Enzymes as Drug Targets - a Closer Look

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Enzymes as Drug Targets - a Closer Look
Transition state analogs and suicide substrates
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Enzymes as Drug Targets - a Closer Look
Transition state analogs and suicide substrates
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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!
P (uncatalyzed)
S
(enzyme catalyzed)
Relative transition-state stabilization lowers
kinetic barrier, increases rate (k2)!
S
P
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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
Etot).
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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

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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)

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Reversible enzyme inhibitors decrease enzyme
activity reversibly
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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?
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Reversible Inhibitors (continued). The transition
state is stabilized more than the substrate
Example 1 Isopentyl Diphosphate isomerase
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Reversible Inhibitors (continued).
Example 2 Purine nucleoside phosphorylase.
Lower activity causes T-cell immunodeficiency.
Potential therapy for T-cell cancer and T-cell
autoimmune disorders.
Inhibitors were designed with KD in the nanomolar
range
Transition state structure was determined with
analogs of substrates
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Reversible Inhibitors (Ex. 2 continued).
Ki 23pM
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Reversible Inhibitors (Ex. 2 continued).
Structures of bovine enzyme target
Transition-state mimic
Substrate analogs
Products
These show how the enzyme binds the transition
state more strongly than the substrate.
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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!!
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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 cant
  be designed, because they dont resemble the
  substrate or transition state.

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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.

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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.
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Irreversible Inhibitors - affinity label examples
(continued)
Ex. 2. TPCK (Tosyl-phenylalanyl-chloromethyl-keto
ne). 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!
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Irreversible Inhibitors - Suicide substrates
Ex. 1. Halo enol lactones and serine proteases
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Irreversible Inhibitors - Suicide substrates
Ex. 2. Vigabatrin, an anticonvulsant that
inhibits a pyridoxyl phosphate-dependent enzyme
that degrades GABA (neurotransmitter).
Part of mechanism for amine substrates in
pyridoxal-dependent enzymes
(Intermediate 4.19 can lose H, CO2, and may
undergo further reactions).
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Irreversible Inhibitors - Suicide substrates Ex.
2 Vigabatrin, (cont)
Normal substrate for aminotransferase
One new electrophilic center
Suicide substrate for aminotransferase
TWO new electrophilic centers!
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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
Michael addition
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Irreversible Inhibitors - Suicide substrates Ex.
2 Vigabatrin, (cont)
Normal substrate final products. Enzyme
unchanged and active
Suicide substrate two pathways for products,
one which inactivates the enzyme!
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Recap Irreversible Inhibitors affinity
labels, suicide substrates

• Affinity labels
• Contain a reactive electrophile that reacts with
  an enzymes 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

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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
beta-lactam 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.
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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.
Beta-lactamase intermediate
A third strategy
Ki 20nM
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.
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Additional Example 1 (cont)
Best inhibitor
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 lactamWill resistance be
a problem?
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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
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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

• c. Ser attacks carbonyl of amide, forming a
  tetrahedral intermediate
• Asp-his-H helps makes amine a better leaving
  group (Peptide strand is broken one part is
  released from enzyme)

1. Substrate binds to active site
2. Asp-his help make ser a better Nu.

e. Asp-his make water a better Nu that attacks
carbonyl of ester
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Additional Example 2 (cont)
f. New tetrahedral intermediate is formed g.
Asp-his-H help make ser a better leaving group.
h. Enzyme is back to original state. Other part
of peptide is released.
Suicide substrate for a serine protease Alpha
keto-amide may be attacked by serine, trapping
the enzyme
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Additional Example 2 (cont)
Note No leaving group attached to the carbonyl,
so serine -OH will not cleave the drug.
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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.
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Appendix Enzyme kinetics
No inhibitor
Simplification of kinetic scheme (by rapid
equilibrium or steady state approaches) leads to
the Michealis-Menten equation.
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Competetive Inhibition
V
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Noncompetetive (mixed)
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Uncompetetive Inhibition
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• 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)
• Traditional (synthetic target-oriented know
  structure of product one product in one reaction
  vessel)
• Solution phase or Solid phase (beads)
• Protecting groups used, high yields desirable
• Parallel (can be solid or solution phase
  simultaneous synthesis of many compounds)
• Location of active compound in a grid allows
  determination of structure of active compound
• Combinatorial (many different products in one
  vessel)
• Use of solid phase, protecting groups, and mix
  and split is most common synthetic approach
• Deconvolution or encoding is required to
  determine structure of active compound
• Chemical structure space versus biological
  structure space how to improve your chances of
  getting a hit?
• Natural product-guided combichem
• Diversity-oriented synthesis (smaller libraries
  of more complex structures that look more like
  natural products than the simpler compounds made
  in standard combinatorial libraries.
• Click chemistry
• Dynamic combinatorial chemistry