounds made in standard
combinatorial libraries.
C. Click chemistry
D. Dynamic combinatorial chemistry